A preliminary characterization of the polypeptide components of the plasma membrane of the L5178y murine lymphatic leuke...

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Title:
A preliminary characterization of the polypeptide components of the plasma membrane of the L5178y murine lymphatic leukemia cell
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vii, 76 leaves. : illus. ; 28 cm.
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Hammond, John Enoch, 1946-
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Mouse leukemia complex   ( lcsh )
Cell membranes   ( lcsh )
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Cytology -- Research   ( lcsh )
Biochemistry and Molecular Biology thesis Ph. D
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF
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Thesis--University of Florida.
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Bibliography: leaves 72-75.
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Typescript.
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Vita.

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Table of Contents
    Title Page
        Page i
        Page i-a
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
    Abbreviations
        Page v
    Abstract
        Page vi
        Page vii
    Chapter 1. Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
    Chapter 2. Methods and materials
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
    Chapter 3. Results and discussion
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
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        Page 46
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        Page 49
        Page 50
        Page 51
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        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
    Chapter 4. Conclusion
        Page 68
        Page 69
        Page 70
        Page 71
    References
        Page 72
        Page 73
        Page 74
        Page 75
    Biographical sketch
        Page 76
        Page 77
        Page 78
        Page 79
Full Text













A PRELIMINARY CHARACTERIZATION OF THE POLYPEPTIDE COMPONENTS OF THE PLASMA MEMBRANE OF THE
L5178y MURINE LYMPHATIC LEUKEMIA CELL







By

JOHN ENOCH HAMMOND














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

1 974











ACKNOWLEDGMENTS

I would like to express my gratitude to my research director, Dr. Waldo R. Fisher, for providing the financial support, for his encouragement during the course of the investigation, and, above all, for his enthusiasm and interest in my research problem.

I cannot adequately express my appreciation to my wife and

colleague, Mary Ethel, for her support and encouragement throughout the course of this investigation. Her editorial comments and suggestions were a great as-set in the preparation of this manuscript. Also, I am extremely grateful for all the years of support and encouragement given me by my parents.

I would like to thank Dr. Kenneth Noonan of the Department of

Biochemi-stry for his assistance in the development of the plasma membrane isolation procedure. Dr. Carl Feldherr was of invaluable assistance in the preparation of the plasma membrane fragments for electron microscopy and of expert assistance in the interpretation of the electron micrographs.

I am grateful for the expert assistance of Mrs. Deborah Truitt in the preparation of this manuscript. Also, I would like to thank Mr. Robert Truitt for his invaluable photographic assistance.

I would like to thank Dr. Elroy Bacallao of the University of Florida Primate Center for his assistance in the production of antiserum in the goat, Red I.

Finally, I would like to remember the one that made this investigation possible and made the ultimate sacrifice: the goat, Red I. May his spirit always graze in green pastures.

ii














TABLE OF CONTENTS


CHAPTER PAGE

Acknowledgments................................................ ii

Abbreviations .................................................. v

Abstract ...................................................... vi

I. Introduction................................................ 1

II. Methods and Materials...................................... 10

Cell Cult ure Technique .................................... 10

Isolation of Plasma Membrane ............................... 11

Electron Microscopy ....................................... 14

Polyacrylamide Gel Electrophoresis Procedure ................ 15

Enzymatic Iodination of Intact L5l78y Cells ................. 16

Liquid Scintillation Counting of 1251 ....................... 16
TrypsinizationProcedure................................... 18

Immunization Procedure..................................... 18

Cytotoxicity Inhibition Assay .............................. 19

Immunoglobulin Purification Procedure....................... 21

Preparation of Fab Fragments ............................... 24

Enzyme Assays............................................. 24

Protein Determinations..................................... 26

Materials................................................. 26









CHAPTER PAGE

III. Results and Discussion.................................... 28

Plasma Membrane Isolation Procedure........................ 28

Characterization of the Isolated Plasma Membrane Fragments.... .35

Preliminary Characterization of the Polypeptide Components
of the Plasma Membrane.................................. 46

Iodination of the L5l78y Cell ............................. 48

Proteolytic Digestion Studies on the L5l78y Cell............ 54

Immunoglobulin Protection Experiment....................... 59

IV. Conclusion............................................... 68

References .................................................... 72

Biographical Sketch............................................ 76





























iv








ABBREVIATIONS




SDS Sodium dodecyl sulfate

PBS Phosphate buffered saline

PGS Puck's saline G

SITS Stilbene-4-acetamino-4'-thiocyano
disulfonate
HL-A Human histocompatibility antigen system

FMA Fluorescein mercuric acetate

BLLA Below lower limits of assay






























v








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.


A PRELIMINARY CHARACTERIZATION OF THE POLYPEPTIDE
COMPONENTS OF THE PLASMA MEMBRANE OF THE
L5178y MURINE LYMPHATIC LEUKEMIA CELL By

John Enoch Hammond

June, 1974

Chairman: Dr. Waldo R. Fisher Major Department: Biochemistry

The objective of this investigation was to &develop a general

technique capable of determining which of the p&'ypeptide components of the plasma membrane acts as an antigenic determinant. To do this it was necessary to characterize the plasma membrane of the cell as to the number, distribution, and organization of its polypeptide components. The cell utilized to develop this technique was the L5178y murine lymphatic leukemia cell. The cell surface antigenic determinants were recognized by the immunological system of a goat.

It was possible to isolate plasma membrane fragments from the L5178y cell with minimal contamination by other cellular organelles. The purity of the isolated fragments was judged by enzyme markers and electron microscopy.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
demonstrated that the plasma membrane fragments were composed of a complex array of polypeptide components. Nine najor components, ranging from a molecular weight of 24,000 to 180,000 daltons, were

vi








resolved. Of these polypeptide components, one was shown to be localized on the surface by both lactoperoxidase iodination and trypsinization. This polypeptide had a molecular weight of approximately 180,000 daltons. It was not possible to detect carbohydrate in any of the polypeptide components by periodic acidSchiff technique.
It was possible to combine the biological specificity of antibody and biochemical techniques to tentatively identify directly an antigenic determinant from the cell surface. It was found that the 180,000 dalton polypeptide component was an antigenic determinant or part of an antigenic determinant complex on the surface of the L5178y cell.




























vii














.CHAPTER I
INTRODUCTION

The plasma membrane is one of the multifunctional organelles of the cell. Its functions range from the maintenance-of the internal environment of the cell to the extremely complex interactions involved in the immunological system. Its transport functions, such as passive and facilitated diffusion and active transport, act so as to maintain the proper internal environment through the movement of nutrients into, and the removal of wastes fromthe cell. Although extremely important, they can be thought of as housekeeping functions. A second function of the plasma membrane is intercellular communication. This function is extremely important in the formation and maintenance of multicellular organisms. The plasma membrane is intimately involved in the secretion and reception of hormones, neurotransmission, tissue formation, and contact inhibition. Finally, the plasma membrane is involved in the function of the immunological defense system. It is through the plasma membrane that -the cells of the immunological system act. In effect, the membrane acts as the sensor for the immunological system, and hence it can tell friend from foe, and respond in the appropriate manner. After many years of work, vie are now just beginning to understand the macromolecular structure of the plasma membrane and how its structure relates to the function of this organelle.

Over the years as experimental data have accumulated numerous

models for the plasma membrane have been proposed. For almost fifty






2
years the basic structure of the plasma membrane was thought to be a bimolecular lipid leaflet (Gorter and Grendel, 1925). In 1935 it was suggested that globular proteins be incorporated into the bimolecular lipid leaflet and this became a representation known as the DanielliDavson model (Danielli and Davson, 1935). In 1957 Robertson modified the Danielli-Davson model as follows:

1) The protein components were in the form rather than

globular.

2) Allowance was made for possible asymmetry of the inner

and outer surfaces.
3) The dimensions of the plasma membrane were further

refined (Robertson, 1957).
This model was widely accepted until the middle 1960's when new experimental evidence cast serious doubt on the fundamental assumptions on which it was based. For example, it was found that the protein conformation in biological membranes was predominantly a helix and random coil; that possible artifacts may have been introduced in preparing membranes for electron microscopy; and that, on repeating the work of Gorter and Grendel, the lipid-area to cell-area ratio was 1.4 or 1.56 instead of 2. This plus other evidence led to the rejection of this model (Hendler, 1971).
Perhaps the most widely accepted plasma membrane model today is the fluid mosaic model which was proposed in the early 1970's (Singer and Nicolson, 1972). Essentially, this model proposes a mosaic structure in which molecules of integral globular proteins alternate with sections of the phospholipid bimolecular leaflet in the cross section of the membrane. These proteins are amphipathic in nature,






3
and hence asymmetric. The polar region of these proteins is postulated to be exposed to aqueous phase and the hydrophobic or apolar region of these proteins is embedded in the hydrophobic interior of the membrane. Presumably, the polar region would then be made of hydrophillic amino acids and also provide the site for covalent attachment of carbohydrate residues. This model also allows for a protein molecule-of suitable size and structure to transverse the entire membrane, and hence be in contact with both the interior and exterior of the cell. Therefore, this model is consistant with and can accomodate most of the accepted functions of taie plasma membrane.

