Citation
Lipoprotein structure

Material Information

Title:
Lipoprotein structure apoprotein interactions in human plasma high density lipoproteins
Creator:
Grow, Thomas Ellis, 1945-
Publication Date:
Copyright Date:
1977
Language:
English
Physical Description:
xi, 137 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Apoproteins ( jstor )
Cholesterols ( jstor )
Crosslinking ( jstor )
Gels ( jstor )
HDL lipoproteins ( jstor )
Incubation ( jstor )
Lipids ( jstor )
Lipoproteins ( jstor )
Molecules ( jstor )
Reagents ( jstor )
Biochemistry and Molecular Biology thesis Ph. D
Blood lipoproteins ( lcsh )
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF
City of Pensacola ( local )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 132-136.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Thomas Ellis Grow.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
022577089 ( ALEPH )
04673926 ( OCLC )
AAH9681 ( NOTIS )

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LIPOPROTEIN STRUCTURE: APOPROTEIN INTERACTIONS
IN HUMAN PLASMA HIGH DENSITY LIPOPROTEINS














By

THOMAS ELLIS GROW


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THEII UNIVERSITY OF FrLORIDA
IN PARTIAL FULFILLMENT OF THEI REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY









UNIVERSITY OF FLORIDA



















DEDICATION

I dedicate this dissertation to my best friend William

A. Patterson and the Krenu Construction Company.


















.ACKNOWLEDGEMENTS

The author would like to express his deep appreciation to

Professor Melvin Fried, his research director, for giving him guidance

and freedom of thought and action during the completion of this work.

The environment of Professor Fried's laboratory was exactly what the

author required for productive work.

The author also wishes to thank his wife, Susan, for her patience,

understanding, and sacrifices made during the last four years.

Special thanks are given to the author's advisory committee and

members of the faculty of the Department of Biochemistry for their

suggestions and criticisms during the execution of this research.

The author also thanks his parents for their understanding during

his years of education.




















TABL.E OF CONTENTS

ACKNOWLEDGEMENTS ...............


. . i


LIST OF TABLES . . . . . . . . . . . . .

LIST OF FIGURES . . . . . . . . . . . .

KEY TO SYMBOLS AND ABBREVIATIONS NOT DEFINED IN TEXT ......

ABSTRACT . . . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . .

Plasma Lipoproteins ... ... .. .... .........


Apoprotein Structure and Function ..............

Lipoprotein Structure ....................

Alternative Classification of Lipoproteins ..........

Exchange Reactions of Lipoproteins .............

Experimental Rational ....................

Research Objectives ... ..................

MATERIALS AND METHODS .....................

Materials . . . . . . . . . . . . .

Methods . . . . . . . . . . . . . .

RESULTS AND DISCUSSION .....................


Isolation and Purity of Lipoprotein Preparations ......


Crosslinking of Total HDL (HDLT) with DFDNB .........

Effect of Crosslinking on HDL Structure ...........


Crosslinking of HDL2 and HDL3 with DFDNB ..........

Crosslinking of HDLT with DMS and DFDNDPS ..........

Temperature Dependence of Crosslinking. ...........


.vi

vil

.ix

.x

1

1

4

S6

11

.12

.15

.18

.20

.20

.21

.31

.31

.36

.42

.42

.43

.50











Interpretation of Crosslinking Results. ... .. .. .. .59

Apoprotein Exchange Between HDL2 and HDL3 . .. .. ... .. .61

Effect of Crosslinking and Monofunctional Labeling
on Apoprotein Exchange. .. .. .. .. .. .. .. ... .66

Effect of Temperature and Dilution on Peptide Exchange .. .69

Rate of Peptide Exchange ... .. ... .. .. .. .. .69

Effect of Bromide lan on Protein Exchange .. .. .. .. .. .80

SDS-Polyacrylamide Gel Analysis of Exchanged Proteins .. .. .80

Interpretations of Protein Exchange Experiments .. . ... .85

Lipid Exchange Between HDL2 and HDL3 in Solution .. .. .. .88

Binding of Labeled HDL to Sepharose .. .. . .. .89

Peptide Exchange Using Sepharose-Bound HDL .. .. .. .. .91

Ultracentrifugal Analysis of Peptide Exchange Using
Sepharose-Bound HDL . .. ... . .. . . . . 91

SDS-PAGE Analysis of Exchanged Peptides Using
Sepharose-Bound RDL . .. .. .. .. ... . 96

Incorporation of Labeled Lipid into RDL2 and HDL3 ... .. 103

Lipid Exchange Between HDL3 and Sepharose-HDLj .. .. .. 106

Effect of Temperature and Dilution on Lipid Exchange .. .. 112

Effect of Saturating Cold Lipid on Labeled Lipid Exchange .. 112

Effect of Crosslinking on Lipid Exchange .. .. .. . 118

Effect of Added Organic Solvent on Exchange of Lipids
and Proteins . .. .. .. .. . .. . .. . 118

Interpretations of Sepharose-HDL Peptide and Lipid
Exchange Experiments ... .. .. .. .. . . . 125

CONCLUSIONS AND SPECULATIONS . ... .. .. ... . .. 128

BIBLIOGRAPHY .. .. .. .. .. .. . .. . . . 32

BIOGRAPHICAL SKETCH .... .. .. ... .. .. . .. 137


















LIST OF TABLES


TABLE

1

2



3



4

5


6


PAGE


Bifunctional Reagents ...........

Amino Acid Analysis of 46,000 Dalton
Crosslinked Product from RDLT Cross-
linked with DFDNB... .........

Effect of Amount of CNBr Activation
on the Amount of Binding of HDL3 by
Sepharose 48 ........

Peptide Exchange Using Sepharose-Bound HDL

Removal of Labeled Lipid from Sepharose-
Bound HDL3 by SDS Wash ..........

Effect of Temperature and Dilution on
Lipid Exchange Between S-HDLg and RDL3 ..

Effect of Saturating Cold Lipid on Lipid
Exchange . . . . . . . . .

Effect of Crosslinking on Lipid Exchange .


. 90

. 92


. . .107


. . ..117


. .. .119

. ...120





























































. . .68

. . .71


. . .73

. . .75


LIST OF FIGURES


FIGURE


PAGE


1 SDS-PAGE of solubilized apoproteins
of IlDLy . . . . . .

2 Separation of HDL2 and HDL3 A d=1.125gm/cm3. ..

3 SDS-PAGE of RDLT crosslinked with DFDNB ......

4 Experimental approach to amino acid analysis
of crosslinked products .............

5 Comparison of the SDS-PAGE methods of
Fairbanks and Lammli for the separation


..33

..35

..38


..40


of highly crosslinked RDLT ............

,S-PAGE of HDL2 crosslinked with DFDNB .......

,S-PAGE of HDL3 crosslinked with DFDNB .......

IS-PAGE of HDLT crosslinked with DMS ........

IS-PAGE of HDLT crosslinked with DMS (5% BME). ...

IS-PAGE of HDLT crosslinked with DFDNDPS ......

S-PAGE of HDLT crosslinked with DFDNDPS
(5% BME). . . . . . . . . . . .

change of apoproteins between [12I]-HDL3
and HDL2 . . . . . . . . . .
125
change of apoproteins between [ I]-HDL2


. . .


.45

.47

.49

.52

.54

.56


.58


.63


.65


6 SD

7 SD

8 SD

9 SD

10 SD

11 SD


12 Ex


13 Ex


d na HD
@


14 Effect of crosslinking on apoprotein exchange .

15 Effect of FDNB labeling on apoprotein exchange

16 Effect of crosslinking the unlabeled subclass
on apoprotein exchange ...........

17 Effect of temperature on apoprotein exchange .











18 Effect of temperature and dilution on
apoprotein exchange .. . .. .. . .77

19 Trime course of upoprotein exchanglpe .. .. .. .79

20 Effect of KBr on apoprotein exchange .. .. .. .82

21 Polyacrylamide gel analysis of exchanged apoprotein .84

22 Polyacrylamide gel analysis of exchanged apo-
proteins: Effect of crosslinking with DFDNB. .. .87
23 Suunitexchnge etwen S-125I]HL n D 9
24 Submnit exchange between S-[12I]-HDL2 and HDL2 9


25 Polyacrylamide gel analysis of available
apoproteins from S-HDL .. .. .. .. .. .. 100

26 Polyacrylamide gel analysis of apoproteins
exchanged from S-[125I]-HD) L ..... 102

27 Uptake of labeled lipids by HDL3 .. .. .. .. 105

28 Effect of increasing amounts of cold HDL, on
lipid exchange .. .. .. .. .. .. 109

29 Effect of increasing amounts of cold HDL3 on
lipid exchange .......... 111

30 Rate of lipid exchange between S-HDL3 and HDL .. 114

31 Time course for lipid exchange between
S-HD~g and HDL3 .. .. .. ... .. .. .. 116

32 Effect of ethanol of the exchange of lipids
from S-HDL~ to HDL ........ 122

33 Effect of ethanol on the exchange of lipid
and protein from S-HDL~ to RDL3 .. ... .. .' 124

34 Model for HDL3 HDL2 interconversion .. . ... 131


















KEY TO SYMBOLS AND ABBREVIATIONS NOT DEFINED IN TEXT


Ai angstrom

Apo apoprotein

CD circular dichroism spectroscopy

Ci curie

dpm disintegrations per min.

IR infrared spectroscopy

LCAT Lecithin cholesterol acyl transferase

leu leucine

MW molecular weight

NMR nuclear magnetic resonance
spectroscopy

ORD Optical rotory dispersion

val valine










Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy





LIPOPROTEIN STRUCTURE: APOPROTEIN INTERACTIONS
IN HUMAN PLASMA HIGH DENSITY LIPOPROTEINS

By

Thomas Ellis Grow

June, 1977

Chairman: Melvin Fried
Major Department: Biochemistry

The modes of association of the major apoprotein -species of human

plasma high density lipoprotein (HDL) was investigated using bifunctional

crosslinking reagents. How these associations help to govern the struc-

ture of the RD)L subclass, HDL2 and HDL was studied by observing both

apoprotein and lipid exchange between HDL particles.

Crosslinking of HDL with a 5 to 10-fold molar excess of difluoro-

dinitrobenzene resulted in the production of high molecular weight

crosslinked products with the major product shown to be the combination

of the two major apoprotein species ApoA-1 and ApoA-2. The composition

of this product was confirmed by amino acid analysis.

Crosslinking with other bifunctional reagents indicatesspecificity

of initial reaction sites as well as some freedom of movement of the

apoproteins within the particle. However, the crosslinking patterns

obtained with the RDL subclasses were identical, suggesting specific

structural properties of HDL2 and HDL3

Apoproteins, labeled with 12i, were shown to exchange between and

among the RIDL subclasses. This bidirectional exchange process was










inhibited by crosslinking with bifunctional reagents and appeared

to depend upon collision complex formation. By the utilization of

Sepharose bound IIDL it could be shown that both free apoprotein mole-

cules, as well as subunits consisting of lipid-apoprotein combinations,

could exchange between HDL2 and HDL3. The exchange appears to involve

a conformational change in the apoproteins during the transfer process.

Radioactive cholesterol, cholesterol palmitate, and phosphatidy1

choline, bound to HDL after adsorption to celite particles, could be

shown to undergo rapid exchange between MDL particles. This exchange

also appears to depend on collision complex formation but was not

inhibited by chemical crosslinking of the apoproteins.

The rate of these exchange processes is significant in the life-

time of the lipoprotein particles in vivo: 2.5%/hour for apoprotein

exchange and >50%/hour for lipid exchange.

It was concluded that ApoA-1 and ApoA-2 must lie very close to one

another in the intact lipoprotein particle. The reproducibility of

the crosslinking experiments on HDL obtained from several donors suggests

that in the intact HDL particle the apoprotein components may be found

in a rather fixed spatial orientation to one another. It is also

suggested that there is a dynamic relationship between HDL2 and HDL .

Even though these two subclasses can exist as stable separate entities,

when they are present together in solution, significant interaction

between particles may occur. Exchange of lipids and apoproteins occurs

between KDL2-HDL2 and HDL -HDL as well as between HDL2 and HDL3 mole-

cules.

It is suggested that the interconversion of RDL2 and HDL3 may be

determined by the availability of certain lipids.

















INTRODUCTION

Plasma Lipoproteins

Plasma lipoproteins play an important role in mammalian energy

metabolism and also serve as the major transport particles for many

physiologically important molecular species. It has been suggested

that they are causally related to atherosclerosis since they are the

carriers for those substances deposited in the atheromatous plaque.

Circulatory system pathology found in hyperlipoproteinemics may result

from quite small alterations in lipoprotein structure; differences that

may be due to altered protein-protein or protein-lipid interaction.

Human serum contains four major types of lipoproteins: chylo-

microns (CHYL), very low density lipoproteins (VLDL), low density

lipoproteins (LDL), and high density lipoproteins (HDL). Although

these four classes appear to-differ in both lipid and protein compo-

sition as well as function, there are distinct interrelationships

between the particles. The presence of one class directly influences

the other classes (1).

Chylomicrons, formed in the intestinal mucosa and released into

the blood from the major lymphatic duct, function as the carrier of

many lipid types. Lypolytic enzymes remove lipids from these particles~

as they circulate. VLDL and LDL appear to be closely related as LDL

may be the result of VLDL delipidation (2). LDL functions as the

major cholesterol carrier in blood. HDL can be divided into two











subgroups; HDL2 and RDL3. HDL may be important in cholesterol

deposition disease (3).

The isolation of lipoproteins from serum or plasma is relatively

simple and may be accomplished by flotation in the ultracentrifuge.

As the density (d) of serum lipoproteins ranges from approximately

0.95 gm/cm3 to 1.21 gm/cm3, centrifugation of serumo, the density of

which has been raised to 1.21 gm/cm3 by the addition of salt, will

result in the lipoproteins floating to the top of the centrifuge tube

while the other proteins sink (4). Each lipoprotein class is charac-

terized by its own density range for isolation: CHYL and VLDL < 1.006,

LDL 1.006 1.063, and HDL 1.063 1.21. By stepwise increases in

density (4), or by density gradient centrifugation (5), these lipo-

protein classes may be prepared in relatively pure form. Although

other methods such as molecular sieve chromotography (6) and chemical

precipitation (7) have been described, the ultracentrifugal floatation

method remains the most widely used.

The protein and lipid compositions of these lipoprotein classes

have been studied and the following have been reported (1,8) (all

percentages on a dry weight basis).

HDL

HDL, whose molecular weight ranges from 180,000 to 350,000,

consists of approximately 50% protein, 22% phospholipids, 3% free

cholesterol, 14% cholesterol esters, and 8% triglycerides. Of the

phospholipid portion, 70% is phosphatidylcholine 14% is spingomyelin.,

5% phosphatidy1 serine and phosphatidyl ethanolamine, and the remain-

der other minor phospholipids. The protein portion of the RDL particle










consists of two major polypeptides, ApoA-1 (ApoGln-I) and ApoA-2

(ApoGln-II). The amino acid sequences of both polypeptides are known

(9,10,11) and ApoA-2 is known to be a dimer of two identical subunits

connected by a single disulfide bridge at position 6 (10). Another

group of smaller proteins, designated ApoC or the C-peptides, comprises

approximately 5% of the total HDL protein. At least three C-peptides,

C-I, C-II, and C-III, are recognized in HDL (2). The protein of HDL

contains 3-4% covalently bound carbohydrate including glucosamine,

fructose, galactose, mannose, and sialic acid (8). In addition to the

C-peptides, KDL also may contain the "thin-line" protein and "arginine

rich" protein as minor components (1).

LDL

LDL particles are larger than HDL with molecular weights ranging

from 2 to 3 million. They also contain a larger percentage of lipid

than HDL (75-78%); consisting of 36-42% cholesterol esters, 21-23%

phospholipids, 8-9% free cholesterol, and 6-8% triglycerides. Other

lipids may be present in smaller amounts. The proteins make up 20-22%

of the particle. The major polypeptide is denoted apoprotein B (ApoB),

a large protein of molecular weight 225,000. There may also be very

small amounts of the C-poptides described for liDL. Again, 3-5% carbo-

hydrate is found covalently associated with the protein.

VLDL

VLDL particles cover a wide range of densities and molecular

weights (3 128 x 106) with at least two maxima at densities of 0.980

and 0.958 gm/cm3. They contain 90% lipid with an average composition

of 50-60% triglycerides, 10-12% free cholesterol, 4-6% cholesterol










esters, and 18-20% phospholipids. Protein represents only 8-12% of

which 40% is apoprotein B and the remaining 60% is divided among the

three C-peptides described for HDL. A small portion of ApoA may also

be present and it has been demonstrated that different fractions may

contain different amounts of apoproteins (2).

Chylomicrons

Chyl contain mostly lipid with only 1-2% protein. These very

large particles have molecular weights of 5 4300 x 108. The protein

consists of all types of previously described apoproteins found in the

other lipoprotein classes. It has been shown that human lymph ch-ylo-

microns contain approximately 66% ApoC, 22% ApoB, and 12% ApoA. The

lipids are predominately triglycerides (80%) with only about 8%

phospholipids and 5% cholesterol (1,2).

It is important to note the overlapping protein composition in

all of these classes. It is presumed that the specific protein compo-

sition of each is important in determining its total lipid content and

the specific type of particle that is formed. These proteins should

play a very important role in determining the function of the particle

as well (1).


Apoprotein Structure and Function

ApoC

ApoC-I has been sequenced (12) and is a single polypeptide chain

of 57 amino acid residues (MW 8000). ApoC-I is known to function in

lipid binding as well as LCAT activation (13). Lipoprotein lipase

also appears to be activated by ApoC-I (14).











ApoC-II is the largest of the C-peptides withi 100 amino acid

residues and a molecular weight of about 12,500 (15). It also binds

lipids and is a potent activator of lipoprotein lipase (16).

ApoC-III, a single polypeptide of 79 amino acid residues has also

been sequenced (17). Attached to threonine-74,ApoC-III contains an

oligosaccharide moiety consisting of galactose, galactosamine, and

sialic acid (17). Although no direct physiological role is yet known,

ApoC-III has been used often for the study of lipid-protein inter-

actions (18-21).

ApoB

The characterization of ApoB has led to many conflicting statements

concerning the number and size of the polypeptide subunits. Due to

technical problems associated with isolation of a soluble apoprotein,

a variety of physical methods have indicated molecular weights ranging

from 8,000 to 275,000 (22). However,.most investigators support the

idea of a large (275,000) molecular weight peptide. These problems

have also hindered studies on specific functions of ApoB and as yet

no definite physiological role other than lipid binding has been dis-

covered.



ApoA is a term used to describe the two most extensively studied

apolipoproteins, ApoA-1 and ApoA-2. ApoA-1, a single polypeptide of

molecular weight 28,330, contains 245 amino acid residues and has been

sequenced (8,23,24). Although the binding of phospholipids by ApoA-1

has been studied by several investigators, conflicting results on lipid

binding in the absence of ApoA-2 have been reported (25,26,27). These










differences in the extent of lipid binding by ApoA-1 may be due to

self-association of the polypeptides that may mask lipid binding sites

(28). Self-association has been demonstrated by both ultracentrifugal

and chemical techniques (1),

ApoA-2 is a polypeptide dimer of two identical chains with a

molecular weight of 17,500 (29). Each chain contains 77 amino acid

residues. The d-isuffide bond occursu between thec single cysteine

residues that occur at position 6 (29). The disulfide linked dimer

appears to be characteristic of only man and chimpanzee; the dimer form

apparently is not crucial for ApoA-2's lipid carrying role (1). ApoA-2

does bind lipid strongly and may increase the binding capacity of

ApaA-1 as well (2).

Although lipid binding is the only role as yet assigned to ApoA-2,

ApoA-1 appears to have several functions other than lipid binding in

the HDL particle. ApoA-1.serves as an activator of LCAT (30), may be

an acceptor of cholesterol and cholesterol ester during VTLDL catabo-

lism (2), and appears to have some function in regulating the lipid

content of membranes (31,32).


Lipoprotein Structure

The study of lipoprotein structure has been approached utilizing

three basic types of techniques: 1) spectroscopic methods (IR, CD,

ORD, X-ray, neutron scattering, NMR, and fluorescence); 2) physical

techniques (Ultracentrifugation and electron microscopy); and 3) chem-

ical methods (chemical modification, amino acid analysis, bifunctional

reagents, reassembly). The actual composition of lipoprotein particles

has been fairly well described, as indicated above, and only in the










case of L~DL is there significant disagreement on Apoprotein size and

composition (1,8). However, when three-dimensional structure is

considered, little is known at present regarding specific protein-

protein or protein-lipid spatial relationships or interactions in

intact lipoprotein particles.

VLDL and Chyl have proved difficult to study due to the signifi-

cant turbidity of their solutions. This fact has limited the use of

spectroscopic methods and as a result' only the techniques of ultra-

centrifugation, electron microscopy, and ge1 filtration have been

used. This, coupled with the fact that these classes consist of a

spectrum of a~ wide variety of sizes with differing compositions, has

prevented all but the most basic of structural studies. Ultracentri-

fugation techniques indicate the following: Chyl MW 5-4300 x 108

daltons, diameter 1200-11000 A, hydrated density 0.93gm/cm VLDL-

MW 3-128 x 106 daltons, diameter 300-700 A, hydrated density 0.93

1.0gm/cm3 (1). The only structural determinations made thus far for

these particles are based on thec surface activity of the protein

components which seems to indicate thaot some portion of the proteins

are located on the surface of the particle (2), The information now

available has led to the suggestion of a lipid-core model for the

triglyceride richChyland VLDL particles. This model assumes that the

surface of the particle is occupied by phospholipids, cholesterol, and

proteins, while the central core consists of the less polar and more

hydrophobic triglycerides and cholesterol esters (33). The quantities

of protein, phiospholipid and cholesterol in these particles appear to

be sufficient to cover the surface of the particles as predicted (34).










