130mm 0F SPECIFIC POLYRIBOSOMES ' A BYEMMUNOCHEMICALMETHODS“- , . ‘ Thesis for the Degree of M. S. MECHEGAN STATE UNIVERSITY A WILLMM HERMAN ESCHENFELDT- ‘ ' 1975 ' 155818, ' ~ slit-JOHN; BY I HOAG & sun3' :g: 939K mev we L H. a '- ' '; uDL‘ -'~~ “!-"L'L u ABSTRACT ISOLATION OF SPECIFIC POLYRIBOSOMES BY IMMUNOCHEMICAL METHODS BY William Herman Eschenfeldt The total polysome population was isolated from the myeloma cell line MOPC-Zl which synthesizes and secretes an IgGl-like molecule. The polysomes were purified by gel filtration on columns of Sepharose 28, 4B or 6B. This procedure separates the polysomes from the smaller intracellular material. Isolation of the poly- somes synthesizing the myeloma protein (IgGl) was attempted through the use of immunochemical techniques. Antibody directed against the myeloma protein was shown to bind specifically to the purified poly- somes. Specific immune precipitation of the polysomes was demon- strated, although nonspecific background precipitation was consistently high. An affinity chromatography system using the myeloma--anti— myeloma system was shown to be specific for those proteins. However, repeated attempts to bind polysomes to affinity chromatography columns were unsuccessful. Nonspecific binding was high and no significant difference in specific binding could be demonstrated. ISOLATION OF SPECIFIC POLYRIBOSOMES BY IMMUNOCHEMICAL METHODS BY William Herman Eschenfeldt A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1975 ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. Ronald J. Patterson for his guidance and encouragement throughout this study. I also wish to thank the Department of Microbiology and Public Health for financial assistance. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . LIST OF FIGURES. . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . REVIEW OF THE LITERATURE . . . . . . . Description of Polyribosomes. . Binding of Specific Antibody to Polyribosomes Precipitation of Polyribosomes with Specific AntibOdieS o o o o o o o o o o 0 Direct Precipitation . . Indirect Precipitation . Isolation of Polyribosomes by Affinity Chromatography MATERIALS AND METHODS. . . . . . . . . Cells . . . . . . . . . . . . . Protein Preparation . . . . . . Myeloma Protein. . . . . Rabbit Antiserum . . . . Anti-Globulin. . . . . . Bovine Serum Albumin . . l4C- -Labeling of Proteins Pepsin Digestion of Antibody . . . . Reduction and Alkylation of Antibody . Affinity Chromatography . . . . Coupling of Protein to Sepharose . . . Affinity Chromatography Columns. . . . Isolation of the Total Polysome Population from iii Cells Page V1 .5 10 10 11 12 12 12 13 14 15 15 16 16 Separation of Membrane-Bound and Free Polysomes . . . . . . . . . . . . . . . . . Total Polysome Population. . . . . . . . . . . Purification of Total Polysomes . . . . . . . . . . . Pelleting. . . . . . . . . . . . . . . . . . . Discontinuous Sucrose Gradients. . . . . . . . Sepharose Columns. . . . . . . . . . . . . . . Analysis of Polysomes. . . . . . . . . . . . . Isolation of Specific Polysomes . . . . . . . . . . . Specific Binding of Labeled Antiserum. . . . . Direct Precipitation of Polysomes. . . . . . . Affinity Chromatography of Polysomes . . . . . RESULTS. 0 O O O O O O O O O O O O O O O O O O O O I O I O 0 Section I - Article Polysome Isolation by Sepharose Column Chromatography. . . . . . . . . . . . . . . . . Section II Isolation of Free and Membrane-Bound Polysomes Binding of Specific Antibodies to Polysomes. . Direct Immune Precipitation of Polysomes . . . Indirect Immune Precipitation of Polysomes . . Characterization of Affinity Columns with Protein . . . . . . . . . . . . . . . . . . Direct Affinity Chromatography of Polysomes. . Indirect Affinity Chromatography of Polysomes. DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O O 0 O O SUWARY O O I I O O O O O O O O O O O I O O O O O O I O O O 0 LIST OF REFERENCES . . . . . . . . . . . . . . . . . . . . . iv Page l6 l7 l8 18 18 l9 19 20 20 20 21 22 22 37 42 49 52 56 67 67 80 89 9O Table 10 ll 12 LIST OF TABLES Distribution of radioactivity in crude polysomes and Sepharose purified