Study of the erythrocyte plasma membrane has played an extremely important role in our basic understanding of the general structure of these membranes. Since this cell is non-nucleated and hence incapable of reproduction, it could be termed a dead cell, but as one investigator (Bretscher, 1973) has pointed out, "A great deal can be learned from studying fossils." Although the erythrocyte is incapable of reproduction, its membrane still possesses a number of biologically important functions (Stein, 1967) and its molecular organization is important in elucidating the general principles involved in membrane architecture.

Since large quantities of pure plasma membrane can be prepared from erythrocytes (Dodge et al., 1963), it is possible to achieve a very complete biochemical characterization of this membrane. The plasma membrane of the erythrocyte has been shown to be composed of approximately twelve major polypeptide components ranging in size from a molecular weight of 15,000 daltons to over 200,000 daltons (Lenard, 1970). Also, one major glycoprotein has been detected. This glyco-







4

protein has been rigorously characterized (Segrest et al., 1972). It has a molecular weight of approximately 30,000 daltons and is composed of about 87 amino acids and 100 sugar residues. The carbohydrate is attached to the N terminal end of the molecule and is localized on the outer side of the plasma membrane. Next comes a very hydrophobic region of the molecule which spans the membrane, and this is followed by a hydrophillic region and the C terminal amino acid.

The erythrocyte system has been extremely useful in the development of techniques designed to localize the protein components in the lipid bimolecular leaflet. The first technique used to localize polypeptide chains in the plasma membrane was limited proteolytic digestion of the intact cell. This technique assumes that the proteolytic enzyme cannot penetrate the membrane and this has been shown to be the case. The plasma membranes of the digested erythrocytes were isolated and subjected to sodium dodecyl sulfate gel electrophoresis. Certain polypeptide bands were found to disappear from the gel indicating that these polypeptides were on the exterior of the erythrocyte. Later, chemical reagents, such as SITS (stilbene-4acetamino-4'-thiocyano disulfonate) and 35S-labelied formylmethionyl sulfone methyl phosphate,were employed to label the exterior polypeptides specifically (Maddy, 1964; Berg, 1969). A third approach using 125
the enzyme lactoperoxidase to label exterior proteins with I has been developed (Phillips and Morrison, 1970). Through the use of these techniques, the topography of the erythrocyte is being delineated and, perhaps of more importance, the validity of these techniques for studying cell surfaces is becoming established. Therefore, the erythrocyte has served as the test system in which various techniques






5

have been developed for the characterization of membrane components; now it is possible to utilize these techniques to probe other cells.

As noted before, the plasma membrane is intimately involved in the function of the immunological system. As is usually the case, the more complex systems are by far the most interesting to study. It is impossible to summarize succinctly the varied functions of the immunological system; however, one can safely say that the great majority of the actions of the immunological system begin at the plasma membranes of the cells that make up this system. For example, the B lymphocyte is capable of binding an antigen which triggers a series of events that ultimately result in the secretion of antibody molecules directed at this antigen. A great deal more is known about the events that follow the antigen-cell receptor interaction than is known about the actual molecular interactions involved.

Among the capabilities of the immunological system is the ability to discriminate between histocompatibility antigens which are genetically controlled and found on all the cells which are part of the host animal and those histocompatibility antigens of foreign tissue. These histocompatibility antigens are known to be associated with the plasma membrane (Bellanti, 1971a). The function of these antigens in the host is unknown. Recently it has been shown that virally and chemically induced tumors develop antigens which are different from those produced by the non-transformed cell (Bellanti, 1971b).

Except when the donor is an identical twin, transplantation of

organs involves the introduction of large amounts of tissue containing many new antigenic determinants, each capable of eliciting both a






6
humoral and cellular immunological response. A great deal is known about the consequences of histo-incompatibility but little is known of the molecular structure and composition of these antigens which are so intimately involved in the immunological response (Bellanti, 1971c). Thus, elucidation of the structure and composition of these antigens may contribute significantly to the ultimate goal of ensuring long-term survival of grafted tissue and perhaps provide a basis for developing methods for increasing the effectiveness of the immunological response to neoplastic tissue.

There arL significant technological problems which must be

surmounted if one is to study antigenic determinants. Consider the porcine lymphocyte; it has been found that the plasma membrane constitutes approximately 0.1 to 0.2% of the dry weight of the cell. A given antigen might then make up 5% of the membrane protein or approximately 10-13 gram per cell (Allan and Crumpton, 1971). One cannot assume that these figures are valid for other cells, but they clearly indicate that it will be extremely difficult to isolate such small quantities of protein, and consequently, extremely sensitive techniques must be used.

Antigenic determinants have been solubilized from both human and murine tissue by a variety of techniques (Fogerty, 1972). These techniques all have their own limitations and differ in their mode of action. Consequently, the solubilized antigenic determinants are in different physical forms. The only common feature is that the solubilized antigenic determinants retain their biological activity and thus can inhibit cytotoxicity of specific antisera. The solubilization techniques used to date are: detergent extraction, sonication,






7

proteolytic digestion, and simple salt extraction.

For example, consider the biochemical properties of human HL-A2 histocompatibility antigens solubilized from cultured human lymphocytes by papain cleavage (Cresswell et al., 1973) and by high salt extraction (Reisfeld and Kahan, 1970). Using the high salt extraction technique followed by purification by ultracentrifugation and preparative polyacrylamide gel electrophoresis, a single polypeptide was isolated. This peptide had a molecular weight of approximately 34,600 daltons, was found to be free of lipid, and contained less than 1% carbohydrate. Hence, the authors conclude that the antigenic specificity is due only to the primary amino acid structure of the antigenic determinants.

The papain solubilized histocompatibility antigen was purified by centrifugation, gel filtration, and ion exchange chromatography which yielded a single peak from the ion exchange column. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two bands with apparent molecular weights of 12,000 and 31,000 daltons were detected. Carbohydrate was detected by the periodate-Schiff reagent in the 31,000 molecular weight fragment. The molecular weight of the immunologically active glycoprotein was the sum of the two fragments or 43,000 daltons.

Thus, with respect to the HL-A2 determinants isolated from

cultured lymphoid cells by two different methods, one finds that the molecular weight of the determinant as well as its carbohydrate composition depends on the method of isolation. Consequently, the molecular structure of these histocompatibility antigens remains confused. This is primarily due to the lack of a rigorous character-






8

ization of the components of the plasma membrane from which the antigens are extracted and the lack of fundamental understanding of how the various agents employed act to free the antigen from the membrane. For example, there is some evidence that the antigens freed by high salt extraction actually result from autoproteolysis (Mann, 1972). Basically, all these extraction procedures are attempts at avoiding the necessity of isolating the plasma membrane from the cells bearing the antigenic determinant of interest.

The goal of this investigation was to develop techniques which

can be utilized in the characterization of antigenic determinants. In order to do this one must first select a cell line that is stable in tissue culture and capable of being grown in suspension culture to high density. The L5l78y murine lymphatic leukemia cell fulfilled this requirement. This cell line has been maintained in culture for approximately sixteen years and it ca'n be routinely grown to high cell density. In order to produce the greatest immunological response to the L5l78y cell, it was decided to cross species and immunize a goat with these cells. The goat is a convenient animal to utilize for the production of antisera, due to its size, relative ease of handling, and the amount of blood which can be drawn at any one time. Thus wie have an antigen source, the L5178Y cell, and the immunological system of the goat to define the antigenic components on the L5l78y cell surface.

The investigation was designed to proceed in the following manner:

1) Development of a method for isolating the plasma

membrane of the L5l78y cell.






9

2) Separation of the membrane proteins permitting their

identification.

3) Localization of specific proteins as belonging to the

exterior surface of the plasma membrane.

4) The use of immunological and biochemical techniques to

determine which polypeptides are components of the

antigenic determinants.

Hopefully, the techniques developed in this investigation will prove to be useful in later studies of tumor specific antigens or histocompatibility antigens and lead to methods capable of producing large quantities of these antigens. Human histocompatibility antigens isolated in sufficiently large quantities may lead to the development of useful reagents to detect early onset of transplant rejection crises, the production of narrow specificity antisera for tissue typing, and perhaps even to induce imm'unotolerance in transplant recipients. The isolation of human tumor specific antigens may lead to the development of reagents to screen high risk segments of the population for early detection of neoplastic tissue. Also, such reagents may be of use in evaluating the success of surgical removal of neoplastic tissue and monitoring the course of chemotherapy. The next decade of research will hopefully increase our awareness of the therapeutic uses of isolated antigenic material.














CHAPTER II

METHODS AND MATERIALS


Cell Culture Technique


The murine lymphatic leukemia cell line, L5l78y, kindly supplied

by Dr. John W. Cramer of the University of Nevada, was used exclusively in this study.-, This cell was originally described by Law arising as a spontaneous tumor in the DBA/2 mouse (Goldenberg and Thomas, 1967). The name of the cell line gives the type of cell, date and place of isolation; namely, a leukemia cell isolated on July 8, 1951 at Yale University. In 1958, it was adapted to tissue culture (Fischer, 1958). At this writing, the cell line has been in tissue culture for sixteen years.