LDL structure has been studied by many of the different techniques

mentioned earlier (2,8,35,36,37,38). Data from electron microscopy

and X-ray diffraction indicate a spherical particle with neither well

defined nor significant subunit structure (2). Information from

analytical ultracentrifugation, as well as X-ray and electron micro-

scopy, give the following information: MIW 2.3-2.7 x 106 daltons,

hydrated density 1.028-1.034gm/cm3, diameter 216-220 A. In distinction

to Chyl and VLDL, LDL is rather uniform in size and composition. The

protein component appears to have about 25% a-helix, 37% B-structure,

and 37% random coil as determined by CD (38). Although this structure

appears to be temperature dependant, a recent report indicates the CD

spectrum may be greatly complicated by the lipids present in the

particle (39). Both NMR and fluorescence studies indicate that the

protein, although near the surface of the LDL particle, is highly

associated with the lipid components of the molecule (36,37). It has

also been suggested that the core of LDL is mostly composed of hydro-

carbon chains while the outer layer of the particle is sparsely

occupied by proteins that emerge from the lipid core (35); a model

similar to the lipid-core model for VLDL. Other investigators,

utilizing electron microscopy and X-ray scattering, have suggested a

significant degree of subunit structure that includes many protein

subunits (20-60 with MW 8,000 or 27,000) arranged with icosahiedral

symmetry. The protein subunits give a symmetrical appearance with

the surface area occupied by both protein and phospholipid (40,41).

These subunit models do not take into consideration recent findings

that indicate the major protein component consists of only two poly-

peptides of molecular weight 250,000 or greater (42).










HDL has been studied most extensively, probably because it is

the easiest of the particles to work with. As noted above, its pro-

tein composition is well known and the major polypeptides have been

sequenced. RDL can be divided into two subclasses, HDL2 and HDL ,

which appear to differ mainly in their molecular weights (HDL2

184,000) (1).~ X-ray studies have indicated that RDL consists of two

regions of differing electron densities: an outer shell and an inner

core. The inner core has a radius of approximately 43 A in HDL2

(37 A in HDL3) while the outer shell radius is 14 A (11 A in HDL3).

The electron density of the outer shell indicates that is consists of

polar lipid headgroups and proteins (43). The size of a fully extended

phospholipid headgroup is about 11 A, a value that fits in well with

the X-ray data. Fluorescence techniques yield information on porcine

HDL3 consistent with a peripheral location of the protein component,

with the suggestion that as much as 80% of the protein is located at

or near the surface (37). This hypothesis is further supported by the

'finding of Scanu that 90% of the lysine residues of the apoprotein

components of human HDL is accessible to succinylating agents (2).

This would indicate that most of the protein is near the surface as

there are many lysines rather evenly distributed throughout the pro-

tein chains.

CD of the protein portions of HDL indicate 70% a-helix, 11%

B-structure, and 19% random coil (38). The removal or alteration of

11pid components greatly affects the structure of HDL apoproteins.

Delipidation lowers the helicity to 52%. Delipidation followed by

relipidation with egg phosphatidyl choline changes a-helicity to 64%











while thle addition of cholesterol further increases the helicity to

70% (38). These observations indicate strong lipid-protein interactions.

Two basic models of HDnL that are consistent with spectroscopic and

chemical data have been proposed. Assman,et al. and Assman and Brewer

(22,44) have suggested a model similar to the membrane structure pro-

posed by Singer and Nicolson (45) in which the HDL protein is depicted

as an "iceberg" floating in a "sea of lipid." This model takes into

account the fact that portions of the helical regions of the apoproteins

are two-sided; that is, one side of the helix is polar while the other

is non-polar (46). This amphipathic organization of the helix could

allow simultaneous interaction of the helix with lipid, protein, and

the aqueous medium. Whether or not the helices are oriented perpen-

dicular or parallel to the surface of the particle isthe_ essential

difference between this model of Assman,et al. and Assman and Brewer

(22,44) and that of Jackson, et al. (25). Jackson has suggested that

the long axes of the helical regions are oriented perpendicularly to

the fatty acy1 chains of the phospholipids. In this model, carbon

atoms 2-4 of the fatty acy1 chains could interact with the non-polar

side of the helix, allowing the hydrophyllic portions to interact with

the medium or other protein chains. There is evidence to suggest that

little electrostatic interaction occurs between phospholipid polar

headgroups and the charged amino acid residues of intact lipo-

proteins (22,47,48).

Little is known of the protein-protein interactions that may occur

in intact lipoproteins. The only studies reported have been carried

out on human HDL apoproteins in vitro. It has been shown that if one

mixes the RDL apoproteins A-1 and A-2 together in equimolar amounts,











they appear to associate into an aggregate of molecular weight

46,000 (28). This A-1;A-2 complex will bind less lipid than would

be expected, indicating that some of the lipid binding sites are

blocked by protein-protein -interaction. Other studies indicate that

in the absence of lipids and other apoproteins, both A-1 and A-2 will

self-associate (49-53). Although such studies help to understand the

types of interactions that may occur, only work done on the intact

particle can shed light upon the actual associations and interactions

that govern the final structure and function of the lipoproteins.

Recent evidence from Friedberg and Reynolds (54) suggests that

A-1 and A-2 always exist in a molar ratio of 2 A-1 polypeptides to

1 A-2 polypeptide dimer in human HDL. Their study also suggests that

this ratio of 2:1 is constant from individual to individual as well as

from subclass to subclass (HD~L2 to HDL3). This would indicate that

perhaps, for ApoA-1 and ApoA-2, a specific polypeptide composition is

found in HDL2 (4 A-1 + 2 A-2) as well as in HDL3 (2 A-1 + 1 A-2).

It is evident that much is yet unknown concerning the structure

of lipoproteins and that experimental evidence derived from studies

utilizing intact particles and techniques that allow precise definition

of spatial orientation is needed.


Alternative Classification of Lipoproteins

It should be noted at this point that another meth~od of classifi-

cation for plasma lipoproteins has been suggested by Kostner and

Alaupovic (55). Based on immunochemical evidence, they have suggested

that lipoproteins occur in separate families, the composition of which

is determined by the presence of specific apoproteins. The three






12



families they describe, LP-A, LP-B, and LP-C, represent particles

containing ApoA-1 and/or ApoA-2 (LP-A), ApoB (LP-B), and ApoC-I-III

(LP-C). The important difference between the two methods of classi-

ficatiohn is that, according~ to Kostner and Alaupovic, a physically

defined density class, such as HDL, may contain not only particles

with just ApoA-1 and ApoA-2 but also particles with only ApoB and

only ApoC. One should realize, however, that HDL isolated as a density

class will contain mostly LP-A and LP-C with a very small amount of

LP-B (2%). This LP-B is only found associated with the hDL2 density

subclass suggesting the possibility of contamination by remnant LDL

particles. Even the presence of particles containing only LP-C could

be an artifact resulting from the generation of incomplete or altered

particles during the extensive ultracentrifugation required for the

isolation of the HDL density class. In any case, the actual differ-

ence between HDL as a density class and LP-A as described by these

investigators would only be the presence of the small amount of C

peptides generally believed to be associated with the HDL molecule.

Although the possibility of lipoprotein families is a question that

merits consideration, the results of the experiments described herein

would apply to either model of the lipoprotein particles.


Exchange Reactions of Lipoproteins

Plasma lipoproteins are known to be a very dynamic population of

macromolecules. They are continuously being synthesized and degraded

at a high rate. The amount of any individual lipoprotein class present

at a given time is determined by the balance between synthesis and

catabolism. The individual components of lipoproteins, however, are










not degraded or synthesized at the same rate. This is due to the fact

that both lipid and protein components can be exchanged between indi-

vidual lipoprotein molecules as well as between different classes of

lipoproteins (1).

Lipid Exchange

Phospholipids appear to exchange between all classes of serum

lipoproteins at rates dependent upon their relative concentrations in

the individual classes (56,57). When labeled phospholipids are incor-

porated into an individual class of lipoproteins and this class is

allowed to interact with other lipoprotein classes in vivo or in vitro,

an exchange reaction with equilibrium, reached in 4 to 5 hours, is

observed (58). This exchange is independent of any protein exchange

and occurs at significantly different rates for different phospho-

lipid types. Lysalecithin exchanges most rapidly, followed by lecithin

and sphingomyelin (57,59). No significant differences are observed for

subclasses of an individual phospholipid class (59). This exchange can

be increased in vitra by the presence of a phospholipid exchange

protein (60).

Cholesterol is known to exchange rapidly between all classes of

human serulm lipoproteins (61). This rapid exchange occurs both in vivo

and in vitro wFth equilibrium attained after 2 to 6 hours~ (62,63).

Cholesterol ester exchange between plasma lipoproteins in vivo and

in vitra has been both suggested and disputed (62,64,65). There is

evidence that seems to indicate that cholesterol enters can be trans-

ferred from LDL to other lipoproteins in vitro (66).











The exchange of other lipid types has been reported for human as

well as other animal lipoproteins. Triglycerides exchange between

different classes of lipoproteins (67) as do unesterified fatty acids

(68) and a-tocopherol (69)..

Protein Exchange

Although the exchange of lipids does not appear to depend upon

the simultaneous exchange of protein, it has been shown that apoprotein

exchange does occur. Only ApoC of HDL and VLDL has been shown to

exchange in vitro and in vivo (70,71). This exchange is bidirectional

and not like the apparent transfer of protein from VLDL to LDL (2).

Mechanisms of Exchange

The movement of either lipid or protein from one lipoprotein class

to another can represent an exchange, a transfer, or the combination

of the two processes. It is evident that both processes depend upon

the lipoproteins existing in a dynamic state.

The in vitro exchange of lipids has been the most studied and the

process appears to be a physicochemical one (72). Exchange could occur

by at least two mechanisms. First, it has been suggested that exchange

occurs when individual lipid molecules escape the lipoprotein particle

and enter the aqueous medium. They are then picked up by other lipo-

proteins or membranes (73,74). This model for exchange lacks strong

evidence in its support but may be the mechanism for certain specific

lipid types (74).

An alternate mechanism involves the formation of collision.complexes

between lipoproteins (75). This would allow for diffusion of lipid

and/or protein molecules between the particles and at the same time











would not require the thermodynamically unfavorable dissociation of

hydrophobic molecules into an aqueous medium.-

As both mechanisms have been supported with experimental evidence,

it is important that each be considered as a possible explanation for

any exchange reaction. Experiments that will belp distinguish

between the two mechanisms would include the observation of the effect

of factors that influence collision rates between molecules, i.e.

temperature, dilution, etc.

Although only lipid transfer mechanisms have been postulated,

the same types of models should be considered when observing protein

transfer or exchange.


Experimental Rational

In studying either protein structure or protein-protein inter-

actions occurrences of natural crosslinking due to disulfide bridges,

peptide linkages between lysine E-NH2 and glutamic or asparatic acid

y or B-COOH, dr carbohydrate bridges have been valuable in under-

standing the final active structure that is being considered, e.g.,

insulin, ribonuclease, collagen. As such crosslinks are often not

found where one might like to find them, the introduction of stable

covalent bridges or crosslinks can aid in the study of protein

structure (See Ref. (76) for a recent review of bifunctional reagents).

Bifunctional reagents are capable of producing three types of cross-

linked products: intramolecularly crosslinked proteins, intermolec-

ularly crosslinked homopolymers (identical subunits), and inter-

molecularly crosslinked protein complexes nonidenticall subunits or

different proteins). Such reagents have been used in a variety of ways










to determine such things as the spatial geometry of membrane components

(77-81), the mapping of ribosome proteins (82), and the subunit

structure of oligomeric proteins (83).

There are two basic types of crosslinking reagents: those that

are cleavable and those that are not. The advantage of cleavable

crosslinkers is that one may isolate the crosslinked product, cleave

the newly formed bridges, and identify the proteins that were cross-

linked. This technique is particularly useful when studying the

crosslinking of multicomponent systems such as membranes.

Table 1 lists a selection of different types of crosslinkers and

gives certain specific characteristics. The selection of reagents

with differing lengths, degrees of hydrophobic character,or reactive

group specificity can yield products that differ because of the specific

structure or physical environment of the proteins that are crosslinked.

For example, in studying the human erythrocyte membrane, Moxley and

Tsai were able to produce different crosslinking patterns by varying

only the length of the bifunctional reagent used (81).

As all lipoprotein particles are thought to contain two or more

polypeptide chains and since the structure and function of these

particles may depend to a great extent upon specific protein composition

and/or protein-protein inlteractions, investigation wlthl btfunctiolnal

reagents could prove to be a useful method to define some of these

interactions.

Because the function and characteristics of individual lipoprotein

classes differ, and these.differences ultimately must be related to

structure, studies dealing with the interactions and associations of










Table 1.


Bifunctional Crosslinking Reagents


STRUCTURE REACTS WITH

N ztCI NiallI Lysine, Tyrosine

3,C-O--(C-(CH, I C-O-CH,


LENGTH


CLEAVED


REAGENT

1. Imidoesters
(i.e. dimethyl
suberimidate)


2. Dinitro Difluoro-
Benzene


Variable


O
F N(De


5-6 A



9-10 A


Lysine, Tyrosine



Lysine, Tyrosine



SH, E-Amino
(High pH)


3. Difluoro Dinitro- F ~ F
Diphenyl sulfone ( >-0

Cz NCOz
4. N, N-Di (Bromoacetyl)
Phenylhydrazine


Pd + HOAc


5. Dimethyl Dithiobis
propionimidate
Dihydrochoride


8 Mercapto-
ethanol


~E-Amino
NH c

HzC -C-2-(CH,);-5- -(cH,a- c- o-CH,

NHCI











the characteristic apoproteins of a given lipoprotein class should

prove of value in understanding how these characteristics are deter-

mined by the structure.

KDL, the best characterized of the lipoproteins, was used in the

experiments described in this report. Its small size, stable structure,

ease of isolation and handling, as well as its variety of polypeptides

made it the model of choice. Although no definitive physiological

role has yet been assigned to HDL, certain other characteristics make

this an interesting model for study. C-peptides such as are found in

HDL are known activators of LCAT and lipoprotein lipase, and Apo-1 is

known to activate LCAT specific for certain substrates (1). Reynolds

and Simon (28) have reported altered lipid binding upon associations

of the apoproteins of HDL. Perhaps this interaction is a mechanism for

regulation of lipid binding. As the different lipoprotein classes

appear closely related and even share many of the same components, both

lipid and protein, the actual association of the different protein

components may play a very important role in regulating both structure

and function for the different lipoprotein particles.


Research Objectives

The purpose of this research was to gain insight into the structure

of intact serum lipoprotein particles specifically, the high density

lipoprotein particle. This was approached by attempting to understand

the interactions and associations of the individual apoprotein compo-

nents of these macromolecules. To aid in organizing the information

gathered, the following objectives were set forth:










1. To determine if bifunctional reagents are capable of inducing

crosslinks between individual apoproteins in intact HDL

2. To characterize and identify any crosslinked products pro-

duced

3. To evaluate the result of the use of crosslinking reagents of

differing length and hydrophobic or hydrophyllic character

4. To determine and explain any possible differences in the

crosslinking patterns produced from HDL2 vs. RDL3

5. To discover if the apoproteins and lipids of intact HDL

exchange between the separate HDL subclasses

6. To investigate any possible effects of chemical crosslinking

on such exchange processes

7. To gain a greater understanding of the relationships and

structures of the two HDL subclasses, HDL2 and HDL .
















MATERIALS AND METHODS

Materials

Chemicals

1,5-difloro, 2,4-dinitrobenzene (DFDNB) and sodium dodecyl sulfate

(SDS) were purchased from Sigma Chemical Co., St. Louis, Missouri.

4,4'-difluoro 3,3' -dinitradiphenylsulfone (DFDNPS); dimethyl suber-

imidate dihydrochlorida (DMA); and dimethyl 3,3'-dithiobispropionim-

idate dihydrocholoride (DTBP) were purchased from Pierce, Rockford,

Illinois. Reagents for acrylamide gel electrophoresis were obtained

from BioRad Laboratories, Los Angeles, California. All other chemicals

used were reagent grade and purchased from common suppliers.

Radioisotopes and Scintillation Materials

[12IJ-Nal; [choline-methyl-14C]-phosphatidy1 choline, specific

activity 50mCi/mmole; [11-12 3H]-cholesterol, specific activity

60Ci/mmole; [11-12 3H]-cholesterol palmitate, specific activity 294mCi/

mmole; Aquasol-2; and mini-vials were all purchased from New England

Nuclear, Boston, Massachusetts.

Blood

Whole human blood was obtained fresh from healthy male donors or

as outdated blood units from the Civitan Regional Blood Center, Gaines-

ville, Florida. In no instances were the results of any repeated

experiments different depending upon the source of blood.










Methods

Isolation of Lipoproteins

Whole blood was centrifuged for 20 min at 2000 RP:M using an IEC

Model SBV centrifuge to remove red cells. The clear serum or plasma

was removed by decanting and the density then raised to 1.063gm/cm3

by the addition of solid KBr. This solution was centrifuged for

20-24 hrs.at 42,000 using a Beckman Ti60 rotor in a Beckman model L2-65B

ultracentrifuge. After centrifugation, the top 3-5 ml containing

CHYL, VLDL, and LDL were removed by aspiration and discarded. The

remaining solution was adjusted to density = 1.21gm/cm3 by the addition

of more solid KBr. The serum was again centrifuged for 24 hrs. at

42,000 RPM as above. The top 2-3 ml of each tube were then removed

using a Pasteur pipet and pooled. When total HDL (HDLt) was desired,

the pooled lipoprotein solution was further purified by another centri-

fugation step. This was carried out as above after the crude HDL

solution was adjusted to density = 1.063gm/cm3 by the addition of 0.15M

NaC1 containing 10 ME EDTA and 0.02% NaN3. Each tube was underlayered

with 5-10 ml of KBr solution of density = 1.21gm/cm~ before centri-

fugation. After centrifugation, the top 5 ml of each tube (LDL

contamination) was removed by aspiration and discarded. The yellow

band in the tube located above the 1.21 density layer was removed using

a Pasteur pipe and saved as purified total RDL. This HDL solution was

immediately dialyzed against 2 x 4 liters of 0.15M NaCl containing

10 ME EDTA and 0.02% NaN3, and stored at 4'C until use.

When the separate RDL subclasses, HDL2 and HDL were desired, the

following procedure was used. After the three ultracentrifugation










steps described above, the total HDL was dialyzed against a Kllr-NaC1

solution of density = 1.125gm/lcm3 contalining 0.15M Nact, 10-51 ULA

and 0.02% NaN3 The solution was placed in centrifuge tubes and

underlayered with 5 ml. of KBr solution, density = 1.21gm/cm3; then

centrifuged for 36-48 hrs. at 45,000 RPM using a Ti60 rotor. This

step resulted in the formation of two separate yellow bands; one at

the top of the tube, and one just above the 1.21 plug. The top band

was taken as HDL2 and the lower as HDL The bands were removed using

a Pasteur pipet and the region between the bands discarded. The HDL2

and HDL3 solutions were then dialyzed against 2 x 4 liters of 0.15M

NaC1 containing 10-5% EDTA and 0.02% NaN3, and stored at 4"C until

use. The purity of HDL preparations was checked by SDS gel electro-

phoresis (see next section for reference and details). In no prepara-

tion did the contamination by other proteins (usually serum albumin)

exceed 1-2% and usually no contamination could be seen at all unless

the gels were greatly overloaded.

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

Polyacrylamide gel electrophoresis was carried out using a Model

300B or 301 BioRad Electrophoresis Cell powered by a BioRad Model 400

Power Supply. TLwo types of gel formulation systems were used. First,

gels were prepared using the procedure of Lammli (84). The acrylamide

concentration of the separating gel was adjusted to 10-12% by alteration

of the ratio of acrylamide to water during gel formulation. SDS concen-

tration was maintained at 0.1% and for 5 x 100rmm gels electrophoresis

was carried out at 1.5ma/gel. Bromophenol Blue was used as a tracking

dye and electrophoresis continued until the dye band was approximately











Icm from the bottom of the gel. Gels were fixed in 12.5% TCA: 40%

ethanol: 7% acetic acid, stained with 0.05% Coomassie Blue in 10%

ethanol: 7%. acetic acid, and destained using a Bio~ad Model 172A2

diffusion destainer. Gels were stored in 7% acetic acid.

For reasons to be discussed later, the gel system was changed

during the course of the work to that of Fairbanks, et al. (85). A

7.8% acrylamide gel with 1.0% SDS was prepared and run at 8mal/gel for

5 x 10Dam gels. Pyronin Y was used as a tracking dye. After electro-

phoresis the gels were fixed in 40% propanol: 7.5% acetic, and stained

with 0.05% Coomassie Blue in 40% propanol: 7.5% acetic acid. After

destaining as above the gels were stored in 7.5% acetic acid.

Molecular weights were determined from the recorded scans using

added molecular weight markers as well as the location of the apo-

proteins of known molecular weights.

Crosslinking reactions

The desired amount of protein solution to be crosslinked was added

to a 13 x 100mm glass tube and 10X concentrated buffer added until a

final concentration of 0.025M buffer was achieved. Crosslinker

dissolved in ethanol, water, or acetone (1-5mg/ml) was then added with

rapid mixing to attain the desired molar ratio of crosslinker to protein.

The amount of organic solvent added never exceeded 5% of the total

volume of the reaction mixture. For specific crosslinkers the follow-

ing buffer and solvent systems were used: DFDNB 0.025M Na2CO3 buffer,

pH 8.8, ethanol: DFDNPS 0.025M Na2CO3 buffer pH 8.8, acetone; DMA,

DMSDTB .02M N2HPO4 buffer pH 7.5, water. The reaction mixtures

were incubated for at least 2 hrs. at room temperature unless otherwise










indicated. The reaction mixtures were used directly without further

treatment as it could been shown that only very small amounts of the

free hydrolysis products of the crosslinkers were formed.

For labeling with FDNB, the monofunctional analog of DFDNB, the

conditions described for DFDNB were used.

Amino acid analysis of specific crosslinked products

To determine the amino acid content of a specific crosslinked

product it was first necessary to separate that product from all the

other products of the crosslinking reaction. This was achieved by

preparative gel electrophoresis of fluorescent labeled proteins. Using

a BioRad Model 301 gel system and 10% Lammli gels as described above,

0.5 to 1.0 ml of HDL solution containing 1-3mg/m1 protein, that had

been made fluorescent by reaction with fluorescamine (86), was applied

to the top of each gel. Electrophoresis was carried out at 6 ma/gel

until sufficient resolution of the separate bands was achieved. The

bands were easily visualized using a long wave UV light and after

electrophoresis were sliced from the gel using a razor blade. Slices

from duplicate gels were combined, crushed with a glass rod, and

mixed with 5 to 10 ml of 0.1% SDS solution. The mixture was incubated

at 370C with shaking for 12 hrs., filtered, and the procedure repeated.

The combined solutions were filtered through a .45p Millipore filter

and dialyzed against 3 x 4 liters of H20. The dialysis bag was

agitated to suspend the fine precipitate and the suspension lyophyllized.