polysomes. . . . . . . . . . . Binding of 14C-anti-MOPC-Zl to polyribosomes. . . . . Indirect immune precipitation of MOPC-Zl polysomes. . Indirect immune precipitation of 3H-uridine labeled polysomes . . . . . . . . . . . . . . . . . . . . . . Protein specificity of immunoadsorbents . . . . . . . Specificity of immunoadsorbents prepared using six carbon spacers. . . . . . . . . . . . . . . . . . . . Affinity chromatography of extracellular protein from MOPC-Zl cells. . . . . . . . . . . . . . . . . . Affinity chromatography of isolated heavy and light chains... . . . . . . . . . . . . . . . . . . . . . . Binding of nascent chains to immunoadsorbents . . . Direct binding of polysomes to immunoadsorbents . . . Indirect binding of polysomes to immunoadsorbents . . Effect on polysomes of passage over affinity columns. Page 32 48 53 57 58 59 60 63 64 68 7O 73 Figure LIST OF FIGURES Page Sucrose gradient profiles of crude and Sepharose purified polysomes prepared as in Materials and Methods. Arrow indicates monosomes (80 S). A254 ()3[3H]uridine(O-—-O)..............34 Sucrose gradient profiles of Sepharose 6B purified polysomes. The crude polysomes were prepared from MOPC-Zl tissue culture cells maintained in vitro in Dulbecco's Modified Eagle Medium, supplemented with 10% fetal calf serum, in an atmosphere of 85% air, 15% C02. Cells were labeled with [3HJuridine and harvested at 5-8 x 105 cells/ml. The remainder of the isolation procedure was identical to that described in Materials and Methods. A. Analyzed on day of isolation. B. After storage at -85°C for 9 weeks. Arrow indicates monosomes (80 S). . . . . 36 Sucrose gradient profiles of free and membrane- bound polysomes. A. Free polysomes. B. Membrane- bound polysomes. A254 ( ) . . . . . . . . . . . . . 39 Sucrose gradient profiles of total polysomes fol- lowing 30,000 x g centrifugation. A. Supernatant. B0 Pellet. A254 ( )0 O o o o o o o o o o o o o o o 41 Binding of labeled anti-MOPC to myeloma polysomes. Percent CPM calculated as CPM/fraction CPM total x 100% 3H-uridine (polysome profile) (O———-O). Incubated with 14C-anti—MOPC only (O-——O). Pre-incubated with unlabeled anti-MOPC followed by l4C-anti-MOPC (El E1). . . . . . . . . . . . . . . . . . . . . . . 44 Binding of labeled anti-MOPC to Sepharose 2B puri- fied polysomes. A. Pre-incubated with unlabeled anti-MOPC followed by l4C-anti-MOPC. B. Incubated with 14C-anti-MOPC alone. 3H—uridine (polysomes) (o————0). 14C-anti-MOPC (o—-——o) . . . . . . . . . . . 47 vi Figure 10 11 12 13 Page Direct immune precipitation of polysomes. The sucrose gradient profiles are plotted as percent CPM per fraction calculated as in Figure 5. 3H- uridine polysomes (l————O). Immune precipitation is plotted as the percent of radioactivity in each fraction precipitable by antiserum ( 0—0 ) . . . . 51 Indirect immune precipitation of polysomes: titra- tion of specific antisera. Polysomes were incubated with varying amounts of anti-MOPC (O—-—-O) or NRGG (O--—O) followed by GARGG. Specific precipitation .( €,--‘) ) was calculated by subtracting NRGG precipitation from anti-MOPC precipitation. . . . . . . 55 Sucrose gradient profiles of heavy and light chalns. A280 (O--O). . . . . . . . . . . . . . . . . 62 Isolation of nascent chains from MOPC-21 polysomes. MOPC-21 polysomes labeled with 3H—amino acids were treated with Na EDTA and separated on 10-30% linear sucrose gradients. A254 (--0; 3H-amino acids (H) o o o o o o o o o o o o o o o o o o o o o o o o 66 Effect on polysomes of passage over affinity columns. A. Control polysomes incubated with l4C-anti-MOPC. B. Polysomes passed over Sepharose- NRGG column; incubated with 14C-anti-MOPC. 3H- uridine (polysomes) (O-—-O); l4C-anti-MOPC (O--Q). . . . . . . . . . . . . . . . . . . . . . . . 72 Binding of l4C-anti—MOPC to ribosomal subunits. EDTA treated MOPC-Zl polysomes labeled with 3H- uridine (O-—-—O) were incubated with 14C-anti—MOPC (O--O) and analyzed on 10-30% linear sucrose gradients . . . . . . . . . . . . . . . . . . . . . . . 