The cells were cultured in Fischer's Medium for the Leukemic Cells of Mice. The medium was supplemented with 10% horse serum, 100 units! ml of penicillin, and 100 iig/ml of streptomycin (Watanabe and Okada, 1967). The cells were mass cultured in glass bottles containing approximately 200 ml of complete medium, at a temperature of 37 0 and in an atmosphere of 5% carbon dioxide. Cell counts were routinely done in a hemocytometer.

In order to determine the viability of the cell cultures, samples were diluted 1:1 with 1% trypan blue in normal saline. All cells which excluded dye were considered to be viable (Sa-chs et al., 1971).

The stock lines were maintained by serial dilution (1:4) through 10








eight successive tubes in duplicate. This greatly reduced the time and effort required to maintain the cultures. Each day during an eightday cycle one set of tubes in the series would reach a maximum number. Thus, each day it was possible to inoculate a mass culture if large numbers of cells were required.

Figure 1 shows a typical logarithmic growth curve of the L5l78y cell. At time zero, a flask containing fresh medium was inoculated such that the initial c-ell count was 104,000 celis/ml. Aliquots were taken at intervals and the cell count was determined. After a short

lag period, growth became relatively linear until the cell count reached approximately one million cells/ml. At this point growth slowed drastically due to exhaustion of the mediL.

From Figure 1, it is possible to calculate the doubling rate, which was approximately eleven hours for this experiment. -This cell line then was extremely well adapted to tissue culture techniques, could be grown to relatively high densities, and required the minimum amount of "black magic" for growth.

All cells harvested for subsequent experiments were taken at the million cell/ml level. No attempts were made to synchronize the cells.


Isolation of Plasma tlenibrarne

Smith Method

In general, it was found that one could conveniently isolate the plasma membrane from 4 6 x 108 cells (Smith and Crittenden, 1973). The cells were washed two times in phosphate buffered saline (PBS) (Dulbecco, 1954) or Puck's saline G (PSG) (Puck et al., 1958) to remove growth medium and horse serum. Five ml of a hypotonic solution










12














9.


7


S


4 3



2



N




















HRS


10 hoseseum
75

43




2.







B 10 15 2'0 2's 35 40 4s 50 s5s 60 6s 70 HRS.

FIGURE I. Growth curve of the L5178y cell in Fischer's Medium for the Leukemic Cells of Mice supplemented with 10% horse serum.






13
consisting of 0.01 M Tris buffer, pH 7.4 and 0.001 M MgCl 2 were added per 2 x 10 8 cells. -The cells were suspended and allowed to swell for fifteen minutes, and then disrupted with approximately eight strokes of the type B pestle Dounce homogenizer. The entire isolation procedure was monitored by phase contrast microscopiy.

The resulting cell debris was then diluted ith an equal volume of phosphate buffered saline, overlayered on a cush-ion of 45% (W/W) sucrose, and centrifuged at 125g for thirty minutes. The interface, containing plasma membrane fragments and nuclei, was diluted to a total volume of 15 ml with PBS. This suspension was divided into three parts and each part (5 ml) was overlayered on a discontinuous gradient composed of 60., 50, 45, 42.5, and 20% (W/W) sucrose. The gradients were centrifuged for eighteen hours at 23,000 rpm in a Beckman SW 25.1 rotor. All rupture and subsequent procedures were performed at 2"0- 50 C.

Membrane fragments were found to band halfway into the 42.5%

sucrose band. This band was removed from each of the three gradients, pooled, and diluted with 2 4 volumes of PBS. The membrane fragments were concentrated by centrifugation-in the SW 25.1 rotor for one hour at 25,000 rpm. Samples not taken' for microscopy, protein determinations, enzyme a ssays, or polyacrylamide gel electrophoresis were stored at -.20 0 C.

Fluorescein Mercuric Acetate Method

Cells (approximately 6 x 10 8) were washed twice with normal saline (0.16 M NaCl) and the pellet from the final wash was suspended in 1 ml of normal saline (Warren, 1969). The suspension was transfered to a small Dounce homogenizing tube and 3.0 ml of fluorescein mercuric








acetate (0.0022 M, pH .8.1) was added. After five minutes at room temperature, the tube was placed in ice until the suspension reached 30 C. The cells were broken with five to fifteen strokes of the type B pestle. The homogenization process was monitored by phase contrast microscopy.

Four ml of 60% (W/W) sucrose solution were added to the homogenate. The homogenate-sucrose mixture was overlayered on 10 ml of 45% (W/W) sucrose solution and centrifuged at 150g for one hour. The upper phase was removed to within 1 mm of the interface. After the interface was removed, about 0.1 volume of water was added and it was recentrifuged on a solution of 45% sucrose for one hour at 125g. The upper phase was removed and combined with the upper phase from the first centrifugation. The membrane fragments were contained in this solution. It was then diluted with 0.1 volume of water, overlayered on 35% (W/W) sucrose, and centrifuged at 1800g for one hour. The membranes pellet through the 35% sucrose. The pellet was suspended in 35% sucrose and overlayered on a linear gradient running from 45 to 65% (W/W) sucrose. The gradient was centrifuged for one hour at 33,000g. An orange band of membranes formed in the middle of the gradient. The band was removed, diluted with water, and pelleted. The pellet contained the purified membrane fragments.



Electron Microscopy

The plasma membrane isolation procedure was modified for those

*preparations which were to be examined in the electron microscope. It was felt that the final centrifugation at approximately 90,000g would damage the unfixed membranes as well as any unfixed contaminating






15

cell organelles (Shands, 1968). To avoid this possibility, the plasma membrane fragments were removed from the discontinuous gradients in the usual manner, diluted 1:1 with 2% Os04, and fixed for one hour. The fixed membrane fragments were then centrifuged at 90,O00g for one hour. The pellet containing the OsO4 fixed plasma membrane was resuspended in 2% bovine serum albumin to which a drop of 25% glutaraldehyde was added. The fixed plasma membrane fragments- bovine serum albumin-glutaraldehyde suspension was immediately centrifuged. The gel that resulted from the bovine serum albumin, crosslinked with glutaraldehyde, was then removed from the centrifuge tube, sliced, dehydrated, embedded in epoxy resin, sectioned, and stained with 0.5% uranyl acetate for thirty minutes at approximately 500 C. This procedure greatly reduced the chance of loss of plasma membrane fragments during sample preparation for electron microscopy. The electron micrographs were taken at a direct magnification of 16,000.


Polyacrylamide Gel Electrophoresis Procedure

Samples for electrophoresis were prepared by suspending the

plasma membrane fragments in 250 pl of 3% sodium dodecyl sulfate (SDS) in 0.1 M Tris buffer at pH 7.8. To the suspended fragments, 10 Il of 2-mercaptoethanol was added. The plasma membrane suspension was then transfered to a 1 ml Teflon homogenizer and homogenized with 8 10 strokes. The homogenate was then heated to 1000 C for five minutes (Triplett, 1972).

All disc gels were 5% acrylamide and 0.1% SDS in 0.1 M phosphate buffer, pH 7.2 (Lenard, 1970). The gels were approximately 10 cm long. Generally, electrophoresis was conducted for 3 1/2 hours at






16

eight milliamps per gel. Upon completion of electrophoresis, the gels were removed from the tubes and stained in Coomassie Brilliant Blue R (Fairbanks et al., 1971). The gels were destained in 7% acetic acid.

The SDS polyacrylamide gel system was calibrated using the
following proteins: cytochrome C, bovine serum albumin, human yG, and reduced human yG. All electrophoretic mobilities were expressed relative to cytochrome C. From Figure 2, one can see that in this system a linear relationship exists between the log of the molecular weight of the protein and its electrophoretic mobility, from 13,000 to 150,000 daltons.


Enzymatic iodination of Intact L5178y Cells
Generally, each experiment was done using approximately 6 x 108

cells. After washing the cells in PBS or PSG, the cells were suspended in 1 ml of PBS which contained 0.5 mg of lactoperoxidase and 0.5 mCu of 125I1 (Poduslo et al., 1972). The reaction was initiated by the addition of 5 il of 1.56 mM H202. Subsequent additions of 5 pl of

1.56 mM H202 were made at fifteen-second intervals until a total of 100 vl of H202 had been added. The reaction was carried out at 370 C.
The cells were then washed to remove unbound 125I1 and then the plasma membranes were isolated as previously described.


Liquid Scintillation Counting of 1251

All liquid scintillation counting of 1251 was done in modified Bray's solution (Rhodes, 1965). This solution contained four grams of PPO (diphenyloxazole), 0.2 grams of DM-POPOP (1,4-bis [4-methyl-5phenyl-2-oxazolyl] benzene), 60 grams of naphthalene, 333 ml of Triton









17




















20


*IG



10 9 8 7
BSA 06
X5 HC

4
0

3



2 LC




CYT C .1 .2 .3 4 .5 .6 .7 ,8 .9 1 RF







FIGURE 2. Calibration curve for the SDS polyacrylamide gel system. All mobilities were expressed relative to cytochrome C. The proteins used were bovine serum albumin, heavy and light chains from reduced human yG, intact human yG, and cytochrome C.