The powdered residue was dissolved in 5 ml of 6N-HC1 with 10pl of

B-mercaptoethano1 and 10pl of 0.5% phenol. The solution was sealed in

glass ampules in vacuo and then hydrolyzed for 24 hrs. at 110*C. The










hydrolyzate was dried by lyophyllization, redissolved in 0.2M citrate

buffer pH 2.2, and standard amino acid analysis run on a Beckman

Model 120C amino acid analyzer. Standard citrate buffers and a

ninhydrin detection system were used.

Calculation of the amino acid composition of then proteins analyzed

was carried out using the formula below.

residues of leu or val predicted/molecule XmoeAA=residues AA
mrmoles leu or val molecule

As the amina acid composition was used as a method of verification

of predicted composition, this type of calculation proved most useful.

Delipidation of lipoproteins

Ten ml of HDL solution to be delipidated wereadded with stirring

to 250 ml of chloroform:methano1 (2:1) at -10"C. The turbid solution

was allowed to stand at -10DC for 30 min, 250 ml of cold ethyl ether

were added and the solution filtered on a Buchner funnel through

Whatman #1 filter paper. The filtrate was washed with 250 ml of cold

ethyl ether and dried with a stream of N2. The precipitate was removed

from the paper and stored at -10*C under N2 until use. This method

is a modification of the procedure of Lux et al. (87).

Cel filtration chromatography

Sephadex 0-200 was allowed to swell in water and then poured into

a 2.5 x 100cm glass column to a height of 90cm. It was eqluilibrated

with 0.1% SDS, 0.02M tris-HC1, 10mM NaN3, 1mM EDTA buffer pH( 8.0,

30mg of delipidated protein dissolved in this buffer was added to the

column. An ascending flow rate of 10ml/hr was used and 150 drop frac-

tions collected. The OD28 was monitored using an ISCO UA-2 UV monitor.

The column was stopped after 120ml of buffer had passed through the

column.










Iodination of lipoproteins

Five ml of the sample to be labeled were dialyzed against 0.5M

glycine buffer pH 9.4, containing 0.15M NaC1, 10 M EDTA, and 0.02%

NaN3; then labeled using the iodine monochloride (IM) method of
MacFarlane (88). The iodine carrier solution (0.5ml) containing

250plCi of [12I]-Nal was added to the HDL solution with rapid mixing.

After 10 min. the unreacted iodine and glycine buffer were removed by

gel filtration chromotography using Sephadex G-25 equilibrated withl

0.15M NaC1 containing 10 M EDTA and 0.02% NaN3. Protein solutions

with a specific activity of 5,000 to 10,000cpm/pg were routinely

obtained using this method. Unlabeled controls were treated as above

except for the addition of the [12I]-iodine carrier solution. It

could be shown that approximately 99% of the radioactivity was asso-

ciated with the apoproteins while only 1% was found in the lipid

fraction.

Incorporation of labeled lipid into HDL,

Fifty mg of celite (50p1 particles, Johns-Mansville Co.) were mixed

with 1.0ml of chloroform to which 50-100pl of labeled lipid dissolved

in toluene-ethanol (1:1) had been added. After mixing, the solvent vol-

ume was reduced with a stream of N2 and the remainder of the solvent

removed in vacuo. Two ml of the HDL solution to be labeled was added

with gentle mixing and the suspension incubated for the desired time

at 370C. The celite was kept in suspension by occasional swirling of

the tubes. After incubation was complete the suspension was filtered

through a 0.45v Millipore filter to remove the celite. All incubations

were carried out for 2 hrs. unless otherwise indicated.











Exch~ange reactions Incubation of HDL2 and HDL mixtures

Thre desired amoulnts of labeled and unlabeled IlDL2 and HlDL3 in

solution were added directly to 5/8" x 3" cellulose nitrate centrifuge

tubes and incubated for the- time and temperature indicated. SDS-PAGE

of controls was used as a method to detect any possible deterioration

of the RDL during incubation. Unless otherwise indicated, for all

incubations described herein the following volumes and concentrations

were used: controls 200pl containing 300pg of HDL protein, mixtures

400pl containing 300vg labeled HDL protein and 300pg cold EDL protein.

For the chaotropic ion experiments, solid KBr was added directly to

the solution to attain the desired concentration of Br ion.

Ultracentrifugation of HDL, HDL, mixtures

The reseparation of labeled and unlabeled HDL2 HDL3 mixtures was

achieved in the following manner. As the mixture to be resolved had

already been placed directly into centrifuge tubes, sufficient NaBr

solution of density =- 1.3288gm/cm3 was added to the mixture to raise

the density to 1.125gm/cm3. NaBr-NaC1 solution (density = 1.125gm/cm3)

containing 0.15M NnC1, 10-5M EDTA, and 0.02% NaN3 was added to give ai

final volume of 10.Dm1. The solutions were then underlayered with

1.0ml of NaBr-NaCL, density 1.21gm/cm3. The tubes were capped and

placed in a Beckman Ti50 rotor for centrifugation at 42,000 RPM for

44-48 hrs.

Gradient Fractionation

After ultracentrifugation all gradients were fractionated using -an

ISCO Model 183 gradient fractionator modified to collect fractions from

the bottom of the tube. Before fractionation each gradient was over-

layered with 1.Dml of 0.15M Nacl solution. 30 drop (0.6ml) fractions

were collected for all experiments.










Activation of Sep~harose by Cyanagen Bromide

Ten ml of packed and well washed Sepharose 4B were placed in a

150ml beaker, 20ml of H20 were added, the pH adjusted to 11.0 with

LM NaOH, and the temperature adjusted to 20'C by the addition of a

small amount of ice. Finely divided CNBr (200mg) were added with

constant stirring and the reaction allowed to proceed until cessation

of proton release was observed by monitoring the pH. The pH was

maintained at 11 during the reaction by the addition of 1M NaOH when

needed and the temperature was adjusted by the addition of ice. When

the reaction was complete, after approximately 15-20 min., 100ml of

ground ice wereadded to the mixture. To Sepharose-ice slurry was then

placed on a coarse glass filter and the Sepharose washed with several

volumes of ice-cold water followed by 200ml of ice-cold 0.1M NaHCO3:

0.5M NaC1 buffer pH 8.0. The washed "activated" Sepharose was used

immediately according to the following procedure.

Coupling of RDL to "Activated" Sepharose

Ten ml of "activated" Sepharose prepared as above or obtained from

Pharmacia were mixed with 10-15ml of 0.1M NaHCO :0.5M Nac1 buffer pH 8.0

in a 100ml beaker. HDL solution (5-10ml of 1-5mg/ml) was added and the

mixture stirred gently for 2-3 hours at 40C. The mixture was then

filtered on a glass filter and washed for 30 min. with several volumes

of IM ethanolamine. The Sepharose-HDL (S-HDL) was then washed with

4 x 100ml of 0.1M NaOAc:0.5M NaCl buffer pH 4.0; 4 x 100ml of 0.1M

NaHCO3:0.5 NaCl buffer pH 8.0; and finally with 2 x 100ml of 0.15M Napl:

10-5M EDTA:0.025% NaN3 The Sepharose prepared in this manner was

stored wet in the final wash solution at 40C.










Exchange reactions using Sepharose-bound HDL

A measured portion of packed Sepharose-HDL labeled with [125] or

radioactive lipid was pipeted into 13 x 100mm glass tubes and an equal

volume of 0.15M NaCZ was added. Cold HDL was added in solution and

the mixture swirled to suspend the gel. After incubation for the

desired time, with occasional swirling to keep the gel suspended, the

mixture was filtered to remove the gel. The filtrate was then counted

in Aquasol-2 in a liquid scintillation counter for lipid exchange exper-

iments, counted in the gamma counter for peptide exchange experiments,

or subjected to ultracentrifugation or SDS-PAGE. For lipid exchange

experiments the following incubation mixtures and times were used:

2591 of Sepharose-HDLg (3mg HDL/ml gel), 200pl of 0.15M NaC1, 50pl of

HDL3 (4tmg/ml) or HDL2' (3mg/ml) incubated for 15 min. at 37"C. For

peptide exchange experiments larger amounts of gel were used due to the

lower levels of HDL binding to the gel (100pg HDL/ml gel). For most

experiments the conditions used were: 400pl Sepharose-[12I]-HDL

(HDL2 or HDL ), 20011l HDL (HDL2 or HDL ) in solution, 200ul 0.15M MaC1.
Incubations were carried out for 5 hours at 370C,

Determination of radioactivity

Al~l counting of [125] was done using a Nuclear Chicago Model 8725

manual gamma counter. The counting efficiency was approximately 63%

based on a comparison with the efficiency of counting of a'Packard

Auto-Gamma Scintillation Spectrophotometer Model 5130 (76% efficiency).

All samples were counted in 13 x 100mm glass tubes using the same volume

of solution in each tube (0.6ml).

For the measurement of [14C] and [ H], liquid scintillation spectro-

scopy was used. The sample was added to 5.0m1 of Aquasol-2 and the






30



mixture counted in "mini-vials" purchased from New England Nuclear.

All scintillation counting was carried out using a Beckman Liquid

Scintillation Spectrophotometer. The counting efficiency for [14C

was calculated to be 92% while for ( li] the efficiency was 46%.

All scintillation counting values were corrected for background

and counting efficiency by coincidence counting with commercially

available standards.
















RESULTS AND DISCUSSION

Isolation and Purity of Lipoprotein Preparations

As the procedure for isolation of HDL is well established (4,6),

the two factors of importance considered were the purity of HDLT prepa-

rations with respect to contamination by other proteins, and the effec-

tiveness of separation during the preparation of RDL subclasses, HDL2

and HDL .

Figures la and lb show the scan of SDS gels of a typical prepara-

tion of RDLT. It can be seen that the only major polypeptide bands

observed have molecular weights that correspond to the major apoproteins

known for HDL. As expected, the ApoA-2 peak (MW 17,500) is split into

its monomer form upon the addition of B-mercaptoethano1 (Fig. lb). The

peak is reduced in size due to a significant loss of low molecular

weight proteins during the fixation and staining of the gels (89).

The purity of HDL2 and HDL3 preparations was checked by recentri-

fugation of the separate subclasses at the density of 1.125gm/cm3 used

during isolation. Under these conditions HDL3 should sink toward the

bottom of the centrifuge tube while HDL2 should float at the top of the

solution. Figure 2 shows the fractionation patterns after this recen-

trifugation. It can clearly be seen that both HDL2 and HDL3 preparations

are pure by the physical criterion of ultracentrifugal mobility. All

preparations of RDL2 were shown to be pure by this method. .HDL~ prepa-

rations, however, occasionally showed some slight contamination by HDL2'

























Figure 1. SDS-PAGE of SDS solubilized apoproteins of HDLT

A. Pattern obtained in the absence of reducing agent.

B. Pattern observed when 5% B-mercaptoethanol is
included in the preparation.








A-1


A-1


a













MIGRATION DISTANCE


























Figure 2. Distribution of protein after recentrifugation
of purified HDL2 (6) or HDL3 ($) at a density
of 1.125gm/cm3







1000 +


*0





8 i HDL3 HDL2 *

600 i





400 a





S200 + a


ee e
e? e




0 2 4 6 8 10 12 14 16 18 20

FRACTION NUMBER










This contamination never represented more than 5% of the total protein

present, and, when observed, an additional centrifugation step was

used to remove it. This HDL2 contamination did not appear to be the

result of HDL3 dimer formation; as once removed, no new contamninant

appeared under any conditions of treatment.


Crosslinking of Total HDL (R~DLT) with DFDNB

The selection of DFDNB as the initial crosslinking reagent to be

evaluated was based on several factors. First, it has been widely

used in previous crosslinking studies involving a variety of protein

types (76). Second, its monofunctional analog FDNB has also been widely

used (76). Third, it was believed that its hydrophobic nature would

'aid in its ability to penetrate the lipoprotein particles. Finally,

pure DFDNB and FDNB are available from commercial chemical laboratories

at low cost.

Figure 3 shows the ge1 patterns observed when a 2mg/ml protein

solution of HDLT is crosslinked withr a 5 fold molar excess of DFDNB

and then run on SDS-PAGE. Figure 3a shows the pattern obtained in the

absence of any reducing agent while 3b is observed in the presence of

1% 8-mercaptoethanol. Although a variety of high molecular weight

crosslinked products are produced (Figure 3a), the first product to

appear and in the highest quantity is a polypeptide complex with an

apparent molecular weight of approximately 46,000. In the presence of

B-mercaptoethanol this major peak is shifted to an apparent molecular

weight of 37,000. Considering only ApoA-1 (HW 28,330) and ApoA-2

dimerr MW 17,500) the appearance of a crosslinked product with a

molecular weight of 46,000 could only be the result of one A-1 linked
























Figure 3. SDS-PAGE (10% Lammli gels) of SDS solubilized apoproteins
of HDLT crosslinked with a 5-fold molar excess of DFDNB.

(A) DFDNB crosslinked HDLT, no reducing agent.

(B) DFDNB crosslinked HDLT, with 5% B-mercaptoethanol.












































I


A-1


46,000


A-2


MIGRATION DISTANCE


37,000











to one A-2 unit. This conclusion is supported by Figure 3b which shows

that the addition of the disulfide cleaving agent, 8-mercaptoethanal,

results in the loss of half of the A-2 dimer leaving a product con-

sisting of one A-1 linked to onle A\-2 monomefr. Th~e molecular weight

of such a complex would be approximately 36,000.

As thle estimation of: molecular weights by SDS get clectrophonresis

can result in considerable error when considering proteins which may

have both inter- and intra-peptide crosslinks (90), additional evidence

is desirable before one can conclude that an A-1***A-2 crosslinked

product is actually produced. To approach this problem it is necessary

to separate the individual crosslinked products so they can be charac-

terized. Gel filtration chromotography using Sephadex G-200 containing

1.0% SDS was attempted. Although this procedure was capable of a

reasonable separation of ApoA-1 from ApoA-2, the crosslinked products

were eluted as a broad, even band; each fraction containing a mixture

of similar molecular weight products. This procedure was not capable

of sufficient purification of any single product. The problem was

solved using the experimental approach diagrammed in Figure 4. The

procedure is detailed in the Methods section of this dissertation. The

results of the amino acid analysis of the 46,000 dalton crosslinked

product is shown in Table 2. Comparison to the predicted values for

sleected amlino acids for an A-1***A-2 complex indicates the 46,000

dalton product is likely a complex of one A-1 and one A-2. Certain

amino acid values were not shown because they were components of the

ge1 buffer system, gave unexpected high or low values for purified A-1

or A-2, or were destroyed during hydrolysis. That both A-1 and A-2




















HDL (HDL2 or HDL )


Chemical Crosslinking ----- DS PAGE


Fluorescent Labeling


Prep. SDS PAGE


Slice out protein band


Elution of protein


Dialysis


HCL hydrolysis


Amino acid hydrolysis


Figure 4. Experimental approach to amino acid analysis of
crosslinked products





Table 2.

Amino Acid Analysis of the 46,000 Dalton Crosslinked Product
from HDL, Crosslinked with DFDNB


Observeda


A-1:A-2 Predicteda

39.0

5.0
16.0

31.0
25.0

55.0

15.0

14.0
27.0

26.0

2.0

5.0


Amino Acid


Lysine
Histidine

Arginine
Alanine
Valine

Leucine

Tyrosine

Phenylalanine
Aspartic acid
Threonine

Isoleucine
Methionine


36.3

7.0

16.8

31.2
25.7

55.0

15.0

15.2

30.1
25.0

1.8


aAll values expressed as residues per mole
for leucine.


of protein based on the value











are components of this complex is suggested by two observations. First,

the shift in molecular weight upon addition of reducing agent could

only occur if ApoA-2 was present as no disulfide bonds occur in Apo-1.

Second, there is no histidine in ApoA-2 or isoleucine in ApoA-1, but

both occur in the 46,000 dalton product. As ApoA-1 contains 5 histidine

residues/molecules and ApoA-2 dimer contains 2 isoleucine residues/mol-

ecule, those must be the source of the histidine and isoleucine found

in the complex. Although the C-peptides contain both isoleucine and

histidine, the low total amount of C-peptides in RDL excludes these

polypeptides from a significant contribution to the composition of any

major crosslinked product.


Effect of Crosslinking on HDL Str'ucture

In discussing the meaning of the results of crosslinking experi-

ments it is important to establish that the introduction of these

crosslinks does not significantly alter the structure of the macro-

molecular complex that is being studied. In the case of HDL, it could

be shown that crosslinked HDL retains the physical properties of native

RDL and will migrate in the analytical ultracentrifuge as a single peak

essentially identical to native RDL. Although even very high levels

of crosslinking (100-fold molar excess) do not appear to disrupt the

particles, all conclusions presented are based on data obtained at

mruch lower levels of crosslinking.


Crosslinking of HDL~ and RDL_ with DFDNB

Although the peptide composition of the two HDL subclasses, RDL2

and RDL appears to be the same, the amount of total protein is

different (1). HDL2 should contain approximately 160,000 daltons of










protein/molecule while HDL3 should only contain 80,000 daltons/molecule.

Under conditions of increasing amounts of crosslinker, one might expect

to observe larger molecular weight crosslinked products for HDL2 as

compared to HDL However,. using the Lammli gel system with molar

ratios of crosslinker higher than 5 to 10-fold, one observes only a

smear of stain at the top of the gel for each subclass. This phenomenon

has been noted for other types of crosslinkers (91). For this reason

a different gel system was used. Fairbanks, et al. have noted that an

SDS concentration of 1%, rather than the 0.1% found in the Lammli

system, is necessary to dissociate membrane protein complexes (85).

Figure 5 shows the comparison of the pattern obtained for HDLT cross-

linked with a 20-fold molar excess of DFDNB and then run on the two gel

systems (5a-Lammli, 5b-Fairbanks). The 1% SDS in the Fairbanks gel

system is apparently sufficient to cause total dissociation of even

highly crosslinked lipoprotein particles.

Figures 6 (HDL2) and 7 (RDL ) demonstrate that for both HDL2 and

HDL3 the crosslinking patterns are essentially the same. It is inter-

esting to note that no crosslinked products of apparent molecular weight

products occur even if the level of crosslinker is increased up to

100-fold. For this reason no separation of HDL2 and HDL3 was carried

out for other crosslinking experiments; instead, HDLT was used.


Crosslinking of HDL_ with DMS and DFDNDPS

To investigate the hypothesis that the hydrophobic nature and length

of the bifunctional reagent used should have some effect .0n the extent

and type of crosslinking produced, a comparison of the gel patterns

obtained was made after DMS or DFDNDPS was used to crosslink HDLT. These




































Figure 5. Comparison of the SDS-PAGE methods
of Fairbanks (A) and Lammli (B) for
the separation of highly crosslinked
HDLT (25x DFDNB) .





MIGRATION DISTANCE



B


MIGRATION DISTANCE

























Figure 6. SDS-PAGE (7.8% Fairbanks gels) of SDS solubilized HDL2
crosslinked with a 10-fold molar excess of DFDNB.





A-1













A-2 C


MIGRATION DISTANCE



























Figure 7, SDS-PAGE (7.8% Fairbanks gels) of SDS solubilized HDL3
crosslinked with a 10-fold molar excess of DFDNB.





A-2 C


MIGRATION DISTANCE










two reagents have similar distances between the functional groups but

differ greatly in hydrophobic character. DMS is water soluble and car-

ries a positively charged imido group while DFDNDPS is quite insoluble

in water and must be added in an acetone solution. At low levels of

crosslinker (10-fold molar excess) these two reagents appear to

generate significantly different initial crosslinked products (Figures

8-11). Two major differences between DMS and DFDNDPS crosslinking can

be seen. First, the total amount of crosslinking is smaller for the

hydrophyllic reagent DMS. Second, the initial site of reaction for DMS

appears to differ from DFDNDPS with a 65,000 dalton product appearing

first. At higher molar ratios of these reagents the patterns become

very similar with all different products appearing in similar amounts

in each case.

The initial products produced with DFDNDPS crosslinking are

essentially the same as those formed with DFDNB even though the former

reagent is twice as long as the latter.


Temperature Dependence of Crosslinking

When the crosslinking of HDLT with a 10-fold molar excess of

DFDNB is carried out at different temperatures ranging from 40 to 700C,

no significant differences in the crosslinking patterns are produced.

The number and relative amounts of the separate crosslinked products

remains similar at all temperatures studied. The only difference

observed is a reduced amount of crosslinking at the very high temper-

atures, 60-70"C. It should be noted, however, that the rate of

formation of hydrolysis products of this bifunctional reagent increases

significantly at these elevated temperatures. This would lead to























Figure 8. SDS-PAGE (10% Lammli gels) of SDS solubilized HDL,
crosslinked with a 5-fold molar excess of DMS. No
reducing agent present.

































ct

vlC













MIGRATION DISTANCE


65,000
























Figure 9. SDS-PAGE (10% Lammli gels) of SDS solubilized HDLT
crosslinked with a 5-fold molar excess of DMS.
5% B-mercaptoethanal included.
















65,000


MIGRATION DISTANCE


























Figure 10. SDS-PAGE (10% Lammli gels) of SDS solubilized HDL,
crosslinked with a 5-fold molar excess of DFDNDPS.
No reducing agent present.






























Cc










MIGRATION DISTANCE
























Figure 11. SDS-PAGE (10% Lammli gels) of SDS solubilized HDL,
crosslinked with a 5-fold molar excess of DFDNDPS.
5% B-mercaptoethanol included.





A-2


EMIGRATION DISTANCE











decreased availability of reagent as well as an increased probability

of monofunctional labeling.


Interpretation of Cross'linking Results

From the data in this section we conclude that at least some of

the two major apoprotein components must lie very close to one another

in the intact lipoproteln particle since DFDNB will only crosslink

groups about 5A apart. If each HDL particle contains both A-1 and A-2

molecules, a number of possible crosslinking products could result.

From our findings with low levels of DFDNB (5-10 fold molar excess)

several observations can be made. First, the major crosslinked product

appears to comprise one A-1 plus one A-2; second, there is no indication

of any A-2 intramolecular crosslinking which might be expected with a

molecule already crosslinked with a disulfide bridge. .There also

seems to be little significant crosslinking-reactive association of two

A-1 molecules as suggested by Scanu (92). Since both A-1 and A-2

contain numerous reactive residues fairly evenly distributed throughout

their sequence (1), any close association of the chains should make

multiple sites available for possible crosslinking.

