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A preliminary characterization of the polypeptide components of the plasma membrane of the L5178y murine lymphatic leukemia cell

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A preliminary characterization of the polypeptide components of the plasma membrane of the L5178y murine lymphatic leukemia cell
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Hammond, John Enoch, 1946-
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vii, 76 leaves. : illus. ; 28 cm.

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Antibodies ( jstor )
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Cell membranes ( jstor )
Cells ( jstor )
Electrophoresis ( jstor )
Enzymes ( jstor )
Epitopes ( jstor )
Gels ( jstor )
Immunoglobulins ( jstor )
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Biochemistry and Molecular Biology thesis Ph. D
Cell membranes ( lcsh )
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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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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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Barber, A. J. and G. A. Jamieson. (1970), J. Biold. Chem. 245, 6357. Bartlett, G. (1959), J. Biol. Chem. 234, 466. Bellanti, J. (1971a), Immunology, Philadelphia, W. B. Sanders, p 83. Bellanti, J. 4(1971b), Immunology, Philadelphia, W. B. Sanders, p 323. Bellanti, J. (1971c), Immunology, Philadelphis, W. B. Sanders, p 389. Berg, H. (1969), Biochim. Biophys. Acta 183, 65. Bergmeyer, H. U. (1963a), Methods of Enzymatic Analysis, New York,
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Bergmeyer, H. U. (1963b), Methods of Enzymatic Analysis, New York,
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Birne, G., ed. (1972), Subcellular Components, Preparation and
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Fischer, G. A. (1958), Ann. N. Y. Acad. Sci. 76, 673. Fogerty, J. E. (1972), Fed. Proc. 31, 1087. Goldenberg, G. J. and Thomas, C. E. (1967), Nature 214, 1339. Gorter, E. and F. Grendel. (1925), J. Exp. Med. 41, 439. Hendler, R. (1971), Physiol. Rev. 51, 66. Huang, C., C. Tsai, and E. Canellakis. (1973), Biochim. Biophys. Acta
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Hunter, W. and F. Greenwood. (1962), Nature (London) 194, 495. Lenard, J. (1970), Biochemistry 9, 1129. Maddaiah, V., S. Chen, I. Rezvani, R. Sharma, and P. J. Collipp.
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Maddy, A. H. (1964), Biochim. Biophys. Acta 88, 390. Mann, D. L. (1972), Transplantation 14, 398. Marchalonis, J. J. (1969), Biochem. J. 113, 299. Marchalonis, J. J. and R. E. Cone. (1973), Transplant. Rev. 14, 3. Nachlas, M. M., B. Monis, D. Rosenblatt, and A. M. Seligman. (1960),
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Reisfeld, R. and B. Kahan. (1970), Fed. Proc. 29, 2034. Rhodes, B. (1965), Anal. Chem. 37, 995. Robertson, J. D. (1957), J. Biophys. Biochem. Cytol. 3, 1043. Rosen, S. I., G. W. Kelly, and V. B. Peters. (1966), Science 152, 352. Sachs, D. H., H. J. Winn, and P. S. Russell. (1971), J. Immunol. 107,
481.

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Shands, J. W., Jr. (1968), Stain Technol. 43, 15. Shapiro, A. L., E. Vinuela, and J. V. Maizel, Jr. (1967), Biochem.
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Shapiro, A. L. and J. V. Maizel, Jr. (1969), Anal. Biochem. 29, 505. Shin, B. and K. Carraway. (1973), Biochim. Biophys. Acta 330, 254. Singer, S. and G. Nicholson. (1972), Science 175, 720. Smith, E. J. and L. B. Crittenden. (1973), Biochim. Biophys. Acta
298, 608.