18

X-100, and p-dioxane to make the total volume one liter.

To determine the location of labeled proteins in polyacrylamide gels, the gels were frozen and then cut into 87 slices. Each slice was then placed in a liquid scintillation vial and 10 ml of the modified Bray's solution were added.
The samples were counted in a Packard Tricarb Liquid Scintillation Spectrometer. The percent counting error depended on the actual number of decompositions per minute and ranged generally from 1 to 4%.


Trypsinization Procedure
The trypsinization procedure used had been originally used to

study the red blood cell plasma membrane (Triplett and Carraway, 1972). It was adapted for use with the L5178y cell. Generally, 4 6 x 108 cells were used per experiment. The cells were washed in PSG and then resuspended in 10 ml of PSG containing 100 gm/ml or 500 jigm/ml of trypsin; 0.1 M NaOH was added as necessary to maintain pH at 7.2. The cells were exposed to trypsin for one-half hour at room temperature. The reaction was stopped by the addition of a two-fold excess of soybean trypsin inhibitor. The cells were then washed twice in PSG and the plasma membranes were isolated as previously described.


Immunization Procedure
Antibodies to the L5178y cell were made in goats (Williams and

Chase, 1967a). The goat was immunized every day for five days and rested two days the first week. The second week, the goat was immunized every other day for five days and rested two days. One further injection was given and the animal was exsanguinated two days later. Each






19

immunization consisted of 2 x 10 8 cells injected into the jugular vein of the goat. Over the course of the immunization period, blood samples were taken and the antibody titer was determined by measuring the cytotoxicity of the goat to the L5178y cell (Sachs et al., 1971). Essentially, equal volumes of target cells, guinea pig complement, and dilutions of serum were mixed and incubated at 37 0 C for thirty minutes. At the end of the incubation, the samples were centrifuged and the supernatant was discarded. To each sample, trypan blue in normal saline was added. If antibody was present, then cells were killed and hence permeable to trypan blue. The antibody titer was then defined as the highest dilution of serum which produced 10% more dead cells than found in the control. The control contained only cells and complement. Antibody titer is shown in Figure 3.


Cytotoxicity Inhibition Assay

One may use this assay to detect Fab fragments. To do this, Fab fragments and cells were incubated with a solution containing the fragments for thirty minute's at 370 C. Then antibody, from which the fragments were prepared, along with complement, was added and incubated at 37 0 C for an additional thirty minutes. At the end of this period, the cells were removed from the reaction mixture by centrifugation. The cell pellet was resuspended in trypan blue in normal saline and the number of dead cells was determined and expressed as a percentage of the total. If the Fab fragments are present and bound to the surface of the cell, they may block the binding of complement fixing antibody. The results can be expressed as a reduction of the percentage of cell death ove.r.the control with no Fab fragments present








20





















1:5121:'. 2561:1281: 64cr
LU
1:321:16 1:8

1:4

1:2

1


4 8 10 14 18
DAY




FIGURE 3. Cytotoxic antibody titer in goat serum.






21

(Sachs et al., 1971).


Immunoglobulin Purification Procedure

Immunoglobulins of yM and yG classes were partially purified (Williams and Chase, 1967b). The immunoglobulins were precipitated from goat serum by the addition of saturated ammonium sulfate resulting in a final concentration of 33% saturation. The precipitate was .centrifuged, the supernatant was discarded, and the pellet was redissolved in glass distilled water. The precipitation was repeated five times. After the final precipitation, the precipitate was dissolved in borate buffered saline, pH 8.2, and applied to a 5 cm x 50 cm G-200 Sephadex column which was equilibrated in the'same buffer (Williams and Chase, 1967c).

The elution profile is shown in Figure 4. Peaks 1 and 2 were

eluted at fractions 43 and 60 respectively. Peak 1 was eluted in the region of the void volume of the column indicating that it was composed of protein of molecular weights greater than 200,000 daltons. Peak 2 was chromatographed by the column and it eluted in a region which corresponds to a molecular weight of 150,000 daltons. The peaks from the G-200 column were pooled individually, dialized, and lyophilized. Samples of the lyophilized proteins from each of the peaks were solubilized in 3% SDS, with and without 2-mercaptoethanol, and SDS gel electrophoresis. The gels are shown in Figure 5. The gels show that peak 1 is composed of at least four components which are sensitive to 2-mercaptoethanol. This indicates that three of the components are probably immunoglobulins of the yM class. The remaining band was most likely immunoglobulin of the yG class which failed.to resolve on the







22








1.4-~



1.2 1.0



.8
0



.6



A



.21




20 40 60 80 100
FRACTION NO. FIGURE 4. Elution pattern of immunoglobulins isolated from goat serum on a 5 x 50 cm G-200 Sephadex column in 0.17 M sodium borate in normal saline at pH 8.2. Each fraction was approximately 10 ml. Peaks 1 and 2 were eluted at fraction 43 and 60 respectively.






23




















I








1 2 3 4 5 6 7 8

FIGURE 5. SDS polyacrylamide gel electrophoresis of the following proteins: 1, cytochrome C; 2, bovine serum albumin; 3, reduced human yG; 4, human yG; 5, peak 1 proteins from Figure 4; 6, peak 1 proteins from Figure

4 reduced; 7, peak 2 proteins from Figure 4; and 8, peak 2 proteins from Figure 4 reduced.






24

G-200 column. Peak 2 was found to be composed of primarily one band that was sensitive to 2-mercaptoethanol and had a molecular weight of approximately 150,000. This demonstrated that peak 2 was composed primarily of immunoglobulins of the yG class.


Preparation of Fab Fragments

Purified goat yG was subjected to proteolysis using mercuripapain, in 0.1 M phosphate buffer, pH 7.0, 0.01 M cysteine, and 0.002 M EDTA (Williams and Chase, 1967d). The reaction was carried out for sixteen hours at 370 C. The proteolysis was stopped by dialysis against glass distilled water at 40 C. The entire reaction mixture was then subjected to gel filtration of a 5 cm x 50 cm G-200 Sephadex column equilibrated in borate buffered saline at pH 8.2. The elution profile of the reaction mixture is shown in Figure 6. This figure shows three peaks. This first peak is unreacted yG, the second peak contains the Fab and Fc fragments and the third peak is most likely small peptides since it elutes in the included volume of the column. Since the Fab and Fc fragments are about 47,000 and 44,000 daltons respectively, this column cannot resolve these two fragments. The presence of the Fc fragments will not interfere with later experiments.


Enzyme Assays
Three enzyme determinations were done: alkaline phosphatase (Bergmeyer, 1963a), glucose-6-phosphatase (Bergmeyer, 1963b), and succinic dehydrogenase (Earl and Korner, 1965). The substrate utilized in the alkaline phosphatase reaction was p-nitrophenylphosphate. In this reaction, the production of p-nitrophenol was followed by









25




















.8

.7

.6

CD
c .544
.4

.3

.2

.1


20 40 60 80 100 120 140
FRACTION NO.



FIGURE 6. Elution pattern of the mercuripapain digest on a

5 x 50 cm G-200 Sephadex column.






26

measuring the increase in absorbance at 412 nm. Glucose-6-phosphatase was measured by following the release of inorganic phosphate from the substrate glucose-6-phosphate (Bartlett, 1959). The succinic dehydrogenase reaction was followed by measuring the decrease in absorbance of 2,6-dichlorophenolindolephenol at 600 nm.


Protein Determinations

Protein was determined by the procedure of Lowry, using bovine serum albumin as a standard (Bailey, 1962).



Materials

All tissue culture media and horse serum were purchased from GIBCO, Grand Island, N.Y. Antibiotics used in tissue culture were purchased from Eli Lilly, Indianapolis, Indiana.

The Dounce homogenizer with the type B pestle was purchased from Canalco, Rockville, Maryland. Sodium dodecyl sulfate was purchased from Sigma Scientific, St. Louis, Missouri.

Trypsin used in proteolysis of the L5178y cell was purchased from Worthington Biochemical Corp., Freehold, New Jersey.

All liquid scintillation reagents were purchased from Sigma

Scientific except the naphthalene and p-dioxane, which were purchased from Matheson, Coleman and Bell, Norwood, Ohio.

Guinea pig complement was purchased from GIBCO.

Sephadex G-200 was purchased from Pharmacia Fine Chemicals, Piscataway, New Jersey.

Enzyme substrates were purchased from Sigma Scientific.

All proteins used were purchased from Sigma Scientific.







27

All common chemicals were reagent grade and used without further purification.