Experiments using higher molar ratios of crosslinker (20-100X) do

show that most possible combinations of A-1 and A-2 are formed. Still,

even at these high concentrations, no A-2 intramalecular crosslinked

molecules are produced. The C-peptides do not appear to be involved in

the crosslinking reactions. At high levels of reagent the amount of

unreacted C-peptide does not appear to decrease when compared to con-

trols. This suggests little association of C-peptides with A-1 or A-2.











The formation of most of the possible products at high crosslinker

concentrations seems to indicate either a significant degree of mobility

of the apoproteins or significant alteration of the native structure of

the particle. 2Two factors suggest that although some mobility exists,

a high degree of specific association of the apoproteins occurs in the

intact HDL particle. First, the decreased crosslinking observed at

high temperature could be due to increased mobility of the polypeptide

chains. Second, the failure of A-2 to form intramalecular crosslinks

suggests a stable conformation that remains fairly fixed even at these

elevated temperatures. The reproducibility of the crosslinking exper-

iments with respect to order of formation and ratios of products formed,

even when HDL is obtained from varied sources, suggests that in the

intact HDL particle the apoprotein components may be found in a rather

fixed spatial orientation to one another.

That the crosslinking patterns for HDL2 and RDL3 do not differ also

argues for a fixed association of 2A-1 and 1A-2 apoproteins. Even though

RDL2 may have two HDL3 protein subunits (54), these do not appear to

interact. This would lead one to speculate that HDL2 might be a dimer

of 2-RDL3 molecules. This idea will be discussed in detail later in

this work.

An additional argument in favor of fixed associations with flexi-

bility of the apoproteins comes from the experiments using the two

reagents DMS and DFDNDPS. These two reagents consistently yield initial

crosslinking products that.differ. This may occur due to different

initial sites of reaction for these reagents. The initial products do

not differ when one compares DFDNB and DFDNDPS, although the latter is

twice as long as DFDNB. This again suggests some flexibility in the

associations of the apoproteins.










From these observations one might suggest a model in which the

apoproteins lie in a fixed, flexible conformation held in place in

part by protein-protein, lipid-protein, and lipid-lipid interactions.

The ability to bind lipids may in turn be governed by-these inter-

actions; the flexibility allowing for differing degrees of binding of

specific "functional" lipids while certain "structural" lipids remain

fixed.


Aorotein Exchange Between HDL^ and RDL

If the ratio of A-1 to A-2 dimer is 2:1, in both HDL2 and HDL3 as

suggested by Friedberg and Reynolds (54), then the difference between

RDL2 and HDL3 may represent the addition of one A-1:A-2 unit, which
consists of two A-1 plus one A-2 peptide dimer. Because our cross-

linking data suggests a degree of flexibility as well as specific inter-

action within an A-1:A-2 unit but the two units do not appear to cross-

link in intact HDL2, it is necessary to consider possible modes of

association of the apoproteips of the two proposed units in HDL .

To investigate this possible association, as well as the relation-

ship between the apoproteins of the different subclasses of HDL, we

have examined the exchange of labeled apoproteins between HDL2 and HDL3,

in the presence and absence of bifunctional crosslinking reagents.

Figures 12 and 13 show the ultracentrifugal fractionation patterns

obtained for controls (300ug HDL protein in 200pl) of [12I]-labeled

HDL3 and [125I-labeled HDL2 incubated In the absence of any other
protein. The results are expressed as dpm per fraction as it was found

that the radioactivity and protein concentration curves coincided (the

specific activities of control fractions were constant throughout the



















Figure 12. Exchange of apoproteins between [12I]-HDL3 and HDL2'

Distribution of label after centrifugation tosaat
HDL2 and HDL3. $ = [125I]-HDL3 control; O =[1 ]HD
incubated with cold HDL2*







20000 +



125
[ I]-HDL
16000 + 3

[125]-D
+ HDL2

12000O +





8000 +B 8





4000







0 2 4 6 8 10 12 14 16 18 20


FRACTION NUMBER























Figure 13. Exchange of apoproteins between [1250-HDL2 and HDL3.

Distribution of label ~ter centrifugation to a Parate
HDL2 and HDL3. $ = [1 I]-HDL2 control; 8 = [1 DI]-HDL2
incubated with cold HDL3







40000 + e




32000 +


[12I]-HDL2
24000 +-



~ [125]-HDL2
16000 +F21
~ + HDL3



8000 + /"a.






0 2 4 6 8 10 12 14 16 18 20
FRA\CTION NUMBER









tube). Figures 12 and 13 also illustrate the results obtained after

mixing equal amounts (300pg protein in 200)11) of (12I]-labeled HDL3

and cold HDL2 (Figure 12) or (12Ii-labeled HDL2 and cold HDL3 and

incubating the mixture for 7 brs. at 370C (Figure 13). In Figure 12

it can clearly be seen that A transfer of label from HDL3 to HDL2 has

occurred. Further, the specific activity of the HDL3 fraction was

significantly decreased when compared to controls. In a typical

experiment, the values for a selected fraction in the RDL3 region of

the tube (fraction 6) were: [12I]-HDL3 control = 2850 dpm/pg;

112I]-HDL3 incubated with cold HDL2 = 1680 dpm/pg. Similar results

were obtained when labeled HDL2 was incubated with cold HDL3 (Figure 13).

However, due to the breadth of the HDL3 peak as compared with the HDL2

fractions, the results are not as easily visualized.


Effect of Crosslinking and Monofunctional
Labeling on Apoprotein Exchange

If the labeled HDL3 is crosslinked with DFDNB before incubation

with HDL2, the transfer of labeled protein is reduced depending upon
the extent of crosslinking. Figure 14 shows the three fractionation

patterns obtained with : [12I]-HlDL3 incubated with HDL2 (control);

[125I]-HDL3j crosslinked with a 5-fold molar excess of DFDNB and then

incubated with HDL2; and [12I]-HDL3 incubated with HDL2 following

crossl~inking of the HIDL3 with a 25-fold molar excess of DFDNB. It canl

be seen that increased crosslinking results in decreased exchange.

To rule out the possibility that the monofunctional substitution

of the apoproteins with the crosslinking reagent was the cause of this'

decrease in exchange, FDNB, a monofunctional analog of DFDNB, was used

to modify the proteins. Reaction with concentrations of FDNB, comparable






















Figure 14. Effect of crosslinking on the apoprotein exchange between
[125IJ-HDL3 and HDL2

Distribution of label after centrifugation to separate
HDL2 and HDL3. 6 = [125I]-HDL3 incubated with cold HDL2;
$ = [125I] _"L3 crosslinked with a 5-fold molar excess of
DFDNB followed by incubation with HDL2; P = [125I]-HDL3
crosslinked with a 25-fold molar excess of DFDNB then
incubated with HDL2*







20000 +





16000 +





12000; +





soo e e se~e e





4000 + \





0 +********..... ***********************--*+ **.......+....... .. ......4......... ....+

O 2 4 6 8 10 12 14 16
FRACTION NUMBER


CONTROLL


5X DFDNB


25X DFDNB


18 20










to amounts of DFDNB that greatly inhibited exchange, had little effect

on the exchange process (Figure 15).

When HDL2 is crosslinked before incubation with labeled llDL the

exchange process is also Inhi~bited (Figulre 16). 'The reverse also hioldu

true; that is for crosslinked HDL3 incubated with labeled HDL2, the

exchange is reduced.


Effect of Temperature and Dilution on Peptide Exchange

Both low temperature and dilution have an effect upon the rate of

protein exchange in HDL. If the incubation is carried out at 4.0, very

little exchange will occur in the time period in which significant

exchange occurs at 37"C (Figure 17). Using the amounts of HDL2 and

HDL3 described in the Methods section, but diluting this mixture with
a 10-fold excess (5ml) of 0.15M NaC1, the rate of exchange at 370C is

also significantly reduced (Figure 18). These two observations are

consistent with a mechanism of exchange involving a collisional com-

plex.

It is this reduction in exchange rates that permits one to employ

the 48 hour centrifugation requir-ed for reseparation of the HDL2 and

HDL3 mixtures. When equal amounts of labeled HDL3 and cold HDL2 are

mixed, then immediately diluted, cooled to 4.0, and placed in the

ultracentrifuge, essentially no exchange occurs, even after 48 hrs.


Rate of Peptide Exchange

Figure 19 shows a time course plot for the exchange of apoproteins

from [12I]-HDL3 into HDL2. The radioactivity represents the total

transferred into the HDL2 region of the centrifuge tube after subtrac-

tion of the amount in those fractions attributed to HDL Estimation



























Figure 15. Effect of monofunctional labeling with FDNB on the exchange
of apoproteins between [125I]-HDL3 and HDL2*

Distribution of label after centrifugation to separate HDL2
and HDL~3. 6 = [125I]-HDL3 reacted with a 25-fold molar
excess of FDNB; 8 = [125I]-HDL3 reacted with a 25-fold molar
excess of FDNB followed by incubation with HDL2*






00 +




00 +


150(




1200




900




600




300


[12I]-HDL3 25X FDNB


[125 L3 25X FDNB

/ p + HDL2




0/ +




,0 + t
tee *



o +*********+. - -----.:........+......~...+..... ...........+.. .........4.......,.. ......... ... _~

0 2 4 6 8 10 12 14 16 18 20
REACTIONN NUMBER























Figure 16. Effect of crosslinking the unlabeled HDL subclass on
the exchange of apoproteins between [1251]-HDL3 and HDL2.

Distribution of label after centrifugation to separate
HDL2 and HDL3' O = 125I]-HDL3 control; 8 = [12b] -HDL3
incubated with HDL2 crosslinked with a 25-fold molar
excess of DFDNB.







20000 +




16000 +




12000 +F4










4000 125I]3-HDL3

rl5]-HDL + HDL2 25X DFDNB



0 +*********+********+********4 *******.+........-t +.................4 c...... ...4.e ....

0 2 4 6 8 10 12 14 16 18 20
FRACTION NUMBER






















Figure 17. Effect of temperature on the apoprotein exchange between
[125I] HD 3 and HDL2
Distribution of label after centrifugation to se rate
HDL2 and HDL3. 9 = [125I]-HDL3 control; af = 12IJ-HDL3
incubated with HDL2 at 4oC. Compare to Figure 12.






20000 +



16000~


12000 + /



sooo +




0~ * + * * * * . . . . . + . . 4 . . . . o . g
a 0 0 2 4 8 1 1 4 6 18 2
PRACION UMBE
























Figure 18. Effect of temperature and dilution on the apoprotein
exchange between [125I]-HDL3 and HDL2*

Distribution of label after centrifugation to separate
HDL andHDLg 6= 125I]-HDL, and HDL, were incubated
at C after dilution with 5ml30f 0.15M2NaCl.








20000 +





16000 +





12000 +F





8000 +


40(


e e-





e' e


e a
300





0 +*********+*********+***-*****+ ***. .....+.........'.. ................4........4........4.......

0 2 4 6 8 10 12 14 16 18 20
FRACTION NUMBER
























125
Figure 19. Time course for the exchange of [ I]-labeled apoproteins
from [125I]-HDL3 into HDL2. 3mg HDL protein/ml each subclass.







6500 +





5200 +
e a



/ 3900; +





2600+





a1300 +







O 36 72 108 144 180 216 252 288 324 360

TIME (MIN.)











of the initial slope at 2300 dpm/hr out of a total of 90,000 dpm gives

an exchange rate of approximately 2.5%/hr. These calculations are based

upon two separate experiments utilizing concentrations of llDL.2 and

HDL3 companrable to plasma levels (3mng/ml eachc subclass). Th'le rate

can be altered by varying the initial concentrations of either or both

subclasses.


Effect of Bromide lon on Protein Exchange

If the chaotropic Br ion is included in the.incubation mixture at

37PC, the amount of exchange in a given time period is greater when

compared to controls with no Br Figure 20 shows the fractionation

pattern obtained when 2M1 KBr is included in the 7 hour incubation solu-

tion as compared to controls. A significant increase in exchange can

be seen. As chaotropic ions are known to effect protein conformation

and association (93), these factors may be involved in the exchange

process.


SDS-Polyacrylamide Gel Analysis of Exchanged Proteins

Gel electrophoresis of the recipient HDL subclass, into which

labeled apoproteins have been transferred, indicates that ApoA-1,

ApoA-2, and the C-peptides are all exchanged. Figure 21 shows the

radioactivity pattern obtained when RDL2, into which labeled apopro-

teins have been transferred by incubation with [ 2I]-HDL3, is run on

SDS-PAGE. The radioactive bands were identified by staining and

scanning duplicate gels.

If the labeled HDL subclass is subjected to limited crosslinking

with a 10-fold molar excess of DFDNB before mixing and incubation
























Figure 20. Effect of KBr on the exchange of apoproteins between
[125I]-HDL3 and HDL2

Distribution of label after enetrifugation to separate
HDL2 and HDL3. 6 = [125I]-HDL3 incubated with HDL2;
s = [1251]-HDL3 incubated with HDL2 in the presence of
2M KBr.







50000 +




40000 +




30000




S20000;


10000 + e *


0 ****+**********+ **** + **********+ ****+ ******** *****


100 2,B / 0 2 1 1 8 2




FRACTION NUMBER

























Figure 21. Polyacrylamide gel analysis of exchanged apoproteins.
125
SDS-PAGE of HDL2 after incubation with [ I]-HDLa
followed by reseparation. The gel was sliced it
2mm slices and the slices counted.







2500 +






2000 +







A-12

W,1500 +






e ~6












0 4 8 12 16 20 24 28 32 36 40

GEL SLICE NUMBER










with cold HDL, the labeled apoproteins that are exchanged are those

that were not crosslinked. Figure 22 shows the gel pattern obtained

for crosslinked [12I]-HDL3 alone, and for HDL2 that was labeled by

exchange of apoproteins from crosslinked [125]-HDL Although a

significant portion of the HDL3 apoproteins were crosslinked into

higher molecular weight products, only uncrosslinked A-1 and A-2

appear to be exchanged.


Interpretations of Protein Exchange Experiments

The transfer of labeled apoprotein from one HDL subclass to

another does not proceed with a loss of protein content of the orig-

inally labeled fraction. The specific activity of that fraction

decreases but the protein content remains unchanged. This is con-

sistent with an exchange process in which there is a one to one exchange

of protein of one subclass with the other. Such exchange could occur

by the transfer of single polypeptide apoproteins, i.e., A-1 or A-2;

or by the exchange of complexes of apoproteins such as 2A-1:1A-2.

These experimental results do not permit us to rule out either of these

possibilities. It might be suggested that since crosslinking inhibits

the exchange, single polypeptide chains are involved. However, the

crosslinking may also prevent a conformational change that might be

necessary for the exchange process. That a conformational change

might be involved is suggested by the fact that when high concentra-

tions of the chaotropic ion, Br are included in the incubation mixture,

increases in the rates of exchange are observed. An actual exchange

involving a conformation change is also suggested by the finding that

crosslinking the unlabeled subclass also greatly inhibits the exchange























Figure 22. Polyacrylamide gel analysis of exchanged apoproteins:
Effect of crosslinking with DFDNB.

125
SDS-PAGE of HDL2 reisolated after incubation with [ IJ-HDL3
crosslinked with DFDNB. Q = [125I]-HDLgL crosslinked with a3
25-fold molar excess of DFDNB; 6 = HDL2 after incubation with
crosslinked [125I]-HDL3.






400 +



320 +



240 + I



S160 +

*:~ ~ h,
80

0~~t +* *******.....+ .... ....... s


O 4 8 12 16 20 24 28 32 36 40
GEL SLICE NUMBER










process. If a simple transfer of peptide into the recipient class

occurred, one would not expect crosslinking to have much effect.

If HDL2 is made up of two identical subunits as suggested by

Friedberg and Reynolds (54), the transfer of label might be visualized

in terms of the exchange of RDL3 for one of the HDL2 subunits.

Although this is a reasonable possibility, one must again consider the

possibility of a conformational change to explain the results of the

crosslinking experiments.

Even though the two RDL subclasses can exist as stable separate

entities, when they are present together in solution, significant inter-

action between particles may occur. Exchange of apoproteins could

possibly occur between HDL2-HDL2 or HDL3-HDL3, as well. as between HDL2

and RDL3 molecules. If collisional complexes cause exchange and single

polypeptides are capable of exchange, then one would expect HDL -HDL3

exchange to occur. Collisional complex formation should also result

in the rapid exchange of lipids of all types due to their small size

and the fusion required for peptide exchange.


Lipid Exchange Between HDL2 and HDL, in Solution

Whcn IlDL3 Is Ioledcir withl [310j-chleslctcorl, [3Hl]-cholesterol panlm--

tate, or (14C]-phosphatidyl choline, thecn incubated with cold HDL2, as

withi peptide exchange; label is transferred to HDL2. However, even

under conditions of low temperature, dilution, and no incubation, total

exchange occurs during the ultracentrifugal reseparation of R1DL2 and

HDL3. This rapid movement prevents the study of the exchange using the
experimental approach that was possible for peptide exchange. For this

reason an alternative approach was developed.











Binding of Labeled HDL to Sepharose

The rapid rate of lipid exchange places one requirement on the

method employed to study this exchange: the separation of HDL3 and

HDL2 'must be done very quickly. As use of the ultracentrifuge requires
at least 24 hours even at 60,000 rpm, it was decided to approach the

problem by immobilizing one of the subclasses by covalently binding

it to Sepharose. This would allow separation by filtration; a process

that would take less than one minute.

When (12I]-HDL3 was reacted with commercially obtained CNBr-acti-

vated Sepharose 4B (Pharmacia) under the coupling conditions described

in the Methods sections, a Sepharose preparation containing approxi-

mately 0.5mg protein per ml of packed Sepharose was obtained. To

determine what percentage of the protein was bound to the Sepharose, a

portion of the gel was washed with 1% SDS solution. Under these

conditions any protein chains not covalently bound to the gel should

be washed off. Table 3 shows the results of such an experiment. Using

the Pharmacia CNBr-activated Sepharose, no more than 30% of the label

could be removed with SDS. Assuming only 3 polypeptide chains/HDL3

molecule, this indicates that an average of two are linked to the

Sepharose. To obtain a preparation with a lower level of binding, it

was necessary to activate our own Sepharose 4B using the conditions

described in the Methods section. Under these conditions, preparations

with over 50% of the protein removable with SDS could be obtained. The

same conditions also gave acceptable levels of binding for HDL2 prepa-

rations (20-40% bound). For all peptide exchange experiments,

preparations with low levels of binding were used. For lipid exchange




Full Text

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LIPOPROTEIN STRUCTURE: APOPROTEIN INTERACTIONS IN HUMAN PLASMA HIGH DENSITY LIPOPROTEINS By THOMAS ELLIS GROW A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1977

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DEDICATION I dedicate this dissertation to my best friend William A. Patterson and the Krena Construction Company,

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. ACKNOWLEDGEMENTS The author would like to express his deep appreciation to Professor Melvin Fried, his research director, for giving him guidance and freedom of thought and action during the completion of this work. The environment of Professor Fried 's laboratory was exactly what the author required for productive work. The author also wishes to thank his wife, Susan, for her patience, understanding, and sacrifices made during the last four years. Special thanks are given to the author's advisory committee and members of the faculty of the Department of Biochemistry for their suggestions and criticisms during the execution of this research. The author also thanks his parents for their understanding during his years of education.

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TABLE OF CONTENTS ACKNOWLEDGEMENTS ill LIST OF TABLES vi LIST OF FIGURES vii KEY TO SYMBOLS AND ABBREVIATIONS NOT DEFINED IN TEXT ' , ix ABSTRACT x INTRODUCTION 1 Plasma Lipoproteins 1 Apoprotein Structure and Function 4 Lipoprotein Structure 6 Alternative Classification of Lipoproteins 11 Exchange Reactions of Lipoproteins • 12 Experimental Rational 15 Research Objectives 18 MATERIALS AND METHODS 20 Materials 20 Methods 21 RESULTS AND DISCUSSION 31 Isolation and Purity of Lipoprotein Preparations 31 Crosslinking of Total HDL (HDL ) with DFDNB 36 Effect of Crosslinking on HDL Structure 42 Crosslinking of HDL and HDL with DFDNB 42 Crosslinking of HDL with DMS and DFDNDPS 43 Temperature Dependence of Crosslinking 50 Iv

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Interpretation of Crosslinking Results 59 Apoprotein Exchange Between HDL and HDL 61 Effect of Crosslinking and Monofunctional Labeling on Apoprotein Exchange 66 Effect of Temperature and Dilution on Peptide Exchange 69 Rate of Peptide Exchange 69 Effect of Bromide Ion on Protein Exchange 80 SDS-Polyacrylamide Gel Analysis of Exchanged Proteins 80 Interpretations of Protein Exchange Experiments 85 Lipid Exchange Between HDL and HDL in Solution 88 Binding of Labeled HDL to Sepharose 89 Peptide Exchange Using Sepharose-Bound HDL 91 Ultracentrifugal Analysis of Peptide Exchange Using Sepharose-Bound HDL 91 SDS-PAGE Analysis of Exchanged Peptides Using Sepharose-Bound HDL 96 Incorporation of Labeled Lipid into HDL„ and HDL^ 103 Lipid Exchange Between HDL„ and Sepharose-HDL„ 106 Effect of Temperature and Dilution on Lipid Exchange 112 Effect of Saturating Cold Lipid on Labeled Lipid Exchange . . . 112 Effect of Crosslinking on Lipid Exchange 118 Effect of Added Organic Solvent on Exchange of Lipids and Proteins 118 Interpretations of Sepharose-HDL Peptide and Lipid Exchange Experiments 125 CONCLUSIONS AND SPECULATIONS 128 BIBLIOGRAPHY 132 BIOGRAPHICAL SKETCH 137

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LIST OF TABLES TABLE PAGE 1 Bifunctional Reagents .17 2 Amino Acid Analysis of 46,000 Dalton Crosslinked Product from HDLj Crosslinked with DFDNB . . 41 ' 3 Effect of Amount of CNBr Activation on the Amount of Binding of HDL„ by Sepharose 4B 90 4 Peptide Exchange Using Sepharose-Bound HDL 92 5 Removal of Labeled Lipid from SepharoseBound HDL„ by SDS Wash 107 6 Effect of Temperature and Dilution on Lipid Exchange Between S-HDL^ and HDL^ 117 7 Effect of Saturating Cold Lipid on Lipid Exchange 119 8 Effect of Crosslinking on Lipid Exchange 120