Stein, W. D. (1967), The Movement of Molecules Across Cell Membranes,
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de The', G. (1968), in The Membranes, A. J. Dalton and F. Haguenau,
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Triplett, R. B. and K. L. Carraway. (1972), Biochemistry 11, 2897. Warren, L. (1969), in Fundamental Techniques in Virology, K. Habel and
N. P. Salzman, editors, New York, Academic Press, p 66.
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75
Williams, C. A. and M. W. Chase. (1967a), Methods in Immunology and
Immunochemistry, Vol. I, New York, Academic Press, p 216.
Williams, C. A. and M. W. Chase. (1967b), Methods in Immunology and
Immunochemistry, Vol. I, New York, Academic Press, p 319.
Williams, C. A. and M. W. Chase. (1967c), Methods In Immunology and
Immunochemistry, Vol. I, New York, Academic Press, p 234.
Williams, C. A. and M. W. Chase. (1967d), Methods In Immunology and
Immunochemistry, Vol. I, New York, Academic Press, p 418.





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







Full Text
45
FIGURE 20. Electron micrograph of OsO^ fixed and uranyl acetate
stained plasma membrane preparation. Direct magnification 28,000X.


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 Th 15178y 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 ygm P/15 min/mg of protein (Birne, 1972).
Now, if the minimum detectable level of phosphorus is 0.5 ygm, one can
calculate that approximately 11 ygm 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 ygms of
protein was used. Since we were unable to detect any glucose-6-
phosphatase activity in the isolated plasma membrane fragments, the
total endoplasmic reticulum contamination must be less than 16%
(11 ygm/70 ygm 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-dichloro-
phenolindolephenol as the electron acceptor. One measures the decrease
in absorbance at 600 nm. Again, the specific activity is defined as


43
FIGURE 18. Electron micrograph of OsO^ fixed and uranyl acetate
stained plasma membrane preparation. Direct magnification 28,000X.


56
FIGURE 26. Growth curve of L5178y cells after
treatment with 500 ugm/ml of Trypsin for thirty
minutes at pH 7.2.


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.
125
B Incorporation of I in plasma membrane polypeptides in the presence of Fab fragment.


48
TABLE II
Polypeptide
Molecular Weight
1
2
3
4
5
6
7
8
9
180,000
153,000
115,000
100,000
70,000
56,000
47.500
26.500
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.
Iodination 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 chioramine-T method of iodination (Hunter and
Greenwood, 1962). Chioramine-T is a powerful oxidizing agent and,
unfortunately, some degree of protein denaturation always occurs.
However, this method has been used successfully for producing radio-
labeled 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.


31
FIGURE 8. Phase contrast micrograph of cells washed in PSG.
Magnification 320X.
FIGURE 9. Phase contrast micrograph of cells after three
minutes in 0.01 M Tris 0.001 M MgC1^ at pH 7.4.
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


os,
12
CM
FIGURE 31. Results of the yG immunoglobulin protection experiment: yG concentration 2.5 mg/ml.
A Coomassie blue staining pattern of plasma membrane polypeptides.
125
B Incorporation of I in plasma membrane polypeptides.


- i#
(T. ^
FIGURE 14. Phase contrast micrograph of the interface resulting from
the 125g centrifugation. Magnification 320X.
Plasma
Membrane
Fragments
FIGURE 15. Photograph of discontinuous sucrose gradient showing
plasma membrane fragments halfway into the 42.5% sucrose layer.


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 cel 11s volume.
Figures 9, 10, and 11 show the cells after suspension in the
hypotonic swelling 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.


10 4 2 4. 8 3 5


75
Williams, C. A. and M. W. Chase. (1967a), Methods in Immunology and
Immunochemistry, Vol. I, New York, Academic Press, p 216.
Williams, C. A. and M. W. Chase. (1967b), Methods in Immunology and
Immunochemistry, Vol. I, New York, Academic Press, p 319.
Williams, C. A. and M. W. Chase. (1967c), Methods In Immunology and
Immunochemistry, Vol. I, New York, Academic Press, p 234.
Williams, C. A. and M. W. Chase. (1967d), Methods In Immunology and
Immunochemistry, Vol. I, New York, Academic Press, p 418.