76 Binding of l4C-anti-MOPC to ribosomal subunits: specificity. A. EDTA treated polysomes incubated with 14C-anti-MOPC. B. EDTA treated polysomes incubated with unlabeled anti-MOPC prior to incuba- tion with 14C-anti-MOPC. C. EDTA treated polysomes incubated with unlabeled NRGG prior to incubation with l4C-anti-MOPC. 3H-uridine (O-——O); 14C-anti- Mopc(o——-0).....................79 vii INTRODUCTION Since the messenger RNA-ribosomal aggregate model for protein synthesis was first described (Warner et al., 1962, 1963; Wettstein et al., 1963; Staehelin et al., 1963), isolation of the polyribo- somal fraction of cells has been widely used as a tool for the study of protein synthesis. Initially, polyribosomes were isolated by fractionating crude cell lysates on sucrose gradients and collecting the material in the 100—300 S region. In order to study synthesis of specific proteins, however, it was necessary to isolate only specific polysomes from the total population. For certain proteins such as myosin and gamma globulin this could be accomplished in part by isolating polysomes on the basis of their size (Heywood et al., 1967; Shapiro et al., 1966). Since cells synthesize proteins of various sizes and many different proteins of similar size, this method of polysome isolation still yielded a diverse population. Evidence for the presence of specific protein fragments (nascent or growing polypeptides) on ribosomes led to attempts to isolate polysomes synthesizing particular proteins by precipitation with specific antiserum-(Kaneyama et al., 1960; Cowie et al., 1961; Warren and Goldthwaite, 1962; Duerre, 1964). Some workers reported binding of antiserum to polysomes without precipitation (Warren and Peters, 1965; V053 and Bauer, 1966). Indirect precipitation of 1 polysomes was also reported, using antiserum to antibodies (anti- gamma globulin) (Delovitch et al., 1972, 1973a, 1973b). An established method for the isolation of specific proteins involves interaction with other proteins bound to an insoluble matrix (affinity chromatography). In 1971, Miller, Cuatrecasas and Thompson reported a partial purification of ribosomes synthe- sizing tyrosine amino transferase using affinity chromatography. This method relied upon the affinity of the enzyme for its substrate or substrate analog. This study reports attempts to isolate heavy and light chain synthesizing polysomes from myeloma cells (MOPC-Zl). Myeloma protein, bovine serum albumin (BSA), rabbit anti-myeloma protein and rabbit anti-BSA were coupled to Sepharose via cyanogen bromide activation. The specificity of these immunoadsorbents was demonstrated in binding studies utilizing 14C—labeled proteins. Polysomes were isolated from MOPC-21 cells and analyzed on sucrose gradients. Binding and precipitation studies were performed using l4C-labeled rabbit anti- myeloma and unlabeled anti—myeloma, respectively. Several methods were tried to demonstrate binding of myeloma producing polysomes to the Sepharose—protein preparations. REVIEW OF THE LITERATURE Description of Polyribosomes The function of ribosomal aggregates in protein synthesis was described simultaneously by two groups of workers. Wettstein, Staehelin and Noll (1963) and Staehelin et al. (1963) called this functional unit an "ergosome" and postulated that it consisted of five or more 73 S ribosomal particles attached to a strand of messenger RNA. Warner et al. (1962, 1963) described essentially the same thing, calling them polyribosomes (or polysomes). Shortly thereafter, other groups described polyribosomes in bacteria and plants, as well as in animal cells (Howell et al., 1964; Gierer, 1963; Clark et al., 1963). Evidence that the size of the polyribosome corresponded to the size of the peptide synthesized provided one method for partial purification of specific polysomes (Lazerides and Lukens, 1971). Heywood et al. (1967) reported the identification of myosin synthe- sizing polysomes. They found that myosin was synthesized on poly- somes containing 50 to 60 ribosomes. Shapiro et al. (1966) reported on gamma globulin synthesis in mouse plasma cell tumors. This system had the advantage that 20-30% of the protein synthesized by the cell was gamma globulin. They reported light chain synthesis by 4 polysomes