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LIST OF FIGURES FIGURE PAGE 1 SDS-PAGE of solubillzed apoproteins of HDL^ 33 2 Separation of HDL2 and HDL^ A d=l. 125gin/cm"^ 35 3 SDS-PAGE of HDL crosslinked with DFDNB 38 4 Experimental approach to amino acid analysis of crosslinked products 40 5 Comparison of the SDS-PAGE methods of Fairbanks and Lammli for the separation of highly crosslinked HDL 45 6 SDS-PAGE of HDL2 crosslinked with DFDNB 47 7 SDS-PAGE of HDL3 crosslinked with DFDNB . 49 8 SDS-PAGE of HDL crosslinked with DMS 52 9 SDS-PAGE of HDL_ crosslinked with DMS (5% 6ME) 54 10 SDS-PAGE of HDL crosslinked with DFDNDPS 56 11 SDS-PAGE of HDL crosslinked with DFDNDPS (5% 6ME). , J 58 125 12 Exchange of apoproteins between [ I]-HDLand HDL2 63 125 13 Exchange of apoproteins between [ I]-HDL„ and HDL 7 65 14 Effect of crosslinking on apoprotein exchange . . . . .68 15 Effect of FDNB labeling on apoprotein exchange ... .71 16 Effect of crosslinking the unlabeled subclass on apoprotein exchange 73 17 Effect of temperature on apoprotein exchange 75 vii

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18 Effect of temperature and dilution on apoprotein exchange 77 19 Time course of apoprotein excliunge 79 20 Effect of KBr on apoprotein exchange 82 21 Polyacrylamide gel analysis of exchanged apoprotein . .84 22 Polyacrylamide gel analysis of exchanged apoproteins: Effect of crosslinking with DFDNB 87 125 23 Subunit exchange between S-[ I]-HDL„ and HDL» ... .95 125 24 Subunit exchange between S-[ I]-HDL3 and HDL. . . . 98 25 Polyacrylamide gel analysis of available apoproteins from S-HDL100 26 Polyacrylamide gel analysis of apoproteins exchanged from S-[125i]_hdl 102 27 Uptake of labeled lipids by HDL^ 105 28 Effect of increasing amounts of cold HDL^ on lipid exchange 109 29 Effect of increasing amounts of cold HDL. on lipid exchange Ill 30 Rate of lipid exchange between S-HDL* and HDL„. ... 114 31 Time course for lipid exchange between S-HDL* and HDL 116 32 Effect of ethanol of the exchange of lipids from S-HDL* to HDL„ 122 33 Effect of ethanol on the exchange of lipid and protein from S-HDL* to HDL ' 124 34 Model for HDL„ HDL„ interconversion ... 131 viii

PAGE 9

KEY TO SYMBOLS AND ABBREVIATIONS NOT DEFINED IN TEXT o A angstrom Apo apoprotein CD circular dichroism spectroscopy Ci curie dpm disintegrations per min. IR infrared spectroscopy LCAT Lecithin cholesterol acyl transferase leu leucine MW molecular weight IMR nuclear magnetic resonance spectroscopy ORD Optical rotory dispersion val valine ix

PAGE 10

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy LIPOPROTEIN STRUCTURE: APOPROTEIN INTERACTIONS IN HUMAN PLASMA HIGH DENSITY LIPOPROTEINS By Thomas Ellis Grow June, 1977 Chairman: Melvin Fried Major Department: Biochemistry The modes of association of the major apoprotein species of human plasma high density lipoprotein (HDL) was investigated using bifunctional crosslinking reagents. How these associations help to govern the structure of the HDL subclass, HDL„ and HDL , was studied by observing both apoprotein and lipid exchange between HDL particles. Crosslinking of HDL with a 5 to 10-fold molar excess of difluorodinitrobenzene resulted in the production of high molecular weight crosslinked products with the major product shown to be the combination of the two major apoprotein species ApoA-1 and ApoA-2. The composition of this product was confirmed by amino acid analysis. Crosslinking with other bifunctional reagents indicates specif icity of initial reaction sites as well as some freedom of movement of the apoproteins within the particle. However, the crosslinking patterns obtained with the HDL subclasses were identical, suggesting specific structural properties of HDL„ and HDL_. 125 Apoproteins, labeled with I, were shown to exchange between and among the HDL subclasses. This bidirectional exchange process was

PAGE 11

inhibited by crosslinking with bifunctional reagents and appeared to depend upon collision complex formation. By the utilization of Sepharose bound IIDL it could be shown that both free apoprotein molecules, as well as subunits consisting of lipid-apoprotein combinations, could exchange between HDL and HDL„. The exchange appears to involve a conformational change in the apoproteins during the transfer process. Radioactive cholesterol, cholesterol palmitate, and phosphatidyl choline, bound to HDL after adsorption to celite particles, could be shown to undergo rapid exchange between HDL particles. This exchange also appears to depend on collision complex formation but was not inhibited by chemical crosslinking of the apoproteins. The rate of these exchange processes is significant in the lifetime of the lipoprotein particles in vivo ; 2.5%/hour for apoprotein exchange and >50%/hour for lipid exchange. It was concluded that ApoA-1 and ApoA-2 must lie very close to one another in the intact lipoprotein particle. The reproducibility of the crosslinking experiments on HDL obtained from several donors suggests that in the intact HDL particle the apoprotein components may be found in a rather fixed spatial orientation to one another. It is also suggested that there is a dynamic relationship between HDL„ and HDL . Even though these two subclasses can exist as stable separate entities, when they are present together in solution, significant Interaction between particles may occur. Exchange of lipids and apoproteins occurs between HDL^-HDLand HDL -HDL„, as well as between HDLj and HDL molecules. It is suggested that the interconversion of HDL„ and HDLmay be determined by the availability of certain lipids.

PAGE 12

. INTRODUCTION Plasma Lipoproteins Plasma lipoproteins play an important role in mammalian energy metabolism and also serve as the major transport particles for many physiologically important molecular species. It has been suggested that they are causally related to atherosclerosis since they are the carriers for those substances deposited in the atheromatous plaque. Circulatory system pathology found in hyperlipoproteinemics may result from quite small alterations in lipoprotein structure; differences that may be due to altered protein-protein or protein-lipid interaction. Human serum contains four major types of lipoproteins: chylomicrons (CHYL) , very low density lipoproteins (VLDL) , low density lipoproteins (LDL), and high density lipoproteins (HDL). Although these four classes appear to differ in both lipid and protein composition as well as function, there are distinct interrelationships between the particles. The presence of one class directly influences the other classes (1). Chylomicrons, formed in the intestinal mucosa and released into the blood from the major lymphatic duct, function as the carrier of many lipid types. Lypolytic enzymes remove lipids from these particles" as they circulate. VLDL and LDL appear to be closely related as LDL may be the result of VLDL delipidation (2). LDL functions as the major cholesterol carrier in blood. HDL can be divided into two

PAGE 13

subgroups; HDL_ and HDL . HDL may be important in cholesterol deposition disease (3). The isolation of lipoproteins from serum or plasma is relatively simple and may be accomplished by flotation in the ultracentrifuge. As the density (d) of serum lipoproteins ranges from approximately 3 3 0.95 gm/cm to 1.21 gm/cm , centrifugation of serum, the density of 3 which has been raised to 1.21 gm/cm by the addition of salt, will result in the lipoproteins floating to the top of the centrifuge tube while the other proteins sink (4). Each lipoprotein class is characterized by its own density range for isolation: CHYL and VLDL < 1.006, LDL 1.006 1.063, and HDL 1.063 1.21. By stepwise increases in density (4), or by density gradient centrifugation (5), these lipoprotein classes may be prepared in relatively pure form. Although other methods such as molecular sieve chromotography (6) and chemical precipitation (7) have been described, the ultracentrifugal floatation method remains the most widely used. The protein and lipid compositions of these lipoprotein classes have been studied and the following have been reported (1,8) (all percentages on a dry weight basis). HDL HDL, whose molecular weight ranges from 180,000 to 350,000, consists of approximately 50% protein, 22% phospholipids, 3% free cholesterol, 14% cholesterol esters, and 8% triglycerides. Of the phospholipid portion, 70% is phosphatidylcholine, 14% is spingomyelin., 5% phosphatidyl serine and phosphatidyl ethanolamine, and the remainder other minor phospholipids. The protein portion of the HDL particle

PAGE 14

consists of two major polypeptides, ApoA-1 (ApoGln-I) and ApoA-2 (ApoGln-II). The amino acid sequences of both polypeptides are known (9,10,11) and ApoA-2 is known to be a dJmer of two identical subunits connected by a single disulfide bridge at position 6 (10). Another group of smaller proteins, designated ApoC or the C-peptides, comprises approximately 5% of the total HDL protein. At least three C-peptides, C-I, C-II, and C-III, are recognized in HDL (2). The protein of HDL contains 3-4% covalently bound carbohydrate including glucosamine, fructose, galactose, mannose, and sialic acid (8). In addition to the C-peptides, HDL also may contain the "thin-line" protein and "arginine rich" protein as minor components (1). LDL LDL particles are larger than HDL with molecular weights ranging from 2 to 3 million. They also contain a larger percentage of lipid than HDL (75-78%); consisting of 36-42% cholesterol esters, 21-23% phospholipids, 8-9% free cholesterol, and 6-8% triglycerides. Other lipids may be present in smaller amounts. The proteins make up 20-22% of the particle. The major polypeptide is denoted apoprotein B (ApoB) , a large protein of molecular weight 225,000. There may also be very small amounts of the C-peptldes described for HDL. Again, 3-5% carbohydrate is found covalently associated with the protein. VLDL VLDL particles cover a wide range of densities and molecular weights (3 128 x 10 ) with at least two maxima at densities of 0.980 3 and 0.958 gm/cm . They contain 90% lipid with an average composition of 50-60% triglycerides, 10-12% free cholesterol, 4-6% cholesterol

PAGE 15

esters, and 18-20% phospholipids. Protein represents only 8-12% of which 40% is apoprotein B and the remaining 60% is divided among the three C-peptides described for HDL. A small portion of ApoA may also be present and it has been demonstrated that different fractions may contain different amounts of apoproteins (2). Chylomicrons Chyl contain mostly lipid with only 1-2% protein. These very Q large particles have molecular weights of 5 4300 x 10 . The protein consists of all types of previously described apoproteins found in the other lipoprotein classes. It has been shown that human lymph chylomicrons contain approximately 66% ApoC, 22% ApoB, and 12% ApoA. The lipids are predominately triglycerides (80%) with only about 8% phospholipids and 5% cholesterol (1,2). It is important to note the overlapping protein composition in all of these classes. It is presumed that the specific protein composition of each is important in determining its total lipid content and the specific type of particle that is formed. These proteins should play a very important role in determining the function of the particle as well (1) . Apoprotein Structure and Function ApoC ApoC-I has been sequenced (12) and is a single polypeptide chain of 57 amino acid residues (MW 8000). ApoC-I is known to function in lipid binding as well as LCAT activation (13). Lipoprotein lipase also appears to be activated by ApoC-I (14).

PAGE 16

ApoC-IT is the largest of tlie C-peptides with 100 amino acid residues and a molecular weight of about 12,500 (15). It also binds lipids and is a potent activator of lipoprotein lipase (16). ApoC-III, a single polypeptide of 79 amino acid residues has also been sequenced (17). Attached to threonine-74, ApoC-III contains an oligosaccharide moiety consisting of galactose, galactosamine, and sialic acid (17). Although no direct physiological role is yet known, ApoC-III has been used often for the study of lipid-protein interactions (18-21). ApoB The characterization of ApoB has led to many conflicting statements concerning the number and size of the polypeptide subunits. Due to technical problems associated with isolation of a soluble apoprotein, a variety of physical methods have indicated molecular weights ranging from 8,000 to 275,000 (22). However, most investigators support the idea of a large (275,000) molecular weight peptide. These problems have also hindered studies on specific functions of ApoB and as yet no definite physiological role other than lipid binding has been discovered. ApoA ApoA is a term used to describe the two most extensively studied apolipoproteins, ApoA-1 and ApoA-2. ApoA-1, a single polypeptide of molecular weight 28,330, contains 245 amino acid residues and has been sequenced (8,23,24). Although the binding of phospholipids by ApoA-1 has been studied by several investigators, conflicting results on lipid binding in the absence of ApoA-2 have been reported (25,26,27). These

PAGE 17

differences in the extent of lipid binding by ApoA-1 may be due to self-association of the polypeptides that may mask lipid binding sites (28). Self-association has been demonstrated by both ultracentrif ugal and chemical techniques (1), ApoA-2 is a polypeptide dimer of two identical chains with a molecular weight of 17,500 (29). Each chain contains 77 amino acid residues. Tlie disulfide bond occurs betweoii tlie single cysteine residues that occur at position 6 (29). The disulfide linked dimer appears to be characteristic of only man and chimpanzee; the dimer form apparently is not crucial for ApoA-2's lipid carrying role (1). ApoA-2 does bind lipid strongly and may increase the binding capacity of ApoA-1 as well (2). Although lipid binding is the only role as yet assigned to ApoA-2, ApoA-1 appears to have several functions other than lipid binding in the HDL particle. ApoA-1 serves as an activator of LCAT (30), may be an acceptor of cholesterol and cholesterol ester during VLDL catabolism (2), and appears to have some function in regulating the lipid content of membranes (31,32). Lipoprotein Structure The study of lipoprotein structure has been approached utilizing three basic types of techniques: 1) spectroscopic methods (IR, CD, CRD, X-ray, neutron scattering, NMR, and fluorescence); 2) physical techniques (Ultracentrifugation and electron microscopy); and 3) chemical methods (chemical modification, amino acid analysis, bifunctional reagents, reassembly). The actual composition of lipoprotein particles has been fairly well described, as indicated above, and only in the

PAGE 18

case of LDL is there significant disagreement on Apoprotein size and composition (1,8). However, when three-dimensional structure is considered, little is known at present regarding specific proteinprotein or protein-lipid spatial relationships or interactions in intact lipoprotein particles. VLDL and Chyl have proved difficult to study due to the significant turbidity of their solutions. This fact has limited the use of spectroscopic methods and as a result only the techniques of ultracentrifugation, electron microscopy, and gel filtration have been used. This, coupled with the fact that these classes consist of a spectrum of a wide variety of sizes with differing compositions, has prevented all but the most basic of structural studies. UltracentriQ fugation techniques indicate the following: Chyl MW 5-A300 x 10 daltons, diameter 1200-11000 A, hydrated density 0.93gm/cm . VLDLMW 3-128 X 10 daltons, diameter 300-700 A, hydrated density 0.93 3 l.Ogm/cm (1). The only structural determinations made thus far for these particles are based on the surface activity of the protein components which seems to indicate that some portion of the proteins are located on the surface of the particle (2). The information now available has led to the suggestion of a lipid-core model for the triglyceride rich Chyl and VLDL particles. This model assumes that the surface of the particle is occupied by phospholipids, cholesterol, and proteins, while the central core consists of the less polar and more hydrophobic triglycerides and cholesterol esters (33). The quantities of protein, phospholipid and cholesterol in these particles appear to be sufficient to cover the surface of the particles as predicted (34).

PAGE 19

LDL structure has been studied by many of the different techniques mentioned earlier (2,8,35,36,37,38). Data from electron microscopy and X-ray diffraction indicate a spherical particle with neither well defined nor significant subunit structure (2). Information from analytical ultracentrifugation, as well as X-ray and electron microscopy, give the following information: MW 2.3-2.7 x 10 daltons, 3 hydrated density 1. 028-1. 034gm/cm , diameter 216-220 A. In distinction to Chyl and VLDL, LDL is rather uniform in size and composition. The protein component appears to have about 25% a-helix, 37% g-structure, and 37% random coil as determined by CD (38). Although this structure appears to be temperature dependant, a recent report indicates the CD spectrum may be greatly complicated by the lipids present in the particle (39). Both NMR and fluorescence studies indicate that the protein, although near the surface of the LDL particle, is highly associated with the lipid components of the molecule (36,37). It has also been suggested that the core of LDL is mostly composed of hydrocarbon chains while the outer layer of the particle is sparsely occupied by proteins that emerge from the lipid core (35); a model similar to the lipid-core model for VLDL. Other investigators, utilizing electron microscopy and X-ray scattering, have suggested a significant degree of subunit structure that Includes many protein subunits (20-60 with MW 8,000 or 27,000) arranged with icosahediral symmetry. The protein subunits give a symmetrical appearance with the surface area occupied by both protein and phospholipid (40,41). These subunit models do not take into consideration recent findings that indicate the major protein component consists of only two polypeptides of molecular weight 250,000 or greater (42).

PAGE 20

HDL has been studied most extensively, probably because it is the easiest of the particles to work with. As noted above, its protein composition is well known and the major polypeptides have been sequenced. HDL can be divided into two subclasses, HDL„ and HDL_, which appear to differ mainly in their molecular weights (HDL„ = 184,000) (1). X-ray studies have indicated that HDL consists of two regions of differing electron densities: an outer shell and an inner core. The inner core has a radius of approximately 43 A in HDL„ (37 A in HDL ) while the outer shell radius is 14 A (11 A in HDL ) . The electron density of the outer shell indicates that is consists of polar lipid headgroups and proteins (43). The size of a fully extended phospholipid headgroup is about 11 A, a value that fits in well with the X-ray data. Fluorescence techniques yield information on porcine HDL~ consistent with a peripheral location of the protein component, with the suggestion that as much as 80% of the protein is located at or near the surface (37). This hypothesis is further supported by the finding of Scanu that 90% of the lysine residues of the apoprotein components of human HDL is accessible to succinylating agents (2). This would indicate that most of the protein is near the surface as there are many lysines rather evenly distributed throughout the protein chains. CD of the protein portions of HDL indicate 70% a-helix, 11% g-structure, and 19% random coil (38). The removal or alteration of lipid components greatly affects the structure of HDL apoproteins. Delipidation lowers the helicity to 52%. Delipidation followed by relipidation with egg phosphatidyl choline changes a-helicity to 64%

PAGE 21

10 while the addition of cholesterol further increases the heliclty to 70% (38). These observations indicate strong lipid-protein interactions. Two basic models of IIDL that are consistent with spectroscopic and chemical data have been proposed. Assman, et al. and Assman and Brewer (22,44) have suggested a model similar to the membrane structure proposed by Singer and Nicolson (45) in which the HDL protein is depicted as an "iceberg" floating in a "sea of lipid." This model takes into account the fact that portions of the helical regions of the apoproteins are two-sided; that is, one side of the helix is polar while the other is non-polar (46). This amphipathic organization of the helix could allow simultaneous interaction of the helix with lipid, protein, and the aqueous medium. Whether or not the helices are oriented perpen/' ( dicular or parallel to the surface of the particle la the essential difference between this model of Assman, et al. and Assman and Brewer (22,44) and that of Jackson, et al. (25) • Jackson has suggested that the long axes of the helical regions are oriented perpendicularly to the fatty acyl chains of the phospholipids. In this model, carbon atoms 2-4 of the fatty acyl chains could Interact with the non-polar side of the helix, allowing the hydrophyllic portions to Interact with the medium or other protein chains. There is evidence to suggest that little electrostatic interaction occurs between phospholipid polar headgroups and the charged amino acid residues of intact lipoproteins (22,47,48). Little is known of the protein-protein interactions that may occur in intact lipoproteins. The only studies reported have been carried out on human HDL apoproteins in vitro. It has been shown that if one mixes the HDL apoproteins A-1 and A-2 together in equimolar amounts.

PAGE 22

11 they appear to associate into an aggregate of molecular weight 46,000 (28). This A-l:A-2 complex will bind less lipid than would be expected, Indicating that some of the lipid binding sites are blocked by protein-protein Interaction. Other studies Indicate that in the absence of lipids and other apoproteins, both A-1 and A-2 will self-associate (49-53). Although such studies help to understand the types of interactions that may occur, only work done on the intact particle can shed light upon the actual associations and interactions that govern the final structure and function of the lipoproteins. Recent evidence from Friedberg and Reynolds (54) suggests that A-1 and A-2 always exist in a molar ratio of 2 A-1 polypeptides to 1 A-2 polypeptide dimer in human HDL. Their study also suggests that this ratio of 2:1 is constant from individual to individual as well as from subclass to subclass (HDL„ to HDL^) . This would indicate that perhaps, for ApoA-1 and ApoA-2, a specific polypeptide composition is found in HDL^ (4 A-1 + 2 A-2) as well as in HDL (2 A-1 + 1 A-2). It is evident that much is yet unknown concerning the structure of lipoproteins and that experimental evidence derived from studies utilizing intact particles and techniques that allow precise definition of spatial orientation is needed. » Alternative Classification of Lipoproteins It should be noted at this point tliat another method of classification for plasma lipoproteins has been suggested by Kostner and Alaupovic (55). Based on immunochemical evidence, they have suggested that lipoproteins occur in separate families, the composition of which is determined by the presence of specific apoproteins. The three

PAGE 23

12 families they describe, LP-A, LP-B, and LP-C, represent particles containing ApoA-1 and/or ApoA-2 (LP-A), ApoB (LP-B), and ApoC-I-III (LP-C). The important difference between the two methods of classification is that, according to Kostner and Alaupovic, a physically defined density class, such as HDL, may contain not only particles with just ApoA-1 and ApoA-2 but also particles with only ApoB and only ApoC. One should realize, however, that HDL isolated as a density class will contain mostly LP-A and LP-C with a very small amount of LP-B (2%). This LP-B is only found associated with the HDL„ density subclass suggesting the possibility of contamination by remnant LDL particles. Even the presence of particles containing only LP-C could be an artifact resulting from the generation of incomplete or altered particles during the extensive ultracentrifugation required for the isolation of the HDL density class. In any case, the actual difference between HDL as a density class and LP-A as described by these investigators would only be the presence of the small amount of C peptides generally believed to be associated with the HDL molecule. Although the possibility of lipoprotein families is a question that merits consideration, the results of the experiments described herein would apply to either model of the lipoprotein particles. Exchange Reactions of Lipoproteins Plasma lipoproteins are known to be a very dynamic population of macromolecules. They are continuously being synthesized and degraded at a high rate. The amount of any individual lipoprotein class present at a given time is determined by the balance between synthesis and catabolism. The individual components of lipoproteins, however, are

PAGE 24

13 not degraded or synthesized at the same rate. This is due to the fact that both lipid and protein components can be exchanged between individual lipoprotein molecules as well as between different classes of lipoproteins (1). Lipid Exchange Phospholipids appear to exchange between all classes of serum lipoproteins at rates dependent upon their relative concentrations in the individual classes (56,57). When labeled phospholipids are incorporated into an individual class of lipoproteins and this class is allowed to interact with other lipoprotein classes in vivo or in vitro, an exchange reaction with equilibrium, reached in 4 to 5 hours, is observed (58). This exchange is independant of any protein exchange and occurs at significantly different rates for different phospholipid types. Lysolecithin exchanges most rapidly, followed by lecithin and sphingomyelin (57,59). No significant differences are observed for subclasses of an individual phospholipid class (59). This exchange can be increased ui vitro by the presence of a phospholipid exchange protein (60). Cholesterol is known to exchange rapidly between all classes of human serum lipoproteins (61). This rapid exchange occurs both In vivo and in vitro with equilibrium attained after 2 to 6 liours (62,63). Cholesterol ester exchange between plasma lipoproteins in vivo and in vitro has been both suggested and disputed (62,64,65). There is evidence that seems to indicate that cholesterol esters can be transferred from LDL to other lipoproteins Jjn vitro (66) .