69
one specific antigenic region or multiple antigenic regions, each cap
able 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 rang
ing from 30,000 to 45,000 daltons (Fogerty, 1972). It is virtually
impossible to. determine if these histocompatibility antigenic determin
ants 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


47
B
FIGURE 21.
A Gel scan of Coomassie blue staining pattern of solubilized
plasma membrane fragments.
B Photograph of SDS polyacrylamide gel.
tfa*
i
t
t
'* |
* i
. *-. .iv
:'


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).
"t
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 viere 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.


19
O
immunization consisted of 2 x 10 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 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 minutes at 37 C. Then antibody, from which the
fragments were prepared, along with complement, was added and incubated
at 37 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 percen
tage of cell death over the control with no Fab fragments present


520
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 I in plasma membrane polypeptides in the absence of Fab fragments.


CHAPTER III
RESULTS AND DISCUSSION
Plasma Membrane Isolation Procedure
Two methods of isolating the plasma membrane of the L5178y leuke
mia 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 sulfhydra! groups thus having the advantage of inhibiting
proteolytic enzymes which require a free sulfhydra! 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


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 from, the cell. Although extremely impor
tant, 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, we are now just begin
ning to understand the macromolecu!ar 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
1


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 viere 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 vies shown
that polyacrylamide gel electrophoresis done in the presence of the
detergent sodium dodecyl sulfate separated proteins by their molecular
weights (Shapiro et al., 1 967; Shapiro and Maize!, 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.


A B
FIGURE 30. Results of the yG immunoglobulin protection experiment: Control experiment with no yG present.
A Coomassie blue staining pattern of plasma membrane polypeptides.
125
B Incorporation of I in plasma membrane polypeptide.


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 acid-
Schiff technique.
It was possible to combine the biological specificity of anti
body 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.
VI 1


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 37 C. The proteolysis was stopped by dialysis against glass
distilled water at 4 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 dal tons 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


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 Dam'ell i-
Davson model (Danielli and Davson, 1935). In 1957 Robertson modified
the Danielli-Davson model as follows:
1) The protein components were in the B 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 pre
paring 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 hydrophi11ic 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 tne 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 investiga
tor (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-


38
conjunction with electron microscopy has resulted in the ultrastructur-
al 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 concentra
tion. 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 (Poliak and Shorey,
1968; Rosen et al., 1966; Ericsson and Orrenius, 1966; Tice and
Barrnett, 1 962). Consequently, this enzyme is an accepted marker enzyme
for rough endoplasmic reticulum contamination in plasma membrane prep
aration. This enzyme is slightly more difficult to assay since it is a


73
Ericsson, J. L. E. and S. Orrenius. (1966), J. Histochem. Cytochem.
14, 761.
Fairbanks, 6., T. L. Steck. and D. F. H. Wallach. (1971), Biochemistry
10, 2606.
Fischer, G. A. (1958), Ann. N. Y. Acad. Sci. 76, 673.
Fogerty, J. E. (1972), Fed. Proc. 31_, 1087.
Goldenberg, G. J. and Thomas, C. E. (1967), Nature 214, 1339.
Gorter, E. and F. Grendel. (1925), J. Exp. Med. 41, 439.
Hendler, R. (1971), Physiol. Rev. 51, 66.
Huang, C. C. 'Tsai, and E. Canellakis. (1973), Biochim. Biophys. Acta
332, 59.
Hunter, W. and F. Greenwood. (1962), Nature (London) 194, 495.
Lenard, J. (1970), Biochemistry 9, 1129.
Maddaiah, V., S. Chen, I. Rezvani, R. Sharma, and P. J. Collipp.
(1971), Biochem. Biophys. Res. Comm. 43, 114.
Maddy, A. H. (1964), Biochim. Biophys. Acta 88, 390.
Mann, D. L. (1972), Transplantation 14, 398.
Marcha!onis, J. J. (1969), Biochem. J. 113, 299.
Marchalonis, J. J. and R. E. Cone. (1973), Transplant. Rev. 14, 3.
Nachlas, M. M., B.Monis, D. Rosenblatt, and A. M. Seligman. (1960),
J. Biophys. Biochem. Cytol. 7_, 261.
Nachman, R., A. Hubbard, and B. Ferres. (1973), J. Biol. Chem. 248,
2928.
Noonan, K. (1974), Personal Communication.
Orenstein, L. (1964), Ann. N. Y. Acad. Sci. 121, 321.
Phillips, D. R. and M. Morrison. (1970), Biochem. Biophys. Res. Comm.
40, 284.
Poduslo, J. F., C. S. Greenberg, and M. C. Glick. (1972), Biochemistry
H, 2616.
Poliak, J. K. and C. D. Shorey. (1968), Develop. Biol. 17, 536.
Puck, T., S. Cieciara, and A. Robinson. (1958), J. Exp. Med. 108, 945.