of about 190 S and heavy chain synthesis by polysomes of approximately 270 S. Binding_of Specific Antibody to Polyribosomes The discovery of growing nascent peptide chains on polyribo- somes offered another possibility for specific polysome isolation and purification. In 1965, Warren and Peters reported binding anti- body directed against rat serum albumin to rat liver polysomes. However, polysome precipitation did not occur. In 1966, V035 and Bauer reported the binding of antibody directed against the FC region of heavy chains to lymphocyte polysomes. More recently, Palacios et a1. (1972) reported binding of 125I-labeled anti- ovalbumin to polysomes and Taylor and Schimke (1974) reported bind- ing of labeled anti-albumin to rat liver polysomes, both without precipitation. Precipitation of Polyribosomes with Specific Antibodies Direct Precipitation In 1967, Williamson and Askonas reported the isolation of poly- somes synthesizing immunoglobulin light and heavy chains by precipitation with specific antiserum. They found that while they could precipitate polysomes in the 300 S region with antibody to the Fc portion of heavy chain, precipitation of polysomes with anti- 1ight chain antiserum was more difficult to demonstrate. They suggested that this might be due to attachment of light chain to nascent heavy chain, or simply due to the small size of the nascent 5 light chain. Scherr and Uhr (1969) also reported direct precipi- tation of immunoglobulin synthesizing polysomes. Takagi and Ogata (1971) reported direct precipitation of serum albumin synthesizing polysomes from rat liver. Precipitation of polysomes with specific antisera was also reported in other systems: B—galactosidase (Kaneyama and Novelli, 1960; Cowie et al., 1961); triose phosphate dehydrogenase (Warren and Goldthwaite, 1962); glutamic dehydrogenase (Duerre, 1964); and ovalbumin (Palmiter et al., 1972; Palacios and Schimke, 1973). In 1972, Delovitch et al. reported the isolation of light chain synthesizing polysomes by direct precipitation. In a series of papers (Delovitch et al., 1972, 1973a, 1973b) this technique was used to obtain specific messenger RNA. It is inter- esting to note that Delovitch found that the use of the F(ab')2 portions of the antibody significantly reduced nonspecific binding and precipitation, suggesting a nonspecific affinity of the FC region for the polysomes. This problem has not been mentioned in recent reports by other workers. Sarkar and Moscona (1973) reported direct precipitation of glutamine synthetase synthesizing polysomes with no difference in results using pepsin treated antibody (F(ab')2) and whole antibody. It should be pointed out, however, that non- specific precipitation was high in both cases. Indirect Precipitation Delovitch et a1. (1972, 1973b) also used a double antibody technique (indirect precipitation) for isolation of polysomes. In this method, polysomes are first incubated with specific antibody 6 followed by anti-gamma globulin. This has the advantage that the "secondary antigen" (the specific anti-protein) serves as a larger collection of antigenic determinants than the nascent peptide, thus facilitating cross-linkage and precipitation by the anti-antibody. Schechter (1973, 1974a, 1974b) has used this indirect procedure for precipitation of kappa (k) light chain synthesizing polysomes from myeloma cells. He has reported that the technique can be used to process large amounts of polysomes (up to 25,000 A units per 260 batch). Indirect immune precipitation of polysomes synthesizing ovalbumin (Shapiro et al., 1974; Shapiro and Schimke, 1974), catalase (Uenoyama and Ono, 1972), and rat serum albumin (Shapiro et al., 1974) have also been reported. Isolation of Polyribosomes by Affinitprhromatography The use of affinity chromatography for the purification of pro- teins is a standard and widely utilized technique (Cuatrecasas, 1970; Cuatrecasas and Anfinsen, 1971; Turkova, 1974). Specific antibodies can be purified by binding to antigen attached to an insoluble matrix. Antigen can be isolated by binding to an insolu- bilized antibody. The affinity does not have to be an