CHAPTER III
RESULTS AND DISCUSSION

Plasma Membrane Isolation Procedure

Two methods of isolating the plasma membrane of the L5178y leukemia cell were evaluated. The fluorescein mercuric acetate (FMA) method of Warren was developed for the L cell (murine fibroblasts) and from that cell it is possible to isolate intact ghosts (Warren et al., 1966). It has been utilized in the isolation of plasma membrane from a number of other cell lines. The other method was developed by Smith and coworkers as modified by Noonan (Noonan, 1974).

The Warren method uses FMA to fix plasma membrane. The FMA binds covalently to sulfhydral groups thus having the advantage of inhibiting proteolytic enzymes which require a free sulfhydral group for activity. The major disadvantage is that it is impossible to assay for marker enzymes after this fixation. If the Warren method is to work, it is imperative to produce intact ghosts or extremely large fragments which will sediment rapidly during the series of low speed centrifugations which follow rupture. If the plasma membrane ruptures into very small fragments, these fragments are lost in the purification procedure. Unfortunately the L5178y cell has an extremely large nucleus making it difficult to remove the nucleus and leave the plasma membrane intact or in large fragments. Thus every attempt to use this method failed due to loss of sample during preparation.

The method of Smith and Crittenden greatly reduces the number of

low speed centrifugations, relying rather on a discontinuous gradient to
28






29

separate the plasma membrane from other cell organelles. The procedure used is shown schematically in Figure 7. The method produced consistent results between experiments.

Figures 8 17 are all phase contrast micrographs of the various stages of the membrane isolation procedure. Figure 8 shows the intact cells as seen after washing in PSG. At this stage, the cells are round and the nucleus can be seen to occupy a significant portion of

the cell 's volume.

Figures 9, 10, and 11 show the cells after suspension in the hypotonic swel fing solution. The photographs were taken after approximately three, eight, and fifteen minutes in the hypotonic solution respectively. One can readily see the appearance of large vacuoles between the cytoplasm and the nucleus. If treatment continues beyond fifteen minutes the cells rupture.

At this point the cells are ready for homogenization. Figure 12 shows the homogenate after eight strokes with the Dounce homogenizer. In every preparation greater than 99% of the cells were ruptured. The tonicity of the homogenate was then raised by-the addition of an equal volume of PBS.

Figure 13 shows the supernatant fraction. One can see that only particulate material remains suspended in this fraction.

Figure 14 shows the interface. Here one can see nuclei and fragments which may be plasma membrane.

Figure 15 is a photograph of the discontinuous density gradient after centrifugation. This photograph shows the band of membrane fragments halfway into the 42.5% sucrose solution. This material was removed and diluted with PBS.






30

Washed Cells


JSuspend in Hypotonic Solution Figure 9 -after 3 minutes

Figure 10 -after 8 minutes

Figure 11 -after 15 minutes


SHomogenize Figure 12


Overlayer on 45% Sucrose and Centrifuge Supernatant
Discard 4 Figure 13 -kInterface

Figure 14


Overlayer on Discontinuous Sucrose Gradient and Cen~tri fuge


Plasma Membrane

Fragments

Figure 16 Sam pl1e

SDilute and 20%
Centrifuge 4 2 5%0

45%
Plasma Membrane Pellet 50%
Figure 17 60%

Figure 15

FIGURE 7. Diagram of plasma membrane isolation procedure.






31






















FIGURE 8. Phase contrast micrograph of cells washed in PSG. Magnification 320X.






9














FIGURE 9. Phase contrast micrograph of cells after three minutes in 0.01 M Tris 0.001 M MgCl2 at pH 7.4. magnification 320X.






32






















FIGURE 10. Phase contrast micrographs of cells after eight minutes in

0.01 M Tris, 0.001 M MgC12 at pH 7.4. Magnification 320X.























FIGURE 11. Phase contrast micrographs of cells after fifteen minutes in 0.01 M Tris, 0.001 M MgCl2 at pH 7.4. Magnification 320X.







33





















FIGURE 12. Phase contrast micrograph of homogenate after eight strokes of the Dounce type B pestle. Magnification 320X.























FIGURE 13. Phase contrast micrograph of the supernatant from the 125g centrifugation. Magnification 320X.






34
........ - ---- ---- ----- ----FIGURE 14. Phase contrast micrograph of the interface resulting from the 125g centrifugation. Magnification 320X.











41-- Pl a sma
Membrane
Fragments





FIGURE 15. Photograph of discontinuous sucrose gradient showing plasma membrane fragments halfway into the 42.5% sucrose layer.






35

Figure 16 is a micrograph of a sample from the band in the 42.5% sucrose. In the photograph it is difficult to see the detail of fragments. Generally, the fragments appeared to be covered with dense granules.

The micrograph of the pelleted membranes is shown in Figure 17. Here one can see a greater concentration of the fragments seen in Figure 15.



Characterization of the Isolated Plasma Membrane Fragments

Perhaps the most difficult task of the investigator is the demonstration that the isolated material from any biological system is indeed what he thinks it is. One may characterize any subcellular organelle by its morphology, chemical composition, enzymatic composition, and by the use of selective modification reagents. However, none of these techniques are sufficiently definitive that they can stand alone. Taken collectively, a circumstantial case can be made for the identity of the isolated subcellular organelle. Unfortunately, this is also the state of the art with respect to the characterization of plasma membrane fractions.

In order to evaluate the isolated subcellular organelle morphologically, it must have some characteristic feature which can be identified in the phase contrast or electron microscope. Furthermore, this morphology must be preserved in the isolation procedure employed. A number of workers (Boone et al., 1969; Barber and Jamieson, 1970) have found that the plasma mem brane of cells vesiculates upon disruption. Unfortunately, a number of other cell organelles such as lysosomes, smooth endoplasmic reticulum, Golgi apparatus, and peroxi-







36





















FIGURE 16. Plasma membrane fragments as seen in the 42.5% sucrose. Magnification 320X.























FIGURE 17. 90,000g pellet of plasma membrane fragments from the diluted 42.5% sucrose layer. Magnification 320X.






37

somes are smooth vesicles in a homogenate and they look much like plasma membrane vesicles. This makes the morphological identification of plasma membrane vesicles, as opposed to the above mentioned vesicles, very difficult.

Fortunately, a number of cellular organelles, such as the nucleus, mitochondria, and rough endoplasmic reticulum, do have characteristic morphological traits which may be used for identification purposes assuming that they survive the isolation procedure intact. The electron microscope can play a useful role in the evaluation of the degree of contamination with these organelles in a preparation of plasma membrane.

Also, it should be noted that the relative amounts of various

subcellular organelles varies significantly from one cell to another. For example, the problems of contamination of rough endoplasmic reticulum in a plasma membrane preparation would be significantly greater if the membranes were isolated from a cell which is actively secreting protein such as a plasmacytoma cell. Hence by judiciously choosing the cell line for investigation, one can minimize some of the problems involved in isolating plasma membrane.

A second useful technique in the circumstantial identification of a subcellular organelle is the use of marker enzymes known to be associated with the organelle being isolated. Marker enzymes are useful in assessing the degree of contamination of other organelles in the desired preparation. The basic requirement for a good marker enzyme is that it must be present exclusively on and be tightly bound to the organelle.

The development of histochemical techniques suitable for use in






38

conjunction with electron microscopy has resulted in the ultrastructural location of approximately twenty enzymes (DePierre and Karnovskey, 1973). Among the enzymes shown to be plasma membrane markers is alkaline phosphatase. This enzyme has been localized in the plasma membrane of a variety of cells, such as the brush border cells of the small intestine (Nachlas et al., 1960), the proximal convoluted tubule cell of the kidney (de The', 1968), and liver cells (Wilfong and Neville, 1970).

Alkaline phosphatase has the advantage over other plasma membrane marker enzymes in that it can be assayed spectrophotometrically. This greatly simplifies the assay. The enzyme was assayed using the artifical substrate p-nitrophenylphosphate. The reaction results in the release of phosphate from this compound and the concomitant production of p-nitrophenol which absorbs light at 420 nm. The reaction was linear over the period followed. The activity was calculated directly from the change in absorbance as a function of time and the specific activity was the activity divided by the protein concentration. The data are summarized in Table I on the following page.

As can be seen in Table I, a 13.5-fold purification of alkaline phosphatase was obtained in the final membrane pellet. This suggests that plasma membrane fragments were isolated by this procedure.

The enzyme glucose-6-phosphatase has been localized in the rough endoplasmic reticulum by electron cytochemistry (Pollak and Shorey, 1968; Rosen et al., 1966; Ericsson and Orrenius, 1966; Tice and Barrnett,1962). Consequently, this enzyme is an accepted marker enzyme for rough endoplasmic reticulum contamination in plasma membrane preparation. This enzyme is slightly more difficult to assay since it is a






39

non-spectrophotometric assay. Essentially the substrate glucose-6phosphate is incubated with aliquots taken at various stages in the purification procedure. The reaction is followed by measuring the release of inorganic phosphate from the substrate. The enzyme activity was defined as the vgm of phosphorus released per fifteen-minute incubation. The specific activity was then the activity divided by the protein concentration in the reaction mixture. *The results are shown in Table I.