14
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
3 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 homogen
ate. 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 inter
face 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 mem
branes 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 33,000a.
An orange band of membranes formed in the middle of the gradient. The
band was removed, diluted with water, and pelleted. The pellet con
tained 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


74
Reisfeld, R. and B. Kahan. (1970), Fed. Proc. 29, 2034.
Rhodes, B. (1965), Anal. Chem. 37, 995.
Robertson, J. D. (1957), J. Biophys. Biochem. Cytol. 3_, 1043.
Rosen, S. I., G. W. Kelly, and V. B. Peters. (1966), Science 152, 352.
Sachs, D. H., H. 0. Winn, and P. S. Russell. (1971), J. Immunol. 107,
481.
Segrest, J., R. Jackson, V. Marchesi, R. Guyer, and W. Terry. (1972),
Biochem. Biophys. Res. Comm. 49, 964.
Shands, J. W., Jr. (1968), Stain Technol. 43, 15.
Shapiro, A. L., E. Vinuela, and J. V. Maizel, Jr. (1967), Biochem.
Biophys. Res. Comm. 28, 815.
Shapiro, A. L. and J. V. Maizel, Jr. (1969), Anal. Biochem. 29, 505.
Shin, B. and K. Carraway. (1973), Biochim. Biophys. Acta 330, 254.
Singer, S. and G. Nicholson. (1972), Science 175, 720.
Smith, E. J. and L. B. Crittenden. (1973), Biochim. Biophys. Acta
298, 608.
Stein, W. D. (1967), The Movement of Molecules Across Cell Membranes,
New York, Academic Press.
de The', G. (1968), jn_ The Membranes, A. J. Dalton and F. Haguenau,
editors, New York, Academic Press, p 121.
Tice, L. W. and R. J. Barrnett. (1962), J. Histochem. Cytochem. 1_0,
754.
Triplett, R. B. (1972), Ph.D. Thesis, Oklahoma State University,
Stillwater, Oklahoma.
Triplett, R. B. and K. L. Carraway. (1972), Biochemistry 11, 2897.
Warren, L. (1969), j_n Fundamental Techniques in Virology, K. Habel and
N. P. Salzman, editors, New York, Academic Press, p 66.
Warren, L., M. C. Glick, and M. K. Nass. (1966), J. Cell. Phys. 68,
269.
Watanabe, I. and S. Okada. (1967), J. Cell Biol. 32, 309.
Wilfong, R. F. and D. M. Neville, Jr. (1970), J. Biol. Chem. 245,
6106.


60
Washed Cells
1.2 x 103 Cells
6 x 108 Cells
Suspend in PSG
Control Cells
Wash twice in rSG
Control Cells
Label with "^1
Labeled Control Cells
Isolate plasma
membrane
Membrane Fragments
SDS Gel
Electrophoresis
6 x 108 Cells
Suspend in PSG
containing either
Fab fragments or
intact yG for 30
minutes
W
Coated Cells
Wash twice in PSG
V
Coated Cells
Label with "^1
Labeled Coated Cells
Isolate plasma
membrane
Membrane Fragments
SDS Gel
Electrophoresis
"W
Gel Scan
Profile
W
Radioactivity
Profile
FIGURE 29. The immunoglobulin protection 'experiment.