antigen- antibody interaction. Any protein-protein interaction such as enzyme-substrate may be utilized in affinity chromatography. In 1971, Miller, Cuatrecasas and Thompson reported the isolation of tyrosine amino transferase polysomes by affinity chromatography. A substrate analog was attached to Sepharose. When the polysome solution was passed over the material, the nascent peptides of the enzyme synthesizing polysomes were bound to the substrate. After 7 washing the columns, the specifically bound polysomes were eluted. An enrichment for tyrosine amino transferase synthesizing polysomes was reported. Le Goffic, Baca and Moreau (1974) recently reported isolating E. coli ribosomes by affinity chromatography. The intended use of the columns (coupled with gentamicin or streptomycin) was to isolate intracellular enzymes which inactivate these antibiotics. It was noted, however, that ribosomes were also bound by the columns. It should be pointed out that this binding occurs via the ribosomal subunits and not the nascent peptides of the polysomes. Thus, this technique did not isolate specific ribosomes, but merely separated ribosomes in general from the remainder of the intracellular material. Palacios et al. (1973b) have reported isolating ovalbumin synthesizing polysomes by first incubating the polysome preparation with specific antibodies in solution followed by incubation with an ovalbumin matrix formed by treating the protein with glutaraldehyde. The antibodies form cross-links between the polysomes and the insoluble matrix. The entire procedure was done batchwise, with no column chromatography. Palacios et al. (1973b) also reported that a matrix of anti-gamma~globulin can be used to bind the polysome- antibody complex. However, attempts to bind polysomes directly with a matrix of specific antibody were unsuccessful. Sidorova et al. (1974) have reported recently the isolation of rat liver albumin and mouse gamma globulin polysomes through the use of affinity chromatography. The specific protein (albumin or 8 gamma globulin) was attached to aminocellulose. The matrix was saturated with specific antibody and then incubated with the poly- somes. Specific polysomes were bound to the matrix, forming a "sandwich" of polysome-antibody-protein-aminocellulose. MATERIALS AND METHODS 9112 The cells used in this study were mouse plasmacytes obtained from myeloma tumors. The MOPC-Zl line of cells (P3.6.2.8.l) is an IgGl producing line with light chains of the k class. These cells secrete whole immunoglobulin and, although they produce excess light chains intracellularly, they secrete no free light chain (Baumal and Scharff, 1973) . Also used in this study were the XC.l cell line and the $49.1 cell line. The XC.1 line is a synthesis variant of another myeloma line, Cl. XC.1 cells neither secrete nor contain intracellular immunoglobulin. The line was derived from an IgG2 producer. The $49.1 line is a lymphoma cell line. The cells are thymus-derived (T cells) which do not produce detectable amounts of gamma globulin. The MOPC-21 cell line was maintained initially as solid tumors in female BALB/c mice. The tumors were a kind gift of the Cell Distribution Center, Salk Institute. Solid tumors were carried subcutaneously and transferred every 14 days into tumor—free mice. Later, the tumor was carried in an ascites form. The ascites tumor was started by injecting a cell suspension from solid tumors intra- peritoneally into female BALB/c mice. Cells were withdrawn from 10 the peritoneum and reinjected into new mice at approximately 7- to 10-day intervals. The 849.1 and XC.1 cell lines, as well as the MOPC-21 line, were maintained in vitro in tissue culture. These tissue culture cell lines were also provided by the Salk Institute. Cells were maintained in Dulbecco's Modified Eagle Medium (GIBCO) supplemented with antibiotics (75 ug/ml streptomycin, 100 units/ml penicillin, 40 units/ml mycostatin) and 10% serum. Initially, gamma globulin free horse serum was used, but this was later replaced with fetal calf serum. Tissue cultures were maintained in plastic flasks and roller bottles. Flasks were