PAGE 25

14 The exchange of other lipid types has been reported for human as well as other animal lipoproteins. Triglycerides exchange between different classes of lipoproteins (67) as do unesterified fatty acids (68) and a-tocopherol (69).. Protein Exchange Although the exchange of lipids does not appear to depend upon the simultaneous exchange of protein, it has been shown that apoprotein exchange does occur. Only ApoC of HDL and VLDL has been shown to exchange in vitro and in vivo (70,71). This exchange is bidirectional and not like the apparent transfer of protein from VLDL to LDL (2). Mechanisms of Exchange The movement of either lipid or protein from one lipoprotein class to another can represent an exchange, a transfer, or the combination of the two processes. It is evident that both processes depend upon the lipoproteins existing in a dynamic state. The in vitro exchange of lipids has been the most studied and the process appears to be a physicochemical one (72). Exchange could occur by at least two mechanisms. First, it has been suggested that exchange occurs when individual lipid molecules escape the lipoprotein particle and enter the aqueous medium. They are then picked up by other lipoproteins or membranes (73,74). This model for exchange lacks strong evidence in its support but may be the mechanism for certain specific lipid types (74). An alternate mechanism involves the formation of collision, complexes between lipoproteins (75). This would allow for diffusion of lipid and/or protein molecules between the particles and at the same time

PAGE 26

15 would not require the thermodynamlcally unfavorable dissociation of hydrophobic molecules into an aqueous medium.As both mechanisms have been supported with experimental evidence, it is important that each be considered as a possible explanation for any exchange reaction. Experiments that will help distinguish between the two mechanisms would include the observation of the effect of factors that influence collision rates between molecules, i.e. temperature, dilution, etc. Although only lipid transfer mechanisms have been postulated, the same types of models should be considered when observing protein transfer or exchange. Experimental Rational In studying either protein structure or protein-protein interactions occurrences of natural crosslinking due to disulfide bridges, peptide linkages between lysine e-NH_ and glutamic or asparatic acid Y or 3-COOH, or carbohydrate bridges have been valuable in understanding the final active structure that is being considered, e.g., insulin, ribonuclease, collagen. As such crosslinks are often not found where one might like to find them, the introduction of stable covalent bridges or crosslinks can aid in the study of protein structure (See Ref. (76) for a recent review of bifunctional reagents). Bifunctional reagents are capable of producing three types of crosslinked products: intramolecularly crosslinked proteins, intermolecularly crosslinked homopoljoners (identical subunits), and intermolecularly crosslinked protein complexes (nonidentical subunits or different proteins). Such reagents have been used in a variety of ways

PAGE 27

16 to determine such things as the spatial geometry of membrane components (77-81), the mapping of rlbosome proteins (82), and the subunit structure of ollgomerlc proteins (83). There are two basic types of crossllnking reagents: those that are cleavable and those that are not. Tlie advantage of cleavable crossllnkers Is that one may Isolate the crossllnked product, cleave the newly formed bridges, and identify the proteins that were crosslinked. This technique is particularly useful when studying the crossllnking of multicomponent systems such as membranes. Table 1 lists a selection of different types of crossllnkers and gives certain specific characteristics. The selection of reagents with differing lengths, degrees of hydrophobic character, or reactive group specificity can yield products that differ because of the specific structure or physical environment of the proteins that are crossllnked. For example, in studying the human erythrocyte membrane, Moxley and Tsai were able to produce different crossllnking patterns by varying only the length of the bifunctional reagent used (81). As all lipoprotein particles are thought to contain two or more polypeptide chains and since the structure and function of these particles may depend to a great extent upon specific protein composition and/or protein-protein interactions, Investigation with blf iincLlonal reagents could prove to be a useful method to define some of these interactions. Because the function and characteristics of individual lipoprotein classes differ, and these differences ultimately must be related to structure, studies dealing with the interactions and associations of

PAGE 28

^ 17 i

PAGE 29

the characteristic apoproteins of a given lipoprotein class should prove of value in understanding how these characteristics are determined by the structure. HDL, the best characterized of the lipoproteins, was used in the experiments described in this report. Its small size, stable structure, ease of isolation and handling, as well as its variety of polypeptides made it the model of choice. Although no definitive physiological role has yet been assigned to HDL, certain other characteristics make this an interesting model for study. C-peptides such as are found in HDL are known activators of LCAT and lipoprotein lipase, and Apo-1 is known to activate LCAT specific for certain substrates (1) . Reynolds and Simon (28) have reported altered lipid binding upon associations of the apoproteins of HDL. Perhaps this interaction is a mechanism for regulation of lipid binding. As the different lipoprotein classes appear closely related and even share many of the same components, both lipid and protein, the actual association of the different protein components may play a very important role in regulating both structure and function for the different lipoprotein particles. Research Objectives The purpose of this research was to gain insight into the structure of intact serum lipoprotein particles specifically, the high density lipoprotein particle. This was approached by attempting to understand the interactions and associations of the individual apoprotein components of these macromolecules. To aid in organizing the information gathered, the following objectives were set forth:

PAGE 30

19 1. To determine if bifunctional reagents are capable of inducing crosslinks between individual apoproteins in intact HDL 2. To characterize and identify any crosslinked products produced 3. To evaluate the result of the use of crosslinking reagents of differing length and hydrophobic or hydrophyllic character 4. To determine and explain any possible differences in the crosslinking patterns produced from HDL„ vs. HDL. 5. To discover if the apoproteins and lipids of intact HDL exchange between the separate HDL subclasses 6. To investigate any possible effects of chemical crosslinking on such exchange processes 7. To gain a greater understanding of the relationships and structures of the two HDL subclasses, HDLp and HDL„.

PAGE 31

MATERIALS AND METHODS Materials Chemicals 1,5-difloro, 2,4-dinitrobenzene (DFDNB) and sodium dodecyl sulfate (SDS) were purchased from Sigma Chemical Co., St. Louis, Missouri. 4,4'-difluoro 3,3'-dinitrodiphenylsulfone (DFDNPS); dimethyl suberimidate dihydrochlorida (DMA); and dimethyl 3,3'-dithiobispropionimidate dihydrocholoride (DTBP) were purchased from Pierce, Rockford, Illinois. Reagents for acrylamide gel electrophoresis were obtained from BioRad Laboratories, Los Angeles, California. All other chemicals used were reagent grade and purchased from common suppliers. Radioisotopes and Scintillation Materials [ I]-Nal; [choline-methylC] -phosphatidyl choline, specific 3 activity 50mCi/mmole; [11-12 H]-cholesterol, specific activity 3 60Ci/mmole; [11-12 H]-cholesterol palmitate, specific activity 294mCi/ mmole; Aquasol-2; and mini-vials were all purchased from New England Nuclear, Boston, Massachusetts. Blood Whole human blood was obtained fresh from healthy male donors or as outdated blood units from the Civitan Regional Blood Center, Gainesville, Florida. In no instances were the results of any repeated experiments different depending upon the source of blood. 20

PAGE 32

21 Methods Isolation of Lipoproteins Whole blood was centrifuged for 20 min at 2000 RPM using an lEC Model SBV centrifuge to remove red cells. The clear serum or plasma 3 was removed by decanting and the density then raised to 1.063gm/cm by the addition of solid KBr. This solution was centrifuged for 20-24 hrs. at 42,000 using a Beckman Ti60 rotor in a Beckman model L2-65B ultracentrifuge. After centrifugation, the top 3-5 ml containing CHYL, VLDL, and LDL were removed by aspiration and discarded. The remaining solution was adjusted to density = 1.21gm/cm by the addition of more solid KBr. The serum was again centrifuged for 24 hrs. at 42,000 RPM as above. The top 2-3 ml of each tube were then removed using a Pasteur pipet and pooled. When total HDL (HDLj-) was desired, the pooled lipoprotein solution was further purified by another centrifugation step. This was carried out as above after the crude HDL 3 solution was adjusted to density = 1.063gm/cm by the addition of 0.15M NaCl containing 10 M EDTA and 0.02% NaN~. Each tube was underlayered 3 with 5-10 ml of KBr solution of density = 1.21gm/cm before centrifugation. After centrifugation, the top 5 ml of each tube (LDL contamination) was removed by aspiration and discarded. The yellow band in the tube located above the 1.21 density layer was removed using a Pasteur pipet and saved as purified total HDL. This HDL solution was immediately dialyzed against 2x4 liters of 0.15M NaCl containing 10~ M EDTA and 0.02% NaN,, and stored at 4°C until use. When the separate HDL subclasses, HDL„ and HDL„, were desired, the following procedure was used. After the three ultracentrifugation

PAGE 33

22 steps described above, the total HDL was dialyzed against a KBr-NaCl solution of density = 1.125gm/cm containing 0.15M NaCi, 10~ M EDTA, and 0.02% NaN.. The solution was placed in centrifuge tubes and 3 underlayered with 5 ml. of JCBr solution, density = 1.21gin/cin ; then centrifuged for 36-48 hrs. at 45,000 RPM using a Ti60 rotor. This step resulted in the formation of two separate yellow bands; one at the top of the tube, and one just above the 1.21 plug. The top band was taken as HDL„ and the lower as HDL . The bands were removed using a Pasteur pipet and the region between the bands discarded. The HDL„ and HDLsolutions were then dialyzed against 2x4 liters of 0.15M NaCl containing 10 M EDTA and 0.02% NaN , and stored at 4°C until use. The purity of HDL preparations was checked by SDS gel electrophoresis (see next section for reference and details). In no preparation did the contamination by other proteins (usually serum albumin) exceed 1-2% and usually no contamination could be seen at all unJess the gels were greatly overloaded. SDS polyacrylamlde gel electrophoresis (SDS-PAGE) Polyacrylamide gel electrophoresis was carried out using a Model 300B or 301 BioRad Electrophoresis Cell powered by a BioRad Model 400 Power Supply. Two types of gel formulation systems were used. First, gels were prepared using the procedure of Lammli (84). The acrylamide concentration of the separating gel was adjusted to 10-12% by alteration of the ratio of acrylamide to water during gel formulation. SDS concentration was maintained at 0.1% and for 5 x 100mm gels electrophoresis was carried out at 1.5ma/gel. Bromophenol Blue was used as a tracking dye and electrophoresis continued until the dye band was approximately

PAGE 34

23 1cm from the bottom of the gel. Gels were fixed in 12.5% TCA: 40% ethanol: 7% acetic acid, stained with 0.05% Coomassie Blue in 10% ethunol: 7% acetic acid, and destained using a BloRad Model 172A diffusion destainer. Gels were stored in 7% acetic acid. For reasons to be discussed later, the gel system was clianged during the course of the work to that of Fairbanks, et al. (85). A 7.8% acrylamide gel with 1.0% SDS was prepared and run at Sma/gel for 5 X 100mm gels. Pyronin Y was used as a tracking dye. After electrophoresis the gels were fixed in 40% propanol: 7.5% acetic, and stained with 0.05% Coomassie Blue in 40% propanol: 7.5% acetic acid. After destaining as above the gels were stored in 7.5% acetic acid. Molecular weights were determined from the recorded scans using added molecular weight markers as well as the location of the apoproteins of known molecular weights. Crosslinking reactions The desired amount of protein solution to be crosslinked was added to a 13 X 100mm glass tube and lOX concentrated buffer added until a final concentration of 0.025M buffer was achieved. Crosslinker dissolved in ethanol, water, or acetone (l-5mg/ml) was then added with rapid mixing to attain the desired molar ratio of crosslinker to protein. The amount of organic solvent added never exceeded 5% of the total volume of the reaction mixture. For specific crosslinkers the following buffer and solvent systems were used: DFDNB 0.025M Na CO buffer, pH 8.8, ethanol: DFDNPS 0.025M Na CO buffer pH 8.8, acetone; DMA, DMS, DTBP 0.025M Na2HP0^ buffer pH 7.5, water. The reaction mixtures were incubated for at least 2 hrs. at room temperature unless otherwise

PAGE 35

24 indicated. The reaction mixtures were used directly without further treatment as it could been shown that only very small amounts of the free hydrolysis products of the crosslinkers were formed. For labeling with FDNB, the monofunctional analog of DFDNB, the conditions described for DFDNB were used. Amino acid analysis of specific crosslinked products To determine the amino acid content of a specific crosslinked product it was first necessary to separate that product from all the other products of the crosslinking reaction. This was achieved by preparative gel electrophoresis of fluorescent labeled proteins. Using a BioRad Model 301 gel system and 10% Lammli gels as described above, 0.5 to 1.0 ml of HDL solution containing l-3mg/ml protein, that had been made fluorescent by reaction with f luorescamine (86), was applied to the top of each gel. Electrophoresis was carried out at 6 ma/gel until sufficient resolution of the separate bands was achieved. The bands were easily visualized using a long wave UV light and after electrophoresis were sliced from the gel using a razor blade. Slices from duplicate gels were combined, crushed with a glass rod, and mixed with 5 to 10 ml of 0.1% SDS solution. The mixture was incubated at 37°C with shaking for 12 hrs., filtered, and the procedure repeated. The combined solutions were filtered through a .45y Millipore filter and dialyzed against 3x4 liters of H„0. The dialysis bag was agitated to suspend the fine precipitate and the suspension lyophyllized. The powdered residue was dissolved in 5 ml of 6N HCl with lOyl of p-mercaptoethanol and 10\il of 0.5% phenol. The solution was sealed in glass ampules in vacuo and then hydrolyzed for 24 hrs. at llO^C. The

PAGE 36

25 hydrolyzate was dried by lyophyllization, redissolved in 0.2M citrate buffer pH 2.2, and standard amino acid analysis run on a Beckman Model 120C amino acid analyzer. Standard citrate buffers and a ninhydrin detection system were used. Calculation of tlie amino acid composition of tlio proteins analyzed was carried out using the formula below. X mmoles AA = mmoles leu or val molecule As the amino acid composition was used as a method of verification of predicted composition, this type of calculation proved most useful. Delipidation of lipoproteins Ten ml of HDL solution to be delipidated were added with stirring to 250 ml of chloroform:methanol (2:1) at -10°C. The turbid solution was allowed to stand at -10°C for 30 min, 250 ml of cold ethyl ether were added and the solution filtered on a Buchner funnel through Whatman //I filter paper. The filtrate was washed with 250 ml of cold ethyl ether and dried with a stream of N„. The precipitate was removed from the paper and stored at -10°C under N until use. This method is a modification of the procedure of Lux et al. (87). Gel filtration chromatography Sephadex G-200 was allowed to swell in water and then poured into a 2.5 X 100cm glass column to a height of 90cm. It was equilibrated with 0.1% SDS, 0.02M tris-HCl, lOmM NaN^, ImM EDTA buffer pH 8.0, 30mg of delipidated protein dissolved in this buffer was added to the column. An ascending flow rate of lOml/hr was used and 150 drop fractions collected. The CD . was monitored using an ISCO UA-2 UV monitor. 2oU The column was stopped after 120ml of buffer had passed through the column.

PAGE 37

26 lodlnation of lipoproteins Five ml of the sample to be labeled were dialyzed against 0.5M glycine buffer pH 9.4, containing 0.15M NaCl, 10~ M EDTA, and 0.02% NaN^; then labeled using the iodine monochloride (IM) method of MacFarlane (88). The iodine carrier solution (0.5ml) containing 125 250pCl of [ I]-Nal was added to the HDL solution with rapid mixing. After 10 min. the unreacted iodine and glycine buffer were removed by gel filtration chromotography using Sephadex G-25 equilibrated with 0.15M NaCl containing 10~ M EDTA and 0.02% NaN . Protein solutions with a specific activity of 5,000 to 10,000cpm/Mg were routinely obtained using this method. Unlabeled controls were treated as above 125 except for the addition of the [ I]-iodlne carrier solution. It could be shown that approximately 99% of the radioactivity was associated with the apoproteins while only 1% was found in the lipid fraction. Incorporation of labeled lipid into HDL Fifty mg of cellte (50m particles, Johns-Mansville Co.) were mixed with l.Oml of chloroform to which 50-lOOyl of labeled lipid dissolved in toluene-ethanol (1:1) had been added. After mixing, the solvent volume was reduced with a stream of N and the remainder of the solvent removed in vacuo. Two ml of the HDL solution to be labeled was added with gentle mixing and the suspension incubated for the desired time at 37 °C. The cellte was kept In suspension by occasional swirling of the tubes. After incubation was complete the suspension was filtered through a 0.45y Millipore filter to remove the cellte. All Incubations were carried out for 2 hrs. unless otherwise indicated.

PAGE 38

27 Exchange reactions Incubation of HDL and HDL mixtures The desired amounts of labeled and unlabeled HDL and HDL. in solution were added directly to 5/8" x 3" cellulose nitrate centrifuge tubes and incubated for thetime and temperature indicated. SDS-PAGE of controls was used as a method to detect any possible deterioration of the HDL during incubation. Unless otherwise indicated, for all incubations described herein the following volumes and concentrations were used: controls 200^1 containing SOOyg of HDL protein, mixtures AOOyl containing SOOyg labeled HDL protein and 300yg cold HDL protein. For the chaotropic ion experiments, solid KBr was added directly to the solution to attain the desired concentration of Br~ ion. Ultracentrifugation of HDL „ HDL , mixtures The reseparation of labeled and unlabeled HDL^ HDL mixtures was achieved in the following manner. As the mixture to be resolved had already been placed directly into centrifuge tubes, sufficient NaBr 3 solution of density = 1.3288gm/cm was added to the mixture to raise 3 3 the density to 1.125gm/cm . NaBr-NaCl solution (density = 1.125gm/cm ) containing 0.15M NaCl, 10~ M l-DTA, and 0.02% NaN was added to give a final volume of 10.0ml. The solutions were then underlayered with 1.0ml of NaBr-NaCl, density = 1.21gm/cm . The tubes were capped and placed in a Beckman Ti50 rotor for centrif ugation at 42,000 RPM for 44-48 hrs. Gradient Fractionation After ultracentrifugation all gradients were fractionated using an ISCO Model 183 gradient fractionator modified to collect fractions from the bottom of the tube. Before fractionation each gradient was overlayered with 1.0ml of 0.15M NaCl solution. 30 drop (0.6ml) fractions were collected for all experiments.

PAGE 39

28 Activation of Sepharose by Cyanogen Bromide Ten ml of packed and well washed Sepharose 4B were placed In a 150ml beaker, 20ml of H„0 were added, the pH adjusted to 11.0 with IM NaOH, and the temperature adjusted to 20°C by the addition of a small amount of ice. Finely divided CNBr (200mg) were added with constant stirring and the reaction allowed to proceed until cessation of proton release was observed by monitoring the pH. The pH was maintained at 11 during the reaction by the addition of IM NaOH when needed and the temperature was adjusted by the addition of ice. When the reaction was complete, after approximately 15-20 min. , 100ml of ground ice were added to the mixture. To Sepharose-ice slurry was then placed on a coarse glass filter and the Sepharose washed with several volumes of ice-cold water followed by 200ml of ice-cold O.IM NaHCO : 0.5M NaCl buffer pH 8.0. The washed "activated" Sepharose was used Immediately according to the following procedure. Coupling of HDL to "Activated" Sepharose Ten ml of "activated" Sepharose prepared as above or obtained from Pharmacia were mixed with 10-15ml of O.IM NaHCO :0.5M NaCl buffer pH 8.0 in a 100ml beaker. HDL solution (5-lOml of l-5mg/ml) was added and the mixture stirred gently for 2-3 hours at 4°C. The mixture was then filtered on a glass filter and washed for 30 min. with several volumes of IM ethanolamine. The Sepharose-HDL (S-HDL) was then washed with 4 X 100ml of O.IM NaOAc:0.5M NaCl buffer pH 4.0; 4 x 100ml of O.IM NaHCO^.-O.S NaCl buffer pH 8.0; and finally with 2 x 100ml of 0.15M NaCl: 10 M EDTA: 0.025% NaN-. The Sepharose prepared in this manner was stored wet in the final wash solution at 4°C.

PAGE 40

29 Exchange reactions using Sepharose-bound HDL 125 A measured portion of packed Sepharose-HDL labeled with [ I] or radioactive lipid was pipeted into 13 x 100mm glass tubes and an equal volume of 0.15M NaCl was added. Cold HDL was added in solution and the mixture swirled to suspend the gel. After incubation for the desired time, with occasional swirling to keep the gel suspended, the mixture was filtered to remove the gel. The filtrate was then counted in Aquasol-2 in a liquid scintillation counter for lipid exchange experiments, counted in the gamma counter for peptide exchange experiments, or subjected to ultracentrifugation or SDS-PAGE. For lipid exchange experiments the following incubation mixtures and times were used: 25yl of Sepharose-HDL(3mg HDL/ml gel), 200^1 of 0.15M NaCl, 50^1 of HDL„ (4mg/ml) or HDL„ (3mg/ml) incubated for 15 min. at 37°C. For peptide exchange experiments larger amounts of gel were used due to the lower levels of HDL binding to the gel (lOOyg HDL/ml gel). For most 125 experiments the conditions used were: 400pl Sepharose-[ I]-HDL (HDLor HDL„), 200pl HDL (HDL. or HDL ) in solution, 200pl 0.15M MaCl. Incubations were carried out for 5 hours at 37°C. Determination of radioactivity 125 All counting of [ T] was done using a Nuclear Chicago Model 8725 manual gamma counter. The counting efficiency was approximately 63% based on a comparison with the efficiency of counting of a Packard Auto-Gamma Scintillation Spectrophotometer Model 5130 (76% efficiency). All samples were counted in 13 x 100mm glass tubes using the same volume of solution in each tube (0.6ml). 14 3 For the measurement of [ C] and [ H] , liquid scintillation spectroscopy was used. The sample was added to 5.0ml of Aquasol-2 and the

PAGE 41

30 mixture counted in "mlni-vials" purchased from New England Nuclear. All scintillation counting was carried out using a Beckman Liquid 14 Scintillation Spectrophotometer. The counting efficiency for [ C] 3 was calculated to be 92% while for [ H] the efficiency was 46%. All scintillation counting values were corrected for background and counting efficiency by coincidence counting with commercially available standards.