22
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.


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
L517Sy 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 polypeptide 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 major components,
ranging from a molecular weight of 24,000 to 180,000 daltons, were
vi


32
FIGURE 10. Phase contrast micrographs of cells after eight minutes in
0.01 M Tris, 0.001 M MgCl^ at pH 7.4. Magnification 320X.
FIGURE 11. Phase contrast micrographs of cells after fifteen minutes
in 0.01 M Tris, 0.001 M MgCl^ at pH 7.4. Magnification 320X.


REFERENCES
Allan, D. and M. J. Crumpton. (1971), Biochem. J. 123, 967.
Bachmann, E., D. W. Allmann, and D. E. Green. (1966), Arch. Biochem.
Biophys. 115, 153.
Bailey, J. L. (1962), Techniques in Protein Chemistry, Amsterdam,
Elsevier, p 293.
Barber, A. J. and G. A. Jamieson. (1970), J. Biol. Chem. 245, 6357.
Bartlett, G. (1959), J. Biol. Chem. 234, 466.
Bellanti, J. (1971a), Immunology, Philadelphia, W. B. Sanders, p 83.
Bellanti, J. "1 (1971b), Immunology, Philadelphia, W. B. Sanders, p 323.
Bellanti, J. (1971c), Immunology, Phi 1adelphis, W. B. Sanders, p 389.
Berg, H. (1969), Biochim. Biophys. Acta 183, 65.
Bergmeyer, H. U. (1963a), Methods of Enzymatic Analysis, New York,
Academic Press, p 783.
Bergmeyer, H. U. (1963b), Methods of Enzymatic Analysis, New York,
Academic Press, p 788.
Birne, G., ed. (1972), Subcellular Components, Preparation and
Fractionation, 2d ed., London, Butterworths, p 204.
Boone, C. W., E. Ford, H. E. Bond, D. C. Stuart, and D. Lorenz.
(1969), J. Cell Biol. 41_, 378.
Bretscher, M. S. (1973), Science 181 622.
Cresswell, P., M. J. Turner, and J. L. Strominger. (1973), Proc. Nat.
Acad. Sci. U. S. 70, 1603.
Daniel 1 i, J. F. and H. Davson. (1935), J. Cell. Comp. Physiol. J5, 495.
DePierre, J. W. and M. L. Karnovsky. (1973), J. Cell Biol 56, 275.
Dodge, J. T., C. Mitchell, and D. J. Hanahan. (1963), Arch. Biochem.
Biophys. 100, 119.
Dulbecco, R. (1954), J. Exp. Med. 99, 167.
Earl, D. C. N. and A. Korner. (1965), Biochem. J. 94, 721.
72


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
dal tons. 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 erythro
cytes (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


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 radio
autography has shown that the radioactive label was localized exclusive
ly 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 L5178y 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
125
mixture, I, cells, and hydrogen peroxide as described in the
Methods chapter, but no 1actoperoxidase. Under these conditions no
radioactivity above background was detected in an aliquot of the
isolated plasma membrane preparation. This preparation was electro-
phoresed and Figure 25 shows the gel scan of the Coomassie blue


54
staining pattern. Since no radioactivity was detected in the prepar
ation, 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 polypep
tides 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


i'xbf


18
X-100, and p-dioxane to make the total volume one liter.
To determine th 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 Scintilla
tion 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).
O
It was adapted for use with the L5178y cell. Generally, 4 6 x 10
cells were used per experiment. The cells were washed in PSG and then
resuspended in 10 ml of PSG containing 100 ygm/nil or 500 ygm/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 rest
ed two days the first week. The second week, the goat was immunized
every other day for five days and rested two days. One further injec
tion was given and the animal was exsanguinated two days later. Each


50
B
500-
1 25
FIGURE 22. First I labeling experiment.
125
A Coomassie blue staining pattern of I labeled plasma membrane
polypeptides.
B Radioactivity profile of "^^1 labeled plasma membrane polypeptides.