incubated in a C0 incubator at 37°C 2 with a humid atmosphere of 85% air, 15% C0 Roller bottles were 2. flushed with 95% air, 5% C02, sealed and incubated at 37°C at a rotation speed of one-half revolution per minute. Cells were routinely grown to a density of l x 106 cells per ml in both flasks and roller bottles and diluted 1:10 with fresh medium. The genera- tion time at maximum cell growth was about 16-18 hours. Protein Preparation Myeloma Protein Mice bearing subcutaneous tumors were bled through the tail vein daily, for several days before they were sacrificed. The sera were pooled and the gamma globulin fraction prepared by ammonium sulfate precipitation. Ice cold saturated ammonium sulfate was added to the serum slowly with continuous stirring, to a final concentration of 40% (v/v). This solution was centrifuged at 10,000 11 rpm (12,000 x g max.) in a Sorvall RC2—B centrifuge for 15 minutes at 4°C. The pellet was resuspended in distilled water (usually about half the original volume). The entire precipitation procedure was then repeated. The final pellet was resuspended in distilled water and dialyzed overnight at 4°C against buffer (10 mM Tris, pH 7.4, 150 mM NaCl). Protein concentration was determined by spectral absorbance at 280 nm, using an extinction coefficient of 13.6 for gamma globulin (Small and Lamm, 1966). Myeloma protein was also obtained from cells grown in tissue culture. MOPC-21 cells were labeled for 24—36 hours with 1 uCi/ml 3H-proline. After the cells had been pelleted by centrifugation, the supernatant fluid was precipitated with ammonium sulfate as above, with the exception that the first precipitation was 50% saturated ammonium sulfate instead of 40%. Protein concentration was determined as above. Rabbit Antiserum New Zealand White rabbits were immunized with myeloma protein or bovine serum albumin (BSA, Cohn Fraction V). Protein at a con- centration of approximately 5 mg/ml was added to an equal volume of Freund's Complete Adjuvant and mixed until an emulsion was formed. Rabbits were injected subcutaneously with 2 ml of the emulsion, fol- lowed 7 days later by a second injection. The rabbits were bled through the marginal ear vein 7-10 days following secondary injec- tions. The animals were periodically given booster injections and bled 7-10 days hence. Normally, about 30 to 50 m1 of whole blood was obtained from one rabbit in a single bleeding. A single bleeding 12 was also taken from each rabbit prior to the first antigen injection (normal serum). Serum was stored at —20°C. The gamma globulin fraction was obtained in the same manner as for myeloma protein. Anti-Globulin Goat anti-rabbit gamma globulin (GARGG) was the kind gift of Dr. L. F. Velicer and Anthony Conley, Department of Microbiology and Public Health, Michigan State University. GARGG was also pur- chased from GIBCO. The lyophilized serum preparation was reconsti- tuted in 5 ml distilled water according to the company's specifications. The gamma globulin fraction was obtained by ammonium sulfate precipitation. Bovine Serum Albumin BSA (Cohn Fraction V) was purchased from Sigma. The protein was used without further purification. 14C-Labeling of Proteins Proteins were labeled in vitro with l4C-formaldehyde by the procedure of Rice and Means (1971). l4C-formaldehyde was purchased from New England Nuclear in ampules containing 50 uCi of isotope in approximately 3 pl water at a specific activity of 55 mCi per millimole formaldehyde. The complete procedure was done at 0°C. One-tenth milliliter sodium borate buffer (50 mM, pH 9.0) was added to the ampule contain- ing the l4C-formaldehyde. The protein was then added in a volume of 20 ul, the total amount of protein being about 300 ug. This was followed 30 seconds later by 4 sequential 2 ul additions of sodium 13 borohydride at 5 mg/ml. One minute after the final 2 ul addition, an additional 10 ul sodium borohydride was added. The solution was then brought to 1.0 ml with the sodium borate buffer and dialyzed overnight against 10 mM Tris (pH 8.0). Pepsin Digestion of Antibody Peptic digestion of antisera was performed essentially as described by Utsumi