PAGE 42

RESULTS AND DISCUSSION Isolation and Purity of Lipoprotein Preparations As the procedure for isolation of HDL is well established (4,6), the two factors of importance considered were the purity of HDL preparations with respect to contamination by other proteins, and the effectiveness of separation during the preparation of HDL subclasses, HDL„ and HDL.. Figures la and lb show the scan of SDS gels of a typical preparation of HDL . It can be seen that the only major polypeptide bands observed have molecular weights that correspond to the major apoproteins known for HDL. As expected, the ApoA-2 peak (MW 17,500) is split into its monomer form upon the addition of 3-mercaptoethanol (Fig. lb). The peak is reduced in size due to a significant loss of low molecular weight proteins during the fixation and staining of the gels (89). The purity of HDL„ and HDL preparations was checked by recentri3 fugation of the separate subclasses at the density of 1.125gm/cm used during isolation. Under these conditions HDL should sink toward the bottom of the centrifuge tube while HDL„ should float at the top of the solution. Figure 2 shows the fractionation patterns after this recentrifugation. It can clearly be seen that both HDL„ and HDL. preparations are pure by the physical criterion of ultracentrifugal mobility. All preparations of HDL. were shown to be pure by this method. HDL. preparations, however, occasionally showed some slight contamination by HDL.. 31

PAGE 43

on

PAGE 44

33 «l AiiSNaa ivDiido

PAGE 45

c

PAGE 46

35 NOiivainaDNOD NiaioHd aAiiviaa

PAGE 47

36 This contamination never represented more than 5% of the total protein present, and, when observed, an additional centrlfugatlon step was used to remove It. This HDL„ contamination did not appear to be the result of HDLdimer formation; as once removed, no new contaminant appeared under any conditions of treatment. Crosslinking of Total HDL (HDL ) with DFDNB The selection of DFDNB as the initial crosslinking reagent to be evaluated was based on several factors. First, it has been widely used in previous crosslinking studies involving a variety of protein types (76). Second, its monofunctional analog FDNB has also been widely used (76). Third, it was believed that its hydrophobic nature would aid in its ability to penetrate the lipoprotein particles. Finally, pure DFDNB and FDNB are available from commercial chemical laboratories at low cost. Figure 3 shows the gel patterns observed when a 2mg/ml protein solution of HDL is crosslinked with a 5 fold molar excess of DFDNB and then run on SDS-PAGE. Figure 3a shows the pattern obtained in the absence of any reducing agent while 3b is observed in the presence of 1% 3-mercaptoethanol. Although a variety of high molecular weight crosslinked products are produced (Figure 3a), the first product to appear and in the highest quantity is a polypeptide complex with an apparent molecular weight of approximately 46,000. In the presence of g-mercaptoethanol this major peak is shifted to an apparent molecular weight of 37,000. Considering only ApoA-1 (MW 28,330) and ApoA-2 (dimer MW 17,500) the appearance of a crosslinked product with a molecular weight of 46,000 could only be the result of one A-1 linked

PAGE 48

•H FQ

PAGE 49

33 <:l AIISNaa TVDIIJO

PAGE 50

39 to one A-2 unit. This conclusion is supported by Figure 3b which shows that the addition of the disulfide cleaving agent, e-mercaptoethanol, results in the loss of half of the A-2 dimer leaving a product consisting of one A-1 linked to one A-2 monomer. The molecular weight of such a complex would be approximately 36,000. As the estimation of molecular weights by SDS gel elcctroplioresia can result in considerable error when considering proteins which may have both interand intra-peptide crosslinks (90), additional evidence is desirable before one can conclude that an A-l"«A-2 crosslinked product is actually produced. To approach this problem it is necessary to separate the individual crosslinked products so they can be characterized. Gel filtration chromotography using Sephadex G-200 containing 1.0% SDS was attempted. Although this procedure was capable of a reasonable separation of ApoA-1 from ApoA-2, the crosslinked products were eluted as a broad, even band; each fraction containing a mixture of similar molecular weight products. This procedure was not capable of sufficient purification of any single product. The problem was solved using the experimental approach diagrammed in Figure 4. The procedure is detailed in the Methods section of this dissertation. The results of the amino acid analysis of the 46,000 dalton crosslinked product is shown in Table 2. Comparison to the predicted values for sleected amino acids for an A-1 « "A-2 cpmplex indicates the 46,000 dalton product is likely a complex of one A-1 and one A-2. Certain amino acid values were not shown because they were components of the gel buffer system, gave unexpected high or low values for purified A-1 or A-2, or were destroyed during hydrolysis. That both A-1 and A-2

PAGE 51

40 HDL (HDL or HDL ) Chemical Crosslinking — SDS PAGE Fluorescent Labeling I Prep. SDS PAGE Slice out protein band I Elution of protein I Dialysis I HCL hydrolysis I Amino acid hydrolysis Figure 4. Experimental approach to amino acid analysis of crosslinked products

PAGE 52

41 Table 2. Amino Acid Analysis of the 46,000 Dalton Crosslinked Product from HDL Crosslinked with DFDNB Amino Acid Lysine Histidine Arginine Alanine Valine Leucine Tyrosine Phenylalanine Aspartic acid Threonine Isoleucine Methionine a Observed

PAGE 53

42 are components of this complex is suggested by two observations. First, the shift in molecular weight upon addition of reducing agent could only occur if ApoA-2 was present as no disulfide bonds occur in Apo-1. Second, there is no histidine in ApoA-2 or isoleucine in ApoA-1, but both occur in the 46,000 dalton product. As ApoA-1 contains 5 histidine residues/molecules and ApoA-2 dimer contains 2 isoleucine residues/molecule, tliese must be the source of the histidine and Isoleucine found in the complex. Although the C-peptides contain both isoleucine and histidine, the low total amount of C-peptides in HDL excludes these polypeptides from a significant contribution to the composition of any major crosslinked product. Effect of Crosslinking on HDL Structure In discussing the meaning of the results of crosslinking experiments it is important to establish that the introduction of these crosslinks does not significantly alter the structure of the macromolecular complex that is being studied. In the case of HDL, it could be shown that crosslinked HDL retains the physical properties of native HDL and will migrate in the analytical ultracentrifuge as a single peak essentially identical to native HDL. Although even very high levels of crosslinking (100-fold molar excess) do not appear to disrupt the particles, all conclusions presented are based on data obtained at much lower levels of crosslinking. Crosslinking of HDL and HDL with DFDNB Although the peptide composition of the two HDL subclasses, HDL„ and HDL^, appears to be the same, the amount of total protein is different (1). HDLj should contain approximately 160,000 daltons of

PAGE 54

43 protein/molecule while HDL should only contain 80,000 daltons/molecule. Under conditions of increasing amounts of crosslinker, one might expect to observe larger molecular weight crosslinked products for HDL„ as compared to HDL^. However, using the Lammli gel system with molar ratios of crosslinker higher than 5 to 10-fold, one observes only a smear of stain at the top of the gel for each subclass. This phenomenon has been noted for other types of crosslinkers (91) . For this reason a different gel system was used. Fairbanks, et al. have noted that an SDS concentration of 1%, rather than the 0.1% found in the Lammli system, is necessary to dissociate membrane protein complexes (85). Figure 5 shows the comparison of the pattern obtained for HDL crosslinked with a 20-fold molar excess of DFDNB and then run on the two gel systems (5a-Lammli, 5b-Fairba'nks) . The 1% SDS in the Fairbanks gel system is apparently sufficient to cause total dissociation of even highly crosslinked lipoprotein particles. Figures 6 (HDL-) and 7 (HDL„) demonstrate that for both HDL„ and HDL^ the crosslinking patterns are essentially the same. It is interesting to note that no crosslinked products of apparent molecular weight products occur even if the level of crosslinker is increased up to 100-fold. For this reason no separation of HDL„ and HDL was carried out for other crosslinking experiments; instead, HDL was used. Crosslinking of HDL with DMS and DFDNDPS To investigate the hypothesis that the hydrophobic nature and length of the bifunctlonal reagent used should have some effect on the extent and type of crosslinking produced, a comparison of the gel patterns obtained was made after DMS or DFDNDPS was used to crosslink HDL . These

PAGE 55

Figure 5. Comparison of the SDS-PAGE methods of Fairbanks (A) and Lammli (B) for the separation of highly crosslinked HDL (25x DFDNB).

PAGE 56

45 MIGRATION DISTANCE

PAGE 57

•s

PAGE 58

47 AxiSNaa ivDiido

PAGE 59

•X3

PAGE 60

49 AxiSNaa ivDiido

PAGE 61

50 two reagents have similar distances between the functional groups but differ greatly in hydrophobic character. DMS is water soluble and carries a positively charged imido group while DFDNDPS is quite insoluble in water and must be added in an acetone solution. At low levels of crosslinker (10-fold molar excess) these two reagents appear to generate significantly different initial crosslinked products (Figures 8-11). Two major differences between DMS and DFDNDPS crosslinking can be seen. First, the total amount of crosslinking is smaller for the hydrophyllic reagent DMS. Second, the initial site of reaction for DMS appears to differ from DFDNDPS with a 55,000 dalton product appearing first. At higher molar ratios of these reagents the patterns become very similar with all different products appearing in similar amounts in each case. The initial products produced with DFDNDPS crosslinking are essentially the same as those formed with DFDNB even though the former reagent is twice as long as the latter. Temperature Dependence of Crosslinking When the crosslinking of HDL with a 10-fold molar excess of DFDNB is carried out at different temperatures ranging from 4° to 70°C, no significant differences in the crosslinking patterns are produced. The number and relative amounts of the separate crosslinked products remains similar at all temperatures studied. The only difference observed is a reduced amount of crosslinking at the very high temperatures, 60-70°C. It should be noted, however, that the rate of formation of hydrolysis products of this bifunctional reagent increases significantly at these elevated temperatures. This would lead to

PAGE 62

HO

PAGE 63

52 AlISNaa TVDIXdO

PAGE 64

N a •H H O K-l iH 73
PAGE 65

54 AxisNaa ivDiido

PAGE 66

e^ S to

PAGE 67

56 AiisNaa Tvoiido

PAGE 68

H • @| •a p ^s & o H e

PAGE 69

AXTSNaa TV3IXJ0

PAGE 70

59 decreased availability of reagent as well as an increased probability of monofunctional labeling. Interpretation of Crosslinking Results From the data in this section we conclude that at least some of the two major apoprotein components must lie very close to one another in the intact lipoprotein particle since DFDNB will only crosslink groups about 5A apart. If each HDL particle contains both A-1 and A-2 molecules, a number of possible crosslinking products could result. From our findings with low levels of DFDNB (5-10 fold molar excess) several observations can be made. First, the major crosslinked product appears to comprise one A-1 plus one A-2; second, there is no indication of any A-2 Intramolecular crosslinking which might be expected with a molecule already crosslinked with a disulfide bridge. There also seems to be little significant crosslinking-reactive association of two A-1 molecules as suggested by Scanu (92) . Since both A-1 and A-2 contain numerous reactive residues fairly evenly distributed throughout their sequence (1) , any close association of the chains should make multiple sites available for possible crosslinking. Experiments using higher molar ratios of crosslinker (20-lOOX) do show that most possible combinations of A-1 and A-2 are formed. Still, even at these high concentrations, no A-2 intramolecular crosslinked molecules are produced. The C-peptides do not appear to be involved in the crosslinking reactions. At high levels of reagent the amount of unreacted C-peptide does not appear to decrease when compared to controls. This suggests little association of C-peptldes with A-1 or A-2.

PAGE 71

60 ' The formation of most of the possible products at high crosslinker concentrations seems to indicate either a significant degree of mobility of the apoproteins or significant alteration of the native structure of the particle. Two factors suggest that although some mobility exists, a high degree of specific association of the apoproteins occurs in the intact HDL particle. First, the decreased crosslinking observed at high temperature could be due to increased mobility of the polypeptide chains. Second, the failure of A-2 to form intramolecular crosslinks suggests a stable conformation that remains fairly fixed even at these elevated temperatures. The reproducibility of the crosslinking experiments with respect to order of formation and ratios of products formed, even when HDL is obtained from varied sources, suggests that in the intact HDL particle the apoprotein components may be found in a rather fixed spatial orientation to one another. That the crosslinking patterns for HDL„ and HDLdo not differ also argues for a fixed association of 2A-1 and lA-2 apoproteins. Even though HDL may have two HDL„ protein subunits (54), these do not appear to interact. This would lead one to speculate that HDL„ might be a dimer of 2-HDL„ molecules. This idea will be discussed in detail later in this work. An additional argument in favor of fixed associations with flexibility of the apoproteins comes from the experiments using the two reagents DMS and DFDNDPS. These two reagents consistently yield initial crosslinking products that. differ. This may occur due to different initial sites of reaction for these reagents. The initial products do not differ when one compares DFDNB and DFDNDPS, although the latter is twice as long as DFDNB. This again sugges-ts some flexibility in the associations of the apoproteins.

PAGE 72

61 From these observations one might suggest a model in which the apoproteins lie in a fixed, flexible conformation held in place in part by protein-protein, lipid-protein, and lipid-lipid interactions. The ability to bind lipids may in turn be governed by these interactions; the flexibility allowing for differing degrees of binding of specific "functional" lipids while certain "structural" lipids remain fixed. Apoprotein Exchange Between HDL ^ and HDL If the ratio of A-1 to A-2 dimer is 2:1, in both HDL„ and HDL_ as suggested by Friedberg and Reynolds (54), then the difference between HDL and HDL. may represent the addition of one A-l:A-2 unit, which consists of two A-1 plus one A-2 peptide dimer. Because our crosslinking data suggests a degree of flexibility as well as specific interaction within an A-1: A-2 unit but the two units do not appear to crosslink in intact HDL„, it is necessary to consider possible modes of association of the apoproteins of the two proposed units in HDL„ . To investigate this possible association, as well as the relationship between the apoproteins of the different subclasses of HDL, we have examined the exchange of labeled apoproteins between HDL« and HDL , in the presence and absence of bifunctional crosslinking reagents. Figures 12 and 13 show the ultracentrifugal fractionation patterns 125 obtained for controls (SOOyg HDL protein in 200^1) of [ I]-labeled 125 HDL„ and [ I] -labeled HDL„ incubated in the absence of any other protein. The results are expressed as dpm per fraction as it was found that the radioactivity and protein concentration curves coincided (the specific activities of control fractions were constant throughout the

PAGE 73

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PAGE 76

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PAGE 77

66 tube). Figures 12 and 13 also illustrate the results obtained after mixing equal amounts (300Mg protein in 200pl) of [I] -labeled HDL 125 and cold HDL2 (Figure 12) or [ I] -labeled HDL^ and cold HDL and incubating the mixture for 7 hrs, at 37°C (Figure 13). In Figure 12 it can clearly be seen that a transfer of label from HDL to HDL has occurred. Further, the specific activity of the HDL„ fraction was significantly decreased when compared to controls. In a typical experiment, the values for a selected fraction in the HDL region of 125 the tube (fraction 6) were: [ I]-HDL control = 2850 dpm/yg; 125 f I]-HDL^ incubated with cold HDL. = 1680 dpm/yg. Similar results were obtained when labeled HDL was incubated with cold HDL (Figure 13) . However, due to the breadth of the HDL peak as compared with the HDL fractions, the results are not as easily visualized. Effect of Crosslinking and Monofunctional Labeling on Apoprotein Exchange If the labeled HDL„ is crosslinked with DFDNB before incubation with HDL2, the transfer of labeled protein is reduced depending upon the extent of crosslinking. Figure 14 shows the three fractionation 125 patterns obtained with : [ Ij-HDL^ incubated with HDL2 (control); 125 [ I]-HDL„ crosslinked with a 5-fold molar excess of DFDNB and then 125 incubated with HDL^; and [ I] -HDL incubated with HDL. following crosslinking of the HDL with a 25-fold molar excess of DFDNB. It can be seen that increased crosslinking results in decreased exchange. To rule out the possibility that the monofunctional substitution of the apoproteins with the crosslinking reagent was the cause of this decrease in exchange, FDNB, a monofunctional analog of DFDNB, was used to modify the proteins. Reaction with concentrations of FDNB, comparable

PAGE 78

m 1-) •H U -V O i JS 0) M in S e II 13 T) Bl O t) O •M nJ 4-1 M ,ci I 3 D in hJ (U J3 U c e 4-1 M ^ 4J iH M-im C nj O (UCN -H ^ li-i H H 3 I iH ' — ' CO o m 0) tfl C CN XI II O -H n) ^1 ta H ® O >> I 4-1 1 — I O M Qjin M-ltN M-lrH • i-J XI p-J « 3 Q I O TJin o CCN M-l cdiH i_, pq hJ II p 9 P •a s CO J3 en 3

PAGE 79

68 NOIIDVHi Had Kia

PAGE 80

69 to amounts of DFDNB that greatly inhibited exchange, had little effect on the exchange process (Figure 15). When HDL2 is crosslinked before incubation with labeled HDL , the excliangti process is also Jnjiibitad (Figure 16). The reverse also hoida true; that is for crosslinked HDL incubated with labeled HDL , the exchange is reduced. Effect of Temperature and Dilution on Peptide Exchange Both low temperature and dilution have an effect upon the rate of protein exchange in HDL. If the incubation is carried out at 4°C, very little exchange will occur in the time period in which significant exchange occurs at 37°C (Figure 17). Using the amounts of HDL and HDL^ described in the Methods section, but diluting this mixture with a 10-fold excess (5ml) of 0.15M NaCl, the rate of exchange at 37°C is also significantly reduced (Figure 18). These two observations are consistent with a mechanism of exchange involving a collisional complex. It is this reduction in exchange rates that permits one to employ the 48 hour centrifugation required for reseparation of the HDL„ and HDL mixtures. When equal amounts of labeled HDL and cold HDL„ are mixed, then immediately diluted, cooled to 4°C, and placed in the ultracentrifuge, essentially no exchange occurs, even after 48 hrs. Rate of Peptide Exchange Figure 19 shows a time course plot for the exchange of apoproteins 125 from [ I]-HDLt into HDL^. The radioactivity represents the total transferred into the HDL„ region of the centrifuge tube after subtraction of the amount in those fractions attributed to HDL„. Estimation

PAGE 81

00

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>->

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e

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c

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c

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PAGE 91

80 of the initial slope at 2300 dpm/hr out of a total of 90,000 dpm gives an exchange rate of approximately 2.5%/hr. These calculations are based upon two separate experiments utilizing concentrations of HDI, and HDL comparable to plasma levels (3mg/ml each subclass). The rale can be altered by varying the initial concentrations of either or both subclasses. Effect of Bromide Ion on Protein Exchange If the chaotropic Br ion is included in the incubation mixture at 37°C, the amount of exchange in a given time period is greater when compared to controls with no Br . Figure 20 shows the fractionation pattern obtained when 2M KBr is included in the 7 hour incubation solution as compared to controls. A significant increase in exchange can be seen. As chaotropic ions are known to effect protein conformation and association (93), these factors may be involved in the exchange process. SDS-Polyacrylamlde Gel Analysis of Exchanged Proteins Gel electrophoresis of the recipient HDL subclass, into which labeled apoproteins have been transferred, indicates that ApoA-1, ApoA-2, and the C-peptides are all exchanged. Figure 21 shows the radioactivity pattern obtained when HDL„, into which labeled apopro125 teins have been transferred by incubation with [ I]-HDL_, is run on SDS-PAGE. The radioactive bands were identified by staining and scanning duplicate gels. If the labeled HDL subclass is subjected to limited crosslinking with a 10-fold molar excess of DFDNB before mixing and incubation

PAGE 92

c

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PAGE 96

85 with cold HDL, the labeled apoproteins tha:t are exchanged are those that were not crosslinked. Figure 22 shows the gel pattern obtained 125 for crosslinked [ I]-HDL alone, and for HDL that was labeled by 125 exchange of apoproteins froin crosslinked [ I] -HDL.. Although a significant portion of the HDL apoproteins were crosslinked into higher molecular weight products, only uncrosslinked A-1 and A-2 appear to be exchanged. Interpretations of Protein Exchange Experiments The transfer of labeled apoprotein from one HDL subclass to another does not proceed with a loss of protein content of the originally labeled fraction. The specific activity of that fraction decreases but the protein content remains unchanged. This is consistent with an exchange process in which there is a one to one exchange of protein of one subclass with the other. Such exchange could occur by the transfer of single polypeptide apoproteins, i.e., A-1 or A-2; or by the exchange of complexes of apoproteins such as 2A-l:lA-2. These experimental results do not permit us to rule out either of these possibilities. It might be suggested that since crosslinking inhibits the exchange, single polypeptide chains are involved. However, the crosslinking may also prevent a conformational change that might be necessary for the exchange process. That a conformational change might be involved is suggested by the fact that when high concentrations of the chaotropic ion, Br , are included in the incubation mixture, increases in the rates of exchange are observed. An actual exchange involving a conformation change is also suggested by the finding that crosslinking the unlabeled subclass also greatly inhibits the exchange

PAGE 97

H -a to x; c u ex

PAGE 98

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PAGE 99

88 process. If a simple transfer of peptide Into the recipient class occurred, one would not expect crosslinking to have much effect. If HDL„ is made up of two identical subunits as suggested by Friedberg and Reynolds (54), the transfer of label might be visualized in terms of the exchange of HDL for one of the HDL„ subunits. Although this is a reasonable possibility, one must again consider the possibility of a conformational change to explain the results of the crosslinking experiments. Even though the two HDL subclasses can exist as stable separate entities, when they are present together in solution, significant interaction between particles may occur. Exchange of apoproteins could possibly occur between HDL2-HDL or HDL -HDL , as well as between HDL and HDL molecules. If collisional complexes cause exchange and single polypeptides are capable of exchange, then one would expect HDL -HDL exchange to occur. Collisional complex formation should also result in the rapid exchange of lipids of all types due to their small size and the fusion required for peptide exchange. Lipid Exchange Between HDL and HDL in Solution 3 1 Wlion KDL^ Is labeled wltli [ II)-clu)JestGrol, [ U]-cholestcrol paJmi14 tate, or [ C]-phosphatidyl choline, then incubated with cold HDL , as with peptide exchange; label is transferred to HDL„ . However, even under conditions of low temperature, dilution, and no incubation, total exchange occurs during the ultracentrifugal reseparation of HDL„ and HDLoThis rapid movement prevents the study of the exchange using the experimental approach that was possible for peptide exchange. For this reason an alternative approach was developed.