ACKNOWLEDGMENTS
I would like to express my gratitude to my research director, Dr.
Waldo R. Fisher, for providing the financial support, for his encour
agement 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 asset 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
Biochemistry for his assistance in the development of the plasma mem
brane 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 anti
serum in the goat, Red I.
Finally, I would like to remember the one that made this investi
gation possible and made the ultimate sacrifice: the goat, Red I. May
his spirit always graze in green pastures.
ii


CHAPTER II
METHODS AND MATERIALS
Cell Culture Technique
The murine lymphatic leukemia cell line, L5178y, kindly supplied
by Dr. John W. Cramer of the University of Nevada, was used exclusively
in this studyp* 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 ug/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 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 (Sachs et al., 1971).
The stock lines were maintained by serial dilution (1:4) through
10


27
All common chemicals were reagent grade and used without further
purification.


39
non-spectrophotometric assay. Essentially the substrate glucose-6-
phosphate 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 ugm 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 ^ Succinic c
Phosphatased 6-Phosphataseu DehydrogenaseL
Homogenate
1
.7
(i.o)d
0.9
(i.o)
1.0
(i.o)
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)
BLLA
e
0.8
(0.8)
Specific activity = A absorbance/min/mg protein,
b Specific activity = ygm P/15 min/mg protein.
c Specific activity e a absorbance/hr/mg protein.
d ( ) indicate Purification factor.
e BLLA e Below the Lower Limits of the Assay. Less than 16% of the
samp!e.
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


FIGURE 27.
Trypsin at
57
2 3 4
5 6 7
CM.
8 9 10
1 PR
Results of treatment of I labeled L5178y cells with
a concentration of 100ugm/ml.
A Coomassie blue staining pattern.
B Radioactivity profile.


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 contamin
ated 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 Mi Hi pore filter. The filter was washed un
til the was!) reached background. Then the filter v/as 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 Mi Hi pore filtered.
The Mill ipore 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 compon
ents. The experiment was done as shown in Figure 29, except the ceils
were exposed to 1 mg of Fab fragments per ml of PSG instead of yG
immunoglobulin. The isolated membranes were solubilized as described
in the Methods Chapter and subjected to electrophoresis. The gels
from the control and Fab coated cells were scanned and the radio
activity profile was determined. The results are shown in Figures 32


53
FIGURE 25. Coomassie blue staining pattern of 1actoperoxidase
control experiment.


25
FIGURE 6. Elution pattern of the mercuripapaln digest on a
5 x 50 cm G-200 Sephadex column.


TABLE OF CONTENTS
CHAPTER PAGE
Acknowledgments ii
Abbreviations v
Abstract vi
I. Introduction 1
II. Methods and Materials 10
Cell Culture Technique 10
Isolation of Plasma Membrane 11
Electron Microscopy 14
Polyacrylamide Gel Electrophoresis Procedure 15
Enzymatic Iodination of Intact L5178y Cells 16
125
Liquid Scintillation Counting of I 16
Trypsinization. Procedure 18
Immunization Procedure 18
Cytotoxicity Inhibition Assay 19
Immunoglobulin Purification Procedure 21
Preparation of Fab Fragments 24
Enzyme Assays 24
Protein Determinations 26
Materi al s 26
i i i


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.


30
Washed Cells
Suspend in Hypotonic Solution
Y
Figure 9 after 3 minutes
Figure 10 after 8 minutes
Figure 11 after 15 minutes
Homogenize
Figure 12
Overlayer on 45% Sucrose and Centrifuge
Supernatant
Discard -
Figure 13
zzzzzzz
Interface
Figure 14
Plasma Membrane
Fragments
Figure 16
Dilute and
Centrifuge
Plasma Membrane Pellet
- Figure 17
Overlayer on Discontinuous
Sucrose Gradient and
Centrifuge
V
Sample
20%
42.5%
45%
50%
60%
Figure 15
FIGURE 7. Diagram of plasma membrane isolation procedure.