and Karush (1965). Antisera were dialyzed over— night against 0.2 M acetate buffer (pH 4.0). The final concentration after dialysis was between 15 and 35 mg/ml and the pH was usually between 4.0 and 4.5. The pH was adjusted to 3.5-4.0 with 2 N HCl. To 1.0 m1 of the protein solution was added crystalline pepsin to a final concentration of 2.0-2.5% (w/w). This solution was then incu- bated at 37°C for 20 hours. At the end of this time, samples were placed on ice and the pH was adjusted to 8.0—8.5 with 1.0 N NaOH. The solutions were then dialyzed overnight against buffer (usually 10 mM sodium phosphate, pH 8.0). The protein was analyzed by sucrose gradient centrifugation and sodium dodecyl sulfate (SDS) gel electrophoresis. The gradients were 5-20% sucrose (w/v) in 50 mM potassium phosphate buffer. Samples were centrifuged at 40,000 rpm (286,000 x g max.) in a Beckman SW41 rotor for 35 hours at 4°C. Gradients were collected on an ISCO density gradient fractionator, with continuous monitoring of absorbance at 280 nm and recorded on a Gilford model 24008 recorder. SDS gel electrophoresis was done by the method of Fairbanks et a1. (1971). Gels were 9% acrylamide and 1% SDS. Sample volumes were 50 ul or less. Gels were electrophoresed at a constant voltage 14 of 8.2 volts per cm gel length for 2-3 hours, until the dye marker (Pyronin-Y) reached the bottom of the gels. The gels were scanned- at 280 nm on a Gilford model 2400 recording spectrOphotometer. After staining with Coomassie Blue, the gels were scanned at 540 nm. Reduction and Alkylation of Antibody Antisera were reduced and alkylated by a modification of the procedure of Nies et al. (1973). Protein solution at a concentration of 15-35 mg/ml was added to solution A (1.0 M Tris, pH 8.1, 0.2 M 2-mercaptoethanol, 2% (w/v) SDS) at a ratio of one part protein solution to four parts solution A. The sample was then placed in a boiling water bath for 5 minutes. An equal volume of solution B (1.0 M Tris, pH 8.1, 2% SDS, 0.4 M iodoacetamide) was then added and the sample incubated at room temperature for 5 minutes. The protein was precipitated by adding 5 volumes of absolute ethanol, followed in 5 minutes by centrifugation at 10,000 rpm for 5 minutes. Pellets were redissolved by adding solution C (10 mM Tris, pH 8.1, 1 mM ethylenediaminetetra—acetate (EDTA), 1% SDS, 1% (v/v) 2- mercaptoethanol) and submerging in a boiling water bath for 3 minutes. The protein was analyzed on SDS gels as described above. Heavy and light chains were separated on 5-20% (w/v) linear SDS sucrose gradients in 50 mM sodium phosphate buffer (pH 8.0). Samples were centrifuged at 40,000 rpm in a Beckman SW 41 rotor for 72 hours at 4°C. 15 Affinity Chromatography Coupling of Protein to Sepharose Protein was coupled to Sepharose by the method of Cuatrecasas (1970). Sepharose 6B was obtained from Sigma. Sepharose 4B and 2B were obtained from Pharmacia. Cyanogen bromide was purchased from Aldrich. Thirty milliliters Sepharose was washed with approxi- mately 500 ml distilled water, then resuspended in 30 ml distilled water. Six to nine grams of cyanogen bromide finely ground by mortar and pestle were added at once with continuous stirring. The pH was adjusted to 11.0 with 5 N sodium hydroxide and maintained by subsequent additions. Temperature was maintained at 20-22°C by the addition of ice. This was continued until the pH stabilized (about 20 minutes, depending upon how rapidly the cyanogen bromide dissolved). A large amount of ice was then added, the mixture was vacuum filtered and washed with one liter of ice cold sodium bicarbonate buffer (0.1 M, pH 7.0). The activated Sepharose was then resuspended in 30 ml of the bicarbonate buffer, protein added (30-45 mg) and the pH adjusted to 6.5—7.0 with 2 N HCl. The mixture was allowed to incubate overnight at 4°C with stirring. The coupled Sepharose was vacuum filtered and washed with one liter of buffer (usually 10 mM sodium or potassium phosphate, pH 8.0) and resuspended in 30-50 ml of the same buffer plus .02% (w/v) sodium azide. The first 100 m1 of the wash buffer was monitored for protein concentration in order to estimate the amount of protein coupled to the Sepharose. The degree of coupling was 750-1000 ug protein per ml Sepharose. 