PAGE 100

89 Binding of Labeled HDL to Sepharose The rapid rate of lipid exchange places one requirement on the method employed to study this exchange: the separation of HDL and HDL2 must be done very quickly. As use of the ultracentrifuge requires at least 24 hours even at 60,000 rpm, it was decided to approach the problem by immobilizing one of the subclasses by covalently binding it to Sepharose. This would allow separation by filtration; a process that would take less than one minute. 125 When [ I]-HDL~ was reacted with commercially obtained CNBr-activated Sepharose 4B (Pharmacia) under the coupling conditions described in the Methods sections, a Sepharose preparation containing approximately 0.5mg protein per ml of packed Sepharose was obtained. To determine what percentage of the protein was bound to the Sepharose, a portion of the gel was washed with 1% SDS solution. Under these conditions any protein chains not covalently bound to the gel should be washed off. Table 3 shows the results of such an experiment. Using the Pharmacia CNBr-actlvated Sepharose, no more than 30% of the label could be removed with SDS. Assuming only 3 polypeptide chains/HDL molecule, this Indicates that an average of two are linked to the Sepharose. To obtain a preparation with a lower level of binding, it was necessary to activate our own Sepharose 4B using the conditions described in the Methods section. Under these conditions, preparations with over 50% of the protein removable with SDS could be obtained. The same conditions also gave acceptable levels of binding for HDL„ preparations (20-40% bound). For all peptide exchange experiments, preparations with low levels of binding were used. For lipid exchange

PAGE 101

90 Table 3. Effect of Amount of CNBr Activation on the Amount of Binding 3 of [1^^I]-HDL „ by Sepharose 4 SAMPLE TOTAL PPM PPM of Filtrate % BOUNP SEPHAROSE 4B-HPL3 14,500 3,920 ' 73% (CNBr Activated-Pharmacia) SEPHAROSE 4B-HPL3 12,500 4,000 68% (CNBr Activated-Pharmacia) SEPHAROSE 3B-HPL3 19,590 10,970 44% (Limited Activation See Methods Section)

PAGE 102

91 experiments, where higher levels of peptide-gel linking were permissible, the CNBr-activated Sepharose from Pharmacia was used. Peptide Exchange Using Sepharose-Bound HDL To test the validity of the Sepharose method we repeated the peptide exchange experiments and compared the results to those obtained using the ultracentrifugal separation. Table 4 gives the data for peptide exchange obtained under a variety of conditions similar to those previously described. Several important observations can be made. First, both cold and dilution reduce the amount of exchange that occurred in a given time period. Second, crosslinking of the HDL subclass on the Sepharose or in solution reduces the exchange. Finally, exchange can occur between Sepharose-HDL^ and HDL , as well as between SepharoseHDL2 and HDL„ in solution. These results suggest that the binding to Sepharose does not greatly affect the exchange process. Because this method allows the study of the interaction of like molecules (i.e. HDL^-HDL^) as well as different types of particles, several experiments that could yield information regarding the possibility of subunit exchange became possible. Ultracentrifugal Analysis of Peptide Exchange Using Sepharose-Bound HDL If HDL„ is bound to Sepharose and then incubated with HDL in solution, one would expect different products in solution depending upon the possibility of subunit exchange. Figure 23 shows the ultracentrifugal fractionation pattern obtained after HDL was incubated ' 125 with [ Il-HDL^ bound to Sepharose. It can be seen that both labeled HDL2 and HDL„ particles are formed during the incubation. This could

PAGE 103

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96 only occur if one of the unbound subunits of HDL„ was combined with an HDL molecule to foirm an HDL molecule in solution. If cold HDL 125 is incubated with Sepharose bound [ I]-HDL , only labeled HDL is found in solution (Figure 2A). If subunit exchange occurs, one would also expect both HDL^ and HDL„ to be formed when HDL„ is incubated 125 with [ I]-HDL„ bound to Sepharose. Figure 24 shows this to be the 125 case. Figure 24 also shows that HDL„ incubated with bound [ I]-HDL_ only yields labeled HDL„ in solution. This could only occur by the exchange of single apoprotein molecules as at least one of the HDLpeptides must be covalently bound to the Sepharose. This would obviously prevent the exchange of a whole subunit. SDS-PAGE Analysis of Exchanged Peptides Using Sepharose-Bound HDL To show that all the different types of apoproteins were availa125 125 ble for exchange, [ I] -HDLand [ I] -HDL bound to Sepharose were washed with 1% SDS and the wash solution subjected to SDS-PAGE. Figure 25 shows the radioactivity pattern obtained after slicing and counting the gels. ApoA-1, ApoA-2, and the C-peptides are all capable of being removed and available for exchange. This experiment also indicates that there is no preferential binding of any single apoprotein species to the Sepharose as the ratios obtained are essentially the same as those found in native HDL. That all types of apoproteins are exchanged is indicated when one does SDS-PAGE of either HDL or HDL after incubation with [""^^^IJ-nDL bound to Sepharose (Figure 26). SDS was added to solubilize the apoproteins of the HDL particles found in the filtrate after incubation and

PAGE 108

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103 removal of Sepharose. Again, all three types of labeled apoproteins are present. The only possible source of label is by exchange with labeled HDL bound to the Sepharose. Incorporation of Labeled Lipid into HDL ^ and HDL To study lipid exchange in lipoproteins it is first necessary to incorporate labeled lipid into one of the lipoprotein subclasses. As most lipids are quite insoluble in aqueous solutions and because one must be able to separate any free lipid from the labeled HDL, it is desirable to place the lipid on a solid support that can be easily separated from the solution. Because the physical state of the lipids appears to be important in determining a lipid binding protein's ability to incorporate them (94), the lipid was first adsorbed to celite particles before mixing with the HDL to be labeled. This procedure proved very satisfactory for the incorporation of cholesterol, cholesterol palmitate, and phosphatdyl choline into HDL or HDL . Figure 27 shows the time course for uptake of labeled lipids from celite by HDL„ under the conditions described in the Methods section. It should be noted that the leveling off of the curves represents the uptake of 90% or more of the label available, not a saturation of the HDL by the lipid. Low levels of labeling were used to try and achieve binding of fewer than one labeled lipid molecule per HDL molecule. This was done to prevent such major alterations in the lipoprotein particle's structure as might occur with increased lipid binding. Calculations of the number of lipid molecules bound per HDL molecule gave the following results for the preparations used for all the experiments described: 3 3 [ H]-cholesterol-HDL= 3700 HDL molecules/ [ H] cholesterol molecule.

PAGE 115

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PAGE 117

106 3 1 [ H]-cholesterol palmitate-HDL =5.0 HDL molecules/ [H] cholesterol/ molecule, [ C] -phosphatidyl choline-HDL = 35 HDL molecules/ ["""C]phosphatidyl choline molecule. These preparations of labeled HDL were bound to CNBr-activated Sepharose 4B (Pharmacia) and the resulting products were used for all Sepharose-HDL lipid exchange experiments described. To measure the amount of label available for exchange and not bound to the HDL irreversibly, aliquots of the Sepharose-HDL„ labeled with lipid were washed with 1% SDS solution, the Sepharose removed by filtration, and the filtrate counted in Aquasol (Table 5) . Essentially all of the label is removed with SDS while washing with buffer has no effect. This fact, in addition to the results obtained with SDS removal of labeled peptides, suggests that stable intact particles are bound to the gel and that non-covalently bound materials can be removed only when the lipoprotein particles are disrupted with detergent. Ultracentrifugation of HDL labeled with radioactive lipid at 3 d = 1.125gm/cm shows that the label followed the lipoprotein pattern produced when the peptides were labeled and indicated no free label in the solution. Lipid Exchange Between HDL , and Sepharose-HDL To determine the extent of bulk transfer of lipid by subunit exchange from HDL„, lipid exchange between HDL„ molecules was studied. Because it had been previously demonstrated that lipids will exchange, experiments designed to help to understand the mechanism of exchange were carried out. If lipid labeled, Sepharose-bound HDL„ (S-HDL*) was incubated for 30 min with varying amounts of HDL„ in solution (Figures 28,29). All three lipid types exhibit this behavior suggesting

PAGE 118

P 107 Table 5. Removal of Labeled Lipid from Sepharose-Bound HDL3 (S-HDL?) By SDS Wash IOOmI of S-HDL3 was washed with 1ml of 1% SDS, filtered, and the filtrate counted in 5ml of Aquasol. Labeled S-HDL * DPM Before SDS DPM SDS wash S[ -^H] -Cholesterol-HDLS-[ H]-Cholesterol palmitate-HDL„ lA S-[ C] -Phosphatidyl choline-HDL~ 29,388

PAGE 119


PAGE 120

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112 that collisional complex formation ±s required for lipid exchange as it appears to be for protein exchange. To try to establish the relative rates of exchange of these lipid types, incubations were carried out at various times and the amount of label transferred plotted as a function of time of incubation (Figures 30,31). It appears that at the concentrations of HDL used, cholesterol and cholesterol palmitate both exchange very rapidly with half times of less than 30 min. Higher concentrations of free HDL result in even faster exchange rates. In the case of phosphatidyl choline, an initially rapid exchange with a short half-life (<30 min) is followed by a slower exchange process. Exchange of cholesterol and cholesterol palmitate after 24 hrs. results in exchange with total counts transferred not much greater than the values obtained for 2 hrs. However, for phosphatidyl choline, the 24 hr. value is more than twice the 2 hr. value. Effect of Temperature and Dilution on Lipid Exchange If separate batches of S-HDL* and HDLare mixed and incubated for equal times at different temperatures, the amount of exchange is decreased at lower temperatures for cholesterol and cholesterol ester (Table 6). Dilution (increasing the volume) of the incubation solution also appears to decrease the exchange (Table 6). These observations are consistent with collision complex formation. Effect of Saturating Cold Lipid on Labeled Lipid Exchange If the mechanism of exchange depended upon the dissociation of lipid from the HDL particle into solution and then subsequent reassociation by other lipoprotein molecules, saturation of the aqueous medium

PAGE 125

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117 Table 6. Effect of Temperature and Dilution on Lipid Exchange Between S-HDL* and HDL „. 25m1 S-HDL* (3mg HDL Protein/1 gel) + 150m1 0.15M NaCl containing lOOiig IIDL^. Incubations were carried out for 15 mln. SAMPLE ^ CONDITIONS PPM FILTRATE 3 [ H] -Cholesterol 4° Incubation 3,157 " 20° Incubation 5,473 " 37° Incubation 6,815 " 37° Incubation, . 1,344 Diluted with 3ml of . 0.15M NaCl 3 [ H]-Cholesterol palmitate 4° Incubation 7,733 " 20° Incubation 8,580 " 37° Incubation 9,614 " 37° Incubation, 4,352 Diluted with 3ml of 0.15M NaCl 14 [ C]-Phosphatidyl choline 4° Incubation 286 " 20° Incubation 283 '• 37° Incubation 273 " 37° Incubation, 147 Diluted with 3ml of 0.15M NaCl

PAGE 129

118 with unlabeled lipid of the same type being transferred should greatly decrease the rate of transfer. Table 7 shows the results of such an experiment. The amount of cold lipid added to the solution was approximately 100 times greater than the total contained in all the lipoprotein particles present. The lipid was added dissolved in a small amount of organic solvent. The majority of the lipid precipitated indicating that saturating conditions were reached. Even under these rather extreme conditions, the amount of exchange observed was equal to the controls containing only bound lipid. This strongly suggests that dissociation into the medium is not a significant factor in the exchange process. Effect of Crosslinking on Lipid Exchange When either the labeled or cold HDL is crosslinked with a 25-fold molar excess of DFDNB before mixing, no significant reduction in exchange appears to occur (Table 8) . This observation is not unexpected as fusion of lipids during collision complex formation would not be influenced greatly by protein crosslinking. Effect of Added Organic Solvent on Exchange of Lipids and Proteins When both lipid and apoprotein exchange is studied in the presence of increasing amounts of ethanol in the solution, the rates of exchange increase significantly (Figures 32,33). These increases appear to be the result of factors other than the total disruption of the particles as control experiments indicate that labeled lipid is not removed from S-HDL* by the solvent alone.

PAGE 130

119 Q (U

PAGE 131

120 Table 8. Effect of Crosslinking on Lipid Exchange Between S-HPL -, andJHDL-. 25m1 S-HDL3 (3Mg HDL Protein/ul Gel) + 150yl 0.15M NaCl containing lOOyg HDL^. incubations. lOOyg HDL^. All crosslinking (C.L.) 50x molar excess DFDNB. 15 min. SAMPLE [ H]-Cholesterol 3 [ H]-Cholesterol palmitate 14 [ C] -Phosphatidyl choline CONDITIONS HDL, HDL^ C.L. DFDNB HDL3, S-HDL*-C.L. DFDNB HDL -3 HDL„ C.L. DFDNB HDL3, S-HDL3-C.L. DFDNB HDLo HDLC.L. DFDNB HDLo, S-HDL*-C.L. DFDNB PPM FILTRATE 10,611 9,075 9,069 17,821 17,124 18,323 421 386 394

PAGE 132

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PAGE 133

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125 Interpretations of Sepharose-HDL Peptide and Lipid Exchange Experiments The data obtained in the Sepharose-bound HDL experiments compares well with that obtained using the ultracentrifugal separation procedure. Low temperature, dilution, and crosslinklng all reduce peptide exchange in both systems. In the gel system, however, the observed reduction is not as great as that seen in the non-bound system. This may be ascribed to the fact that the amount of HDL bound to the gel (100-200 lig/ral) is much less than 2-3mg/ml, the concentration of HDL at which the original method was used. To observe significant exchange in a similar time period, the concentration of cold HDL added far exceeds that of the labeled and bound subclass. This condition pulls the exchange process in the direction of removal of label from the bound HDL. To obtain levels of binding comparable to those possible in solution (3mg/ml), highly activated Sepharose is needed. Unfortunately, this level of activation also results in multiple binding to each HDL molecule and subsequently reduced amounts of free peptide are available for exchange. A very interesting observation, not possible with the solution exchange procedure, is that one can demonstrate exchange between individual HDL^ molecules and between HDL molecules. S-HDL ->-HDL exchange could occur by both subunit exchange and exchange involving the movement of single polypeptide chains. However, S-HDL-->-HDLexchange could only occur by transfer of single apoprotein species. An actual exchange is again strongly suggested by the ultracentrifugal analysis of S-HDL^-»-HDL exchange products which shows only HDL molecules are produced. If only a transfer of protein occurred, the resulting particles

PAGE 137

126 would have a higher density than native HDL_ molecules and would sink to the bottom ov the centrifuge tube. The results of this experiment indicate a normal distribution pattern for HDL„. The production of free HDL when HDL is incubated with S-HDLindicates that subunit exchange has occurred. The only possible source of free HDL^ is from the combination of an HDL„ molecule and one of the unbound subunit s of the S-HDL-. The reverse experiment using S-HDLo and free HDL„ results in the formation of free HDL„; a possibility only if a subunit is transferred from HDL„ in solution to S-HDL-, resulting in free HDL^ molecules and bound HDLj molecules. Subunit exchange would have to involve both A-1 and A-2 apoproteins but single peptide exchange between S-HDL and HDL„ could be specific for a particular apoprotein. However, Figures 25 and 26 show that this is not the case. This is interesting in view of the fact that A-1 and A-2 appear to differ significantly in their ability to bind lipids. The results of this experiment also suggest that a dynamic association exists between A-1 and A-2. If one of the members of the A-1: A-2 unit is bound to the Sepharose, a dissociation of those labeled apoproteins that are free, followed by a reassociation with unlabeled peptides must occur. Since the particles do not appear to be disrupted during the exchange process, a significant degree of flexibility must exist within the intact particle. The results of the lipid exchange experiments support the suggestion that collision complex must occur for the exchange process to occur. As with the peptide exchange, lower temperature, lower concentrations of free HDL, and dilution of the reaction mixtures, all reduce

PAGE 138

127 the rate of exchange appreciably. The conclusion is also strongly supported by the experiments using saturating levels of cold lipid. If dissociation of the lipid molecule into the medium was required, surely increasing the amount of free lipid would have a great effect on exchange. Tlie rates of lipid exchange are consistent with their size and mobility in the intact particles. A fusion of the contact surface of the collision complex would result in a fast exchange of lipids and would also provide a hydrophobic environment for movement and rearrangement of the apoproteins and interior lipid molecules. The exchange of phospholipids appears to follow two processes. A very rapid exchange of approximately one-fourth of the label, followed by a much slower exchange of the remaining label; this could be explained by assuming the slowly exchanging phospholipid was strongly associated with the protein while the more rapidly exchanging fraction was free phospholipid. The lack of effect of crosslinking is not unexpected as the incubation time only included the fast exchange portion. By decreasing solvent polarity with the addition of ethanol, one might expect to cause a disruption of the HDL particle. This does not appear to be the case, but the rate of exchange is increased. This could be due to an overall decrease in the strength of interaction of both protein and lipid components. Such a decrease, although reducing the Interactions, still is not sufficient to break apart the particle. This finding suggests that the HDL molecules are held together by strong forces, capable of withstanding conditions which would greatly affect certain other types of molecular aggregations.

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CONCLUSIONS AND SPECULATIONS From the data in these experiments we may reach the following major conclusions: 1) Bifunctional crosslinking reagents of both hydrophobic and hydrophillic character are capable of forming protein-protein cross. links between the major apoprotein components of human HDL. 2) DFDNB crosslinking suggests that ApoA-1 and ApoA-2 must, at least part of the time, lie in close proximity in intact HDL„ and HDL^. 3) HDL, appears to contain two subunit complexes that are not crosslinked with DFDNB, DMS, or DFDNDPS under conditions tested. 4) The separate apoprotein molecules can exchange between and among the HDL subclasses HDL and HDL at a rate rapid enough to be of importance in the biological lifetime of the particles. 5) Subunits of HDL„ can exchange with HDL resulting in a new HDL„ particle containing the original HDL„. 6) Crosslinking with bifunctional reagents apparently prevents a conformational change necessary for exchange of the apoproteins. 7) The three major lipid types of HDL all exchange among and between the HDL subclasses. 8) Both lipid and protein exchange appear to involve formation of collision complexes between the lipoprotein particles. 9) There exists a dynamic relationship between HDL^ and HDL . Even though these two subclasses can exist as stable separate entities, 128

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129 when they are present together In solution, significant interaction between particles may occur. The results of the experiments of Friedberg and Reynolds along with the evidence presented. here suggest that a "lipid binding unit" of 2 ApoA-1 + 1 ApoA-2 molecules form the basis for the structure of both HDL^ and HDL„. However, the combination of two HDL. molecules does not produce an HDL„ molecule. There are two possible explanations; first, the extra lipid present in HDL„ (60% lipid, 40% protein) could come from membranes or other lipoprotein molecules by lipid binding to HDLmolecules. The interaction of a possibly unstable lipid rich HDL_ molecule with another HDL^ molecule could result in the formation of an . HDL„ molecule. HDL„ molecules alone in solution would not have a source of extra lipid and would thus remain as HDL molecules only. Since HDL„ molecules in solution would have no place in which to deposit this extra lipid, they could not produce HDL„ molecules. An alternative explanation would involve the alteration of apoprotein interactions and conformation into a form capable of increased lipid binding. Again, this change may be only possible in the presence of other lipoproteins or cell membranes. Since the exact nature of such subtle apoprotein interaction may govern lipid binding, we suggest that further studies should be undertaken to determine the exact location of chemical crosslinks. Although the exact role of HDL is not known, we suggest that it might serve its major function as a transport vehicle for cholesterol and cholesterol esters. The ease with which HDL adsorbs these lipids from celite particles would seem to indicate that any readily ayailable

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130 lipid of this type would be rapidly picked up by HDL in the circulatory system. This idea could acconnnodate an interconversion of HDL„ and HDL~ by interaction with a lipid source. A source containing phospholipids as well as cholesterol would be required; again other plasma lipoproteins or cell membranes come to mind. The phospholipid would serve to maintain apoprotein structure and perhaps interaction. This model suggests experiments that could determine the effect of different lipid sources on HDL„ HDL„ interconversion and exchange. Such a model might be diagrammed as shown in Figure 34. In both HDL„ and HDL the protein covers the surface of the particles. However, when HDL is placed in the presence of a lipid source, extra cholesterol ester and triglyceride could be accommodated by altering the conformation of the protein in such a way that when two HDL„ molecules fuse, the surface of the new particle is covered with protein and additional inner space for neutral lipid is made available. The larger HDL^ particle formed might also require small increases in phospholipid or cholesterol but the major difference would be increased neutral lipid content.

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131 "LIPID RICH"HDL3 HDL' Figure 34. Model for HDL -HDL Interconversion.

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BIOGRAPHICAL SKETCH Thomas Ellis Grow was born in Pensacola, Florida, on January 6, 1945, the son of Captain Harold B. Grow and Beatrice D. Grow. He completed his primary and secondary education in Pensacola and graduated from Escambia High School in 1962. He then attended Pensacola Junior College from 1962 to 1964 and received an A.S. degree in 1964. After one semester at Florida State University he moved to California where he worked as a research assistant at Scripps Clinic and Research Foundation. In 1967 he returned to Pensacola where he completed his B.S. degree in Biology in 1969 at the University of West Florida. He entered the Master's degree program in Marine Biology in 1969 and while working toward this degree was employed as a research biologist by the Environmental Protection Agency's Pesticide Research Laboratory at Sabine Island, Gulf Breeze, Florida. From 1970 to 1973 he was employed as a teacher at the Pensacola School of Liberal Arts. He left Pensacola in 1973 to enter the University of Florida's Department of Biochemistry to work for a Ph.D. degree. He received a Ph.D. in Biochemistry in June of 1977. 137

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I certify that I have read this study and that, in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. MelVin Fried, Chairman Professor of Biochemistry and Molecular Biology I certify that I have read this study and that, in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Q^QA^iri. Q /.6(/>VVV_Ben M. Dunn Assistant Professor of Biochemistry and Molecular Biology I certify that I have read this study and that, in my opinion, it conforms to acceptaj)le standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Kenneth D. Noonar Assistant Professor of Biochemistry and Molecular Biology

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I certify that I have read this study and that, in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Federic'o" A. Vilallonga Professor of Pharmacy This dissertation was submitted to the Graduate Faculty of the Department of Biochemistry in the College of Arts and Sciences and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. Dean, Graduate School

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UNIVERSITY OF FLORIDA 3 1262 08553 3007