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 dal'tons.
Enzymatic Iodination of Intact L5178y Cells
O
Generally, each experiment was done using approximately 6 x 10
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
125
of I (Poduslo et al., 1972). The reaction was initiated by the
addition of 5 yl of 1.56 mM 1^02. Subsequent additions of 5 yl of
1.56 mM H2O2 were made at fifteen-second intervals until a total of
100 yl of H2O2 had been added. The reaction was carried out at 37 C.
125
The cells were then washed to remove unbound I and then the
plasma membranes were isolated as previously described.
125
Liquid Scintillation Counting of 1 I
125
All liquid scintillation counting of I was done in modified
Bray's solution (Rhodes, 1965). This solution contained four grams
of PP0 (diphenyloxazole), 0.2 grams of DM-P0P0P (1,4-bis [4-methyl-5-
phenyl-2-oxazolyl] benzene), 60 grams of naphthalene, 333 ml of Triton


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.


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% cf 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 lew. 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


13
consisting of 0.01 M Tris buffer, pH 7.4 and 0.001 M MgCl^ were added
g
per 2 x 10 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 pro
cedure was monitored by phase contrast microscopy.
The resulting cell debris was then diluted with an equal volume
of phosphate buffered saline, overlayered on a cushion 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 5 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
viere concentrated by centrifugation in the SW 25.1 rotor for one hour
at 25,000 rpm. Samples not taken for microscopy, protein determina
tions, enzyme assays, or polyacrylamide gel electrophoresis were
stored at -20 C.
Fluorescein Mercuric Acetate Method
O
Cells (approximately 6 x 10u) 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


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 H 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 micro
graphs showed occasional vesicles highly suggestive of mitochondria.
Further speculation on the degree of contamination was not possible
with the present data.


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 L5178y murine lymphatic leukemia cell fulfilled
this requirement. This cell line has been maintained in culture for
approximately sixteen years and it can be routinely grown to high cell
density. In order to produce the greatest immunological response to
the L5178y 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 v/e
have an antigen source, the L5178y cell, and the immunological system
of the goat to define the antigenic components on the L5178y cell
surface.
The investigation was designed to proceed in the following
manner:
1) Development of a method for isolating the plasma
membrane of the L5178y cell.


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 redis
solved 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


63
Also, two new peaks appeared in the radioactivity profile of the anti
body 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 theL5178y cell which
the goat's immunological system recognized. This was based on the
reduction of isotope incorporated into polypeptide 1 and the appear
ance of two new 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,


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 L5178y Cell 48
Proteolytic Digestion Studies on the L5178y Cell 54
Immunoglobulin Protection Experiment 59
IV. Conclusion 68
References 72
Biographical Sketch 76
iv


12
FIGURE 1. Growth curve of the L5178y cell in Fischer's
Medium for the Leukemic Cells of Mice supplemented with
10% horse serum.


17
FIGURE 2. Calibration curve for the SDS polyacryl
amide 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.


n
eight successive tubes in duplicate. This greatly reduced the time
and effort required to maintain the cultures. Each day during an eight-
day 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 L5178y
cell. At time zero, a flask containing fresh medium was inoculated
such that the initial cell count was 104,000 cells/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 cell s/ml. At this point growth
slowed drastically due to exhaustion of the medium.
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 Membrane
Smith Method
In general, it was found that one could conveniently isolate the
plasma membrane from 4 6 x 10 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


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 histo
compatibility 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 immunotolerance 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.


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 carbo
hydrate 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 develop
ment of techniques designed to localize the protein components in the
lipid bimolecular leaflet. The first technique used to localize poly
peptide chains in the plasma membrane was limited proteolytic diges
tion 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 ery
throcyte. Later, chemical reagents, such as SITS (stilbene-4-
acetamino-4'-thiocyano disulfonate) and S-labeled formylmethionyl
sulfone methyl phosphate,were employed to label the exterior polypep
tides 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 deline
ated 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


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 Biochemistry 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.
7 / - -
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


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 art. 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 con
stitutes 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
-13
approximately 10 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 solubil
ized antigenic determinants retain their biological activity and thus
can inhibit cytotoxicity of specific antisera. The solubilization
techniques used to date are: detergent extraction, sonication,


23
W
^ CH U U
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.


58
"i 2 1 4 5 ~~i } 8 5"
CM
1 ?*¡
FIGURE 28. Results of treatment of I labeled L5178y cells with
Trypsin at a concentration of 500 ugm/ml.
A Coomassie blue staining pattern.
B Radioactivity profile.