TABLE I

Alkaline a Glucose b Succinic
Phosphatase 6-Phosphatase Dehydrogenasec

Homogenate 1.7 (1.0)d 0.9 (1.0) 1.0 (1.0)

Supernatant 0.4 (0.2) 1.2 (1.41) 9.1 (9.1)

Interface 1.5 (1.0) 1.4 (1.66) 2.2 (2.2)

Membrane Pellet 20.0 (13.5) BLLAe 0.8 (0.8)

a Specific activity F A absorbance/min/mg protein. b Specific activity = pgm P/15 min/mg protein. c Specific activity E A absorbance/hr/mg protein.
d
( ) indicate Purification factor.
e BLLA = Below the Lower Limits of the Assay. Less than 16% of the
sample.



From Table I, it can be seen that the enzyme glucose-6-phosphatase can be detected in the homogenate, supernatant, and interface. However, no activity could be detected in the plasma membrane pellet. This may be due to lack of sufficient protein in the enzyme assay mixture. It






40

was not possible to increase the yield of the procedure to obtain a greater amount of protein for this assay.

In order to estimate the minimum possible endoplasmic reticulum contamination in the isolated plasma membrane preparation, one has to make the tenuous assumption that the activity of endoplasmic reticulum glucose-6-phosphatase from rat liver approximates the activity of the enzyme ih 1L178y endoplasmic reticulum, which may or may not be the case. This gross assumption was made because this enzyme has not been purified from the L5178y cell or, for that matter, from any other cell (Maddaiah et al., 1971).

The specific activity of glucose-6-phosphatase from rat liver

endoplasmic reticulum is 44 pgm P/15 min/mg of protein (Birne, 1972). Now, if the minimum detectable level of phosphorus is 0.5 vgm, one can calculate that approximately 11 pgm of endoplasmic reticulum could be detected using this assay system. In the glucose-6-phosphate assay mixture done on the plasma membrane fraction, a maximum of 70 Igms of protein was used. Since we were unable to detect any glucose-6phosphatase activity in the isolated plasma membrane fragments, the total endoplasmic reticulum contamination must be less than 16% (11 ugm/70 igm x 100).

In order to assess the degree of mitochondrial contamination in the plasma membrane preparation, the mitochondrial marker enzyme, succinic dehydrogenase, was measured. This enzyme has been localized to the mitochondrion by biochemical techniques (Bachmann et al., 1966). It can be assayed spectrophotometrically using 2,6-dichlorophenolindolephenol as the electron acceptor. One measures the decrease in absorbance at 600 nm. Again, the specific activity is defined as






41

the change in absorbance per unit time per mg protein. The results are presented in Table I.

Table I shows that the supernatant contains the greatest succinic dehydrogenase activity (approximately 90% of the total recovered activity). The interface contains succinic dehydrogenase activity (approximately 9.8% of the total recovered activity), as does the isolated plasma membrane pellet (approximately 0.1% of the total recovered activity). This indicates a limited degree of mitochondrial contamination in the isolated plasma membrane fragments.

The alkaline phosphatase data indicated that plasma membrane fragments were purified by the isolation procedure employed. The succinic dehydrogenase data indicated that the degree of mitochondrial contamination in the isolated plasma membrane fragments was low. From the data it appeared that the greatest succinic dehydrogenase activity was found to be in the supernatant of the 125g centrifugation. This suggested that this low speed centrifugation left the majority of the mitochondria in suspension.

The glucose-6-phosphatase assay showed that this enzyme was

present in the homogenate, the 125g supernatant, and the interface. However, no activity was detected in the isolated plasma membrane fragments. Due to the limited sensitivity of this assay, it is only possible to state that the isolated plasma membrane fragments contained less than 16% endoplasmic reticulum. Thus, we must rely on electron microscopy to demonstrate the presence or absence of endoplasmic reticulum.
A series of sixteen electron micrographs were taken of the uranyl stained sections. Twelve of these micrographs were taken at a direct






42

magnification of 16,000 and the remaining four were taken at a direct magnification of 48,000. Three of the micrographs taken at a direct magnification of 16,000 are shown in Figures 18, 19, and 20. As expected, the isolated material was vesicular in nature. Many of these vesicles had sharp, well defined membranes, characteristic of the plasma membrane.

Also seen in the micrographs were round vesicles studded with projections approximately 170 in diameter. These projections were most likely ribosomes indicating possible endoplasmic reticulum contamination.

There were other vesicles seen, highly suggestive of mitochondria. Some of these vesicles appeared to contain double membranes and cristae-like invaginations. Upon measurement, the average diameter of these vesicles was 0.80 microns. This diameter is somewhat small for a mitochondrion; however, the isolation procedure could significantly alter the structure of the mitochondrion.

The electron micrographic evidence coupled with the enzyme assays indicate that plasma membrane fragments were isolated. Also, there is likely contamination of the plasma membrane preparation with some endoplasmic reticulum. The succinic dehydrogenase assay indicated a low level of mitochondrial contamination; however, the electron micrographs showed occasional vesicles highly suggestive of mitochondria. Further speculation on the degree of contamination was not possible with the present data.







43


































FIGURE 18. Electron micrograph of OsO4 fixed and uranyl acetate stained plasma membrane preparation. Direct magnification 28,OOOX.







44



































FIGURE 19. Electron micrograph of OsO4 fixed and uranyl acetate stained plasma membrane preparation. Direct magnification 28,OOOX.







45























4m










FIGURE 20. Electron micrograph of OsO4 fixed and uranyl acetate stained plasma membrane preparation. Direct magnification 28,OOOX.






46

Preliminary Characterization of the Polypeptide Components of the Plasma Membrane

Since the basic function of the plasma membrane is to define the environment of the cell, it must be made in such a manner as to render it insoluble in an aqueous environment. The inherent insolubility of plasma membrane polypeptides was the primary stumbling block to their characterization. Many different techniques were utilized to solubilize isolated plasma membranes; however, most of these techniques were only partially successful. An additional requirement for adequate characterization was the development of an extremely sensitive analytical technique capable of separating complex mixtures of proteins.

In 1964, the polyacrylamide gel electrophoresis technique was

developed (Orenstein, 1964). This technique was extremely sensitive, requiring only microgram quantities of protein. In 1967, it was shown that polyacrylamide gel electrophoresis done in the presence of the detergent sodium dodecyl sulfate separated proteins by their molecular weights (Shapiro et al., 1967; Shapiro and Maizel, 1969). Since most membranes can be solubilized in SDS, this technique provided a powerful new tool for the preliminary characterization of all the proteins in the membrane.

Figure 21 shows the protein components found in the L5178y plasma membrane. One can see that the plasma membrane contains a complex array of proteins.

The polypeptide components were numbered arbitrarily in decreasing order of molecular weight. Only the major, reproducible polypeptides were numbered. Table II gives the molecular weight of each of the polypeptide components.







47







.8

67 A
.7o 7

59 S4 8
3

.2










1 2 3 4 5 6 7 8 9 10
-2





CM.



I -B



FIGURE 21. A Gel scan of Coomassie blue staining pattern of solubilized

plasma membrane fragments.

B Photograph of SDS polyacrylamide gel.






48



TABLE II

Polypeptide Molecular Weight

1 180,000
2 153,000
3 115,000
4 100,000
5 70,000
6 56,000
7 47,500
8 26,500
9 24,000


As noted in the Methods chapter, the proteins were stained with Coomassie Brilliant Blue R. Several attempts were made to use the periodate-Schiff technique to stain for carbohydrate (Fairbanks et al., 1971). All attempts to detect carbohydrate with this technique were unsuccessful.


lodination of the L5178y Cell

Proteins labeled with radioactive iodine have been very useful

in biochemical research. Proteins of high specific activity have been produced using the chloramine-T method of iodination (Hunter and Greenwood, 1962). Chloramine-T is a powerful oxidizing agent and, unfortunately, some degree of protein denaturation always occurs. However, this method has been used successfully for producing radiolabeled antibodies for use in determinations such as growth hormone and insulin in plasma.

In 1969, a new method for the iodination of proteins was published (Marchalonis, 1969). The method used catalytic amounts of the enzyme lactoperoxidase to iodinate immunoglobulins without denaturation.






49

Since this technique was extremely gentle it was soon recognized that it could be used to label proteins-in plasma membranes of cells without killing the cell (Marchalonis, 1971). Electron micrographic radioautography has shown that the radioactive label was localized exclusively on the surface of the cell Apparently, the large size of the enzyme prevents it from penetrating the membrane and also it is not phagocytized. Thus, this technique, which has been-extremely useful in ordering the protein components in the plasma membrane of the cell, was employed to probe the L5l78y cell surface.

The results of three iodination experiments are shown in Figures 22, 23, and 24. Each experiment was done as described in the Methods chapter. In each figure there is a gel scan of the Coomassie blue staining pattern and the radioactivity profile of the gel. One should note that these experiments were done at different times, consequently the degree of label incorporation varied from experiment to experiment even though every attempt was made to duplicate the labeling conditions. In all experiments, polypeptide 1 showed the highest degree of isotope incorporation. There were also four other regions in the gel which showed the presence of isotope. In these regions, polypeptides 3, 4, 5, 6, and 7 were localized. Very little isotope was detected in the region of polypeptides 8 and 9.

The control for these experiments was the standard labeling mixture, I2 cells, and hydrogen peroxide as described in the Methods chapter, but no lactoperoxidase. Under these conditions no radioactivity above background was detected in an aliquot of the isolated plasma membrane preparation. This preparation was electrophoresed and Figure 25 shows the gel scan of the Coomassie blue










50



.9



7


A
6i














2i









1 3 4 5 6 7 ~ 9 10
CM.











B


600 500 400

300-1




200 100



1 2 3 5 6 9 410
CM,




FIGURE 22. First 125I1 labeling experiment. A Coomassie blue staining pattern of 125I labeled plasma membrane



polypeptides.


B Radioactivity profile of 125I labeled plasma membrane polypeptides.









51





.9



A




6











1+-I
4i
















2 3 4 5 6 7 8 98 10 CM.





180

i B
140!



120 1001






60


40i



20



I 2 1 4 6 8 8 Io
CM,


FIGURE 23. Second 125I labeling experiment. A Coomassie blue staining pattern of 125I1 labeled plasma membrane


polypeptides.


B Radioactivity profile of 125I labeled plasma membrane polypeptides.










52






7



A







,4-'
11




3-1




2!








CM. 280



240 B



200



160 120 8o 40



1 2 3 4 .5 8 7 8 9 10 CM.




FIGURE 24. Third 125I labeling experiment. A Coomassie blue staining pattern of 125I1 labeled plasma membrane


polypeptides.


B Radioactivity profile of 125I labeled plasma membrane polypeptides.











53















.9




.8




.7
.6













.4




.3



.2











CM. FIGURE 25. Coomassie blue staining pattern of lactoperoxidase control experiment.






54

staining pattern. Since no radioactivity was detected in the preparation, the gel was not sliced and counted. Thus the label incorporated into these polypeptides was covalently bound and totally dependent on the presence of lactoperoxidase.

One concludes from this series of experiments that those polypeptides which become labeled must be on the surface of the cell. As noted before, polypeptide 1 was more highly labeled than any other polypeptide component. This high degree of isotope incorporation into the isolated plasma membrane further demonstrates the validity of the isolation method.

The percent recovery of isotope in the gel was determined for

each labeling experiment. For the three experiments done, the percent recovery in the gel averaged 10%. The remainder of the applied isotope could be recovered in the lower buffer of the electrophoresis apparatus. This isotope was thought to be incorporated into the lipid components of the plasma membrane.



Proteolytic Digestion Studies on the L5178y Cell

At this point the polypeptide components of the plasma membrane of the L5178y cell had been characterized with respect to their molecular weight and one polypeptide component had been shown to be localized on the exterior of the plasma membrane by the lactoperoxidase iodination technique. In order to further demonstrate the validity of this localization, additional experiments were conducted.

The use of proteolytic enzymes to probe the cell surface of

erythrocytes has yielded important information about the localization






55
of membrane proteins (Triplett and Carraway, 1972). It was decided to probe the surface of the L5178y cell with trypsin after it had been enzymatically labeled with 125I. This would make it possible to evaluate the disappearance of a protein band not only by a loss of stain in a specific region of the polyacrylamide gel but also by a decrease in radioactivity.

The L5178y cell was found to be more sensitive to trypsin than the erythrocyte. However, it was found that the cell would withstand trypsin up to 500 ligm/ml for thirty minutes. Phase contrast microscopy after suc-, treatment showed that greater than 90% of these cells were intact. In order to show that the cells were not significantly damaged by trypsinization, a control trypsinization experiment was done as described in the Methods chapter, but under sterile conditions. An aliquot of the trypsinized cells was washed one time and inoculated into growth medium. Figure 26 shows the growth curve of these cells. From the growth curve one can calculate a doubling time of about twelve hours and a maximum cell density of about 1.2 x 106 cells/ml. From Figure 26, it appears that the trypsinization procedure did not adversely affect the viability of the cells.

In Figures 27 and 28 one can see the results of two trypsinization experiments. Again in this figure a gel scan and the radioactivity profile are shown. From the gel scan one can see that at trypsin concentrations of 100 pgm/ml and 500 igm/ml polypeptide 1 was almost completely removed. However, the isotope profile shows that a peak of radioactivity still is found in this region although it is significantly lower than in any of the previous labeling experiments. With respect to the other polypeptide components, the Coomassie blue scan is








56













10*
9 8

7

6 0 5
x

-J
-J
w
0 3




2








1
5 10 15 20 25 30 35 40 45
HRS


FIGURE 26. Growth curve of L5178y cells after treatment with 500 ugm/ml of Trypsin for thirty minutes at pH 7.2.










57










A











.4



.3





.2






1 2 3 4 5 6 7 8 9 10 CM.







8




B









4
















2 3 4 5 6 7 8 9 10 CM.




FIGURE 27. Results of treatment of 125I labeled L5178y cells with Trypsin at a concentration of 100lgm/ml. A Coomassie blue staining pattern. B Radioactivity profile.










58







,9




.8- A











.5







.4



























.20 10
CM











B




100
90

S


70

60

SO.
40-'


20


101



CM




FIGURE 28. Results of treatment of 1251 labeled L5178y cells with Trypsin at a concentration of 500 i gm/ml. A Coomassie blue staining pattern. B Radioactivity profile.







59
unchanged. From these experiments it is readily apparent that polypeptide 1 is exposed to the action of both trypsin and lactoperoxidase. Hence it would appear that polypeptide 1 must be exposed on the surface of the cell.



Immunoglobulin Protection Experiment

Presumably, if one incubated the L5178y cells with antibody to

these cells, the antibody would bind specifically to membrane antigens. By washing the cells to remove the unabsorbed antibody, and enzymatically labeling the surface with 125I, then it should be possible to see a reduction in incorporation of label into one or more of the proteins separated on SDS.polyacrylamide gel electrophoresis. This would identify the polypeptide components on the cell surface which the immunological system of the goat recognized as an antigenic determinant. The experiment is shown diagramatically in Figure 29.

Before attempting the immunoglobulin protection experiment, the

agglutination properties of yG immunoglobulin were invest??ied. This was done because it was feared that the cells might be agglutinated and large areas would be shielded from the action of lactoperoxidase due to agglutination and not antibody binding. The yG immunoglobulin was found to be an extremely poor agglutinin. The immunoglobulin protection experiment was done as outlined above using 25 mg yG immunoglobulin per ml of PSG. The results are shown in Figures 30 and 31.

From the data one can see a 2.3-fold reduction in isotope incorporation in polypeptide 1 in the antibody coated cells over the control






60

Washed Cells
1.2 x 109 Cells
I I


6 x 108 Cells 6 x 108 Cells

Suspend in PSG
Suspend in PSG containing either
Suspnd i PSGFab fragments or intact yG for 30
minutes

Control Cells Coated Cells


Wash twice in {-SG Wash twice in PSG

Control Cells Coated Cells


Label with Label with 1251


Labeled Control Cells Labeled Coated Cells

Isolate plasma Isolate plasma
membrane membrane


Membrane Fragments Membrane Fragments



SDS Gel SDS Gel
Electrophoresis Electrophoresis






.Radioactivity Gel Scan Gel Scan Radioactivity
Profile Profile



FIGURE 29. The immunoglobulin protection experiment.






























,7 700



.6 600



.5 500



4 2 4003 3001



.2 2001001
,i



1 2 3 4 6 7 110 ~i
CM CM


A B




FIGURE 30. Results of the yG immunoglobulin protection experiment: Control experiment with noyG present. A Coomassie blue staining pattern of plasma membrane polypeptides. B Incorporation of 125I in plasma membrane polypeptide.






























700 600






500 .7 400



300



200




A



L 2 3 4 + 8, 0

CM


,2


A B







C M



FIGURE 31. Results of the yG immunoglobulin protection experiment: yG concentration 2.5 mg/ml.



A Coomassie blue staining pattern of plasma membrane polypeptides.



B Incorporation of 1251 in plasma membrane polypeptides.






63

Also, two new peaks appeared in the radioactivity profile of the antibody coated cells in'the region of the polyacrylamide gel which corresponded to molecular weights of 55,000 and 23,000 daltons. This is the molecular weight range of heavy and light chains resulting from the reduction of yG immunoglobulin by 2-mercaptoethanol.

This suggests that polypeptide 1 is one, or a component of one, of the antigenic determinants on the surface of the L5178y cell which the goat's immunological system recognized. This was based on the reduction of isotope incorporated into polypeptide 1 and the appearance of two nde labeled polypeptides in the region of immunoglobulin heavy and light chains. The reduced immunoglobulin chains could only have become labeled if the antibody had been bound to the cell surface during the labeling procedure.