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
125
enzymatically labeled with I. 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 ygm/ml for thirty minutes. Phase contrast micros
copy after sucvi 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
r
hours and a maximum cell density of about 1.2 x 10 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 trypsiniza
tion 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 ygm/ml and 500 ygm/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


52
. t 1 1 1 1 1 1 1 1 r~
11 2 34 56 789 10
CM.
FIGURE 24. Third labeling experiment.
125
A Coomassie blue staining pattern of I labeled plasma membrane
polypeptides.
125
B Radioactivity profile of I labeled plasma membrane polypeptides.


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% OsO^, and fixed for one hour.
The fixed membrane fragments were then centrifuged at 90,000g for one
hour. The pellet containing the OsO^ fixed plasma membrane was
resuspended in 2% bovine serum albumin to which a drop of 25% glutar-
aldehyde was added. The fixed plasma membrane fragmentsbovine serum
albumin-glutaraldehyde suspension was immediately centrifuged. The
gel that resulted from the bovine serum albumin, cross! inked 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 50 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 yl of 3% sodium dodecyl sulfate (SDS)
in 0.1 M Tris buffer at pH 7.8. To the suspended fragments, 10 yl 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 100 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


ABBREVIATIONS
SDS Sodium dodecyl sulfate
PBS Phosphate buffered saline
PGS Puck's saline G
SITS Sti1bene-4-acetamino-41-thiocyano
disulfonate
HL-A Human histocompatibility antigen system
FMA Fluorescein mercuric acetate
BLLA Below lower limits of assay
v


59
unchanged. From these experiments it is readily apparent that poly
peptide 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 enzymat
ically labeling the surface with 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 if vied. 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 incor
poration in polypeptide 1 in the antibody coated cells over the control.


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
1974


20
FIGURE 3. Cytotoxic antibody titer in goat serum.


51
1 23456789 10
CM.
t or
FIGURE 23. Second iDI labeling experiment.
125
A Coomassie blue staining pattern of I labeled plasma membrane
polypeptides.
125
B Radioactivity profile of I labeled plasma membrane polypeptides.


7
proteolytic digestion, and simple salt extraction.
For example, consider the biochemical properties of human HL-A2
histocompatibility antigens solubilized from cultured human lympho
cytes by papain cleavage (Cresswell et al., 1973) and by high salt
extraction (Reisfeld and Kahan, 1970). Using the high salt extrac
tion 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
Mo 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 polyacryla
mide 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-


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


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 demon
stration 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 composi
tion, 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 morpho
logically, 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 membrane of cells vesiculates upon disrup
tion. Unfortunately, a number of other cell organelles such as
lysosomes, smooth endoplasmic reticulum, Golgi apparatus, and peroxi-



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FIGURE 19. Electron micrograph of OsO^ fixed and uranyl acetate
stained plasma membrane preparation. Direct magnification 28,Q0QX.


I
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.


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 immuno
logical. 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
125
the enzyme lactoperoxidase to label exposed polypeptides with I.
Also, limited proteolysis of the intact cell was used to verify the
lactoperoxidase localization of the polypeptides in the plasma mem
brane. By these two criteria one polypeptide was demonstrated to be
exposed on the cell surface.
The demonstration that polypeptide 1, 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


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^ Fab fragments per cell. Another possibil
ity is that th' 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.


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
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.
l ie l V I I I 111 cu
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.
n // j
/y V -ff
Owen M. Rennert
Professor of Pediatrics and
Biochemistry