In order to ensure that the reduction in isotope incorporation in polypeptide 1 was due to antibody and not agglutination, the experiment was repeated with Fab fragments derived from the same immunoglobulin source used in the previous experiment.

For a number of years it has been general knowledge that papain cleaves immunoglobulins of the yG class into two Fab fragments containing one binding site each, and one Fc fragment containing the complement binding site. Thus, the Fab fragment retains its antigenic binding capabilities but since it has been rendered monovalent it is no longer able to agglutinate cells. Fab fragments were then made as described in the Methods chapter.

Next it was necessary to show that Fab fragments would bind to the cell. It was thought that these fragments would inhibit the cytotoxicity of the intact immunoglobulins. After several attempts,






64

no inhibition of cytotoxicity could be demonstrated in the presence of the Fab fragment. SDS polyacrylamide gel electrophoresis showed that the prepared fragments were of the proper size, that they were sensitive to reduction with 2-mercaptoethanol and that they were not contaminated by intact yG immunoglobulins. A second attempt to demonstrate binding was made using Fab fragments which had been enzymatically iodinated (Marchalonis, 1969). The labeled Fab fragments were incubated for thirty minutes with cells. An aliquot of the cells was removed and applied to a Millipore filter. The filter was washed until the wash reached background. Then the filter was removed from the holder and counted. A control which consisted of the same volume of buffer without cells and with the same volume of labeled Fab fragments was also Millipore filtered.

The Millipore filter cells had an activity of 25,659 cpm. The filter control contained 12,154 cpm. It appeared that the cells bound Fab fragments. The control filter had adsorbed nonspecifically a large amount of protein; however, the filter with cells contained twice as many counts as the control.

On the basis of this experiment it was decided to attempt to coat the cells with Fab fragments and determine if there would be a reduction of label incorporation into any of the polypeptide components. The experiment was done as shown in Figure 29, except the cells were exposed to 1 mg of Fab fragments per ml of PSG instead of yG immunoglobulin. The isolated membranes were solub ilized as described in the Methods Chapter and subjected to electrophoresis. The gels from the control and Fab coated cells were scanned and the radioactivity profIle was determined. The results are shown in Figures 32






65

and 33. From these data we see that there is no significant difference between the amount of isotope incorporated into the control and the Fab-coated cells. Thus, this experiment did not indicate which protein or proteins were antigenic determinants recognized by the goat's immunological system.

Several possible explanations exist for the failure of this

experiment. There may not have been enough Fab fragments per cell to cover all the sites, although a crude calculation indicated that there were approximately 10 7 Fab fragments per cell. Another possibility is that thk' Fab fragment could not adsorb all the isotope and did not protect the antigenic determinant from being labeled. However, if this were indeed the case, one would expect to see a new peak in the lower portion of the gel corresponding to the reduced iodinated fragments of the parent Fab molecule. This was not observed. These two factors may account for insufficient protection from enzymatic iodination being afforded the antigenic determinant.




















12.


112 10



















it. 10

9.




79





7.


A B


0

.54


4 Y

4


3 3















CM ~ 4 ~ 1

L M




FIGURE 32. Results of Fab fragment protection experiment: Control no Fab fragments. A Coomassie blue staining pattern of plasma membrane polypeptides in the absence of Fab fragments. B Incorporation of 1251 in plasma membrane polypeptides in the absence of Fab fragments.
























12


1.1
11 to

10



.9




.8



A B 7 .7 AB 7




&y



05

S


.4
4



.33





2










SCM





FIGURE 33. Results of Fab fragment protection experiment: Fab fragments at concentrations of 1 mg/ml. A Coomassie blue staining pattern of plasma membrane polypeptides from cells exposed to Fab fragments. B Incorporation of 125I in plasma membrane polypeptides in the presence of Fab fragment.













CHAPTER IV
CONCLUSION

The proteins exposed on the surface of the plasma membrane of mammalian cells are of critical biological importance. An unknown number of the proteins serve as antigenic determinants. The objective of this investigation was to develop a technique which could be employed to identify directly the antigenic determinants on the cell surface. The L5178y murine lymphatic leukemia cell and the immunological system of a male goat served as the model system for the development of this technique.

First it was necessary to determine which of the plasma membrane polypeptides were exposed on the cell surface. This was done by using the enzyme lactoperoxidase to label exposed polypeptides with 1 Also, limited proteolysis of the intact cell was used to verify the lactoperoxidase localization of the polypeptides in the plasma membrane. By these two criteria one polypeptide was demonstrated to be exposed on the cell surface.

The demonstration that polypeptide I, only, is shielded from

enzymatic iodination by prior incubation with goat yG antibody produced against the L5178y cell is strongly suggestive that this polypeptide has the properties of an antigenic determinant. From the present data it is not possible to determine if this polypeptide is an antigen of the cell or if it is part of a complex of polypeptides which together form the antigen. It is not known whether this protein is composed of
68






69

one specific antigenic region or multiple antigenic regions, each capable of eliciting an immunological response. Further characterization of this polypeptide could contribute significantly to our knowledge of the molecular structure of antigenic determinants.

To date, a great deal of effort has been expended in isolating and characterizing histocompatibility antigens (Reisfeld and Kahan, 1970; Cresswell et al., 1973). Generally, it has been possible to isolate a polypeptide that could inhibit the cytotoxicity of a specific antisera. In the case of the papain cleavage procedure the antigen is known to be released by proteolysis (Cresswell et al., 1973). There is some evidence that histocompatibility antigens solubilized by detergents and high salt may actually result from autoproteolysis (Mann, 1972). In any case histocompatibility antigens isolated in this manner have been found to be relatively small polypeptides with molecular weights ranging from 30,000 to 45,000 daltons (Fogerty, 1972). It is virtually impossible to.determine if these histocompatibility antigenic determinants were part of one large polypeptide or part of several polypeptides on the surface of the cell. This and other questions will not be resolved until better experimental techniques are available.

Another group of surface proteins has been characterized. They are the antigenic receptors found on T and B lymphocytes (Marchalonis and Cone, 1973). The best evidence to date indicates that these receptors are large immunoglobulin-like molecules embedded in the cell surface. They have been solubilized and shown to cross-react with anti-yM serum, hence, they are thought to be of the yM immunoglobulin class. Since polypeptide 1 was solubilized and subjected to gel electrophoresis in the presence of 2-mercaptoethanol, it is highly






70

unlikely that it is an immunoglobulin-like molecule.

Since polypeptide 1 has not been isolated, little can be said of its properties. The SDS polyacrylamide gel electrophoresis indicated that the apparent molecular weight of this polypeptide was 180,000 daltons. The implication of this finding is that polypeptide 1 consists of a linear polymer of approximately 1800 amino acid residues. By comparison to well characterized water soluble proteins this protein is extremely large; however, with respect to membrane proteins this high molecular weight is not at all unusual.

A number of other investigators have demonstrated the presence of similarly high molecular weight polypeptides on cells. Shin and Carraway have found that one major polypeptide on the surface of murine sarcoma 180 cells can be labeled using the lactoperoxidase technique (Shin and Carraway, 1973). Poduslo and coworkers have demonstrated high molecular weight polypeptides on the surfaces of both the BHK and the L cells (Poduslo et al., 1972). Other cells such as the HeLa cell (Huang et al., 1973), platelets (Nachman et al., 1973), and erythrocytes (Phillips and Morrison, 1970) have been found to have a high molecular weight component present in their plasma membranes. In each of the cases the high molecular weight polypeptide was found to be associated with carbohydrate. However, it was not possible to detect the presence of carbohydrate in polypeptide 1 of the L5178y plasma membrane with the periodic acid-Schiff staining technique. One cannot rule out the possibility that polypeptide 1 does contain carbohydrate. A more sensitive method seems to be needed in order to detect the carbohydrate if it is present.

Having identified polypeptide 1 as a possible antigen, it would






71

be most interesting to establish its relationship to the known histocompatibility antigens of this cell. It may be possible to design a series of immunoglobulin protection experiments which could yield significant information about the molecular structure of these antigenic determinants. Perhaps other workers will be able to continue this most interesting problem and bring it to a definitive conclusion.








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76


BIOGRAPHICAL SKETCH


John Enoch Hammond was born in Holden, Massachusetts, on

February 27, 1946. He and his family moved to northwest Florida where he attended public schools. In 1964 he graduated from Milton High School, Milton, Florida.

In 1968 he received his B.S. degree in chemistry from Florida State University, Tallahassee, Florida. After graduation he was employed by Vitro Services, Fort Walton Beach, Florida as an analytical chemist.

In September 1969 he entered graduate school in the Department of Biochemi-stry at the University of Florida.

His wife is Mary G. Hammond, now a fourth year medical student in the College of Medicine at the University of Florida.








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.




Waldo R. Fisher, Chairman Associate Professor of Medicine and Bi-ochemistry









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
Professor of Biochemistry









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.

117


Owen M. Rennert Professor of Pediat'rics and
Biochemistry








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.




Carl M. Feldherr
Associate Professor of Pathology







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.

June, 1974


Dean,,Graduate School