16 Affinity Chromatography Columns Unless otherwise noted, all columns were poured in disposable pasteur pipettes to a final packed volume of 0.2-0.3 m1 Sepharose. Columns were washed with 1-2 ml 10% normal rabbit serum or horse serum to saturate nonspecific binding sites. They were then washed with 5-8 ml buffer. (Specific buffers are described in Results.) Protein was applied to columns in volumes no larger than 0.1 ml and incubated at room temperature for approximately 5 minutes, unless noted otherwise. This was followed by washing with 5-10 ml buffer, collecting fractions and precipitating with 10% trichloro- acetic acid (TCA). Precipitated fractions were collected by vacuum filtration on Whatman GF/C glass fiber filters (2.4 cm) and counted in 10 ml toluene-Omnifluor (New England Nuclear) scintillation fluid. In some cases, columns were washed with elution buffers to remove specifically bound proteins. Fractions were collected and precipitated in the same manner as above. Isolation of the Total Polysome Pppulation from Cells Separation of Membrane-Bound and Free Polysomes Cells were grown in roller bottles to a density of 5-8 x 105 cells/m1. If labeled polysomes were desired, 3H—uridine was added at a concentration of 1 uCi/ml during the last 2-3 hours of incubation. All isolation procedures were performed on ice or at 4°C unless noted otherwise. Cells were washed once in normal saline and once in RSB (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgC12) and then resus- pended in an equal volume of RSB (or to a total volume of 1.0 ml, l7 whichever was greater). Following incubation on ice for 10 minutes, the cells were lysed in a glass Dounce homogenizer and the nuclei were pelleted at 2000 rpm (900 x 9 max.) in an International refrigerated centrifuge. The supernatant fluid was removed and centrifuged at 16,000 rpm (30,000 x g max.) for 10 minutes in a Sorvall RC2-B centrifuge. The supernatant fluid contained free polysomes. The pellet was resuspended in TKM buffer (50 mM Tris, pH 7.6, 25 mM KCl, 5 mM MgC12) (Miller et al., 1971) and treated with sodium desoxycholate (DOC) and Triton X—100, final concentra- tions 0.5% (w/v) and 0.5% (v/v), respectively. This solution was then centrifuged at 30,000 x g max. for 10 minutes. The supernatant contained membrane-bound polysomes. Total Polysome Population Cells were poured over frozen crushed saline to facilitate rapid cooling. This procedure was first employed late in the study and seemed to yield a better population of polysomes. Pelleted cells were washed once in saline, resuspended in RSB and incubated on ice for 5 minutes. The cells were lysed by adding non-ionic detergent Nonidet P-40 (NP-40, Shell) to the suspension to a final concentration of 0.5% (v/v). Nuclei were pelleted and the super— natant treated with DOC—Triton X-100 as above. This solution con- tained both free and membrane—bound polysomes and is referred to as crude polysomes. In later experiments, the pelleted nuclei were washed in a small volume of RSB, recentrifuged and the supernatant fluid combined with the original supernatant before treatment with DOC-Triton X-100. 18 Purification of Total Polysomes Pelleting In this procedure, the crude polysome fraction was transferred to polyallomer centrifuge tubes, brought to tube volume with RSB and pelleted by centrifugation for 2 hours at 50,000 rpm (226,000 x 9 max.) in a Type SOTi rotor in the Beckman Model L5-50 ultra- centrifuge. The pellet was washed once with saline and either frozen at -80°C or resuspended in buffer. Discontinuous Sucrose Gradients Another procedure used was the method of Schimke et al. (1974). Crude polysomes brought to 6 ml with polysome buffer (25 mM Tris, pH 7.6, 25 mM NaCl, 5 mM MgC12) were layered over a dis- continuous sucrose gradient consisting of 4.0 ml 1.0 M sucrose over 2.0 ml 2.5 M sucrose, both in polysome buffer. This was then centrifuged at 41,000 rpm for 1.5 hours in a Beckman SW 41Ti rotor. The polysomes banded at the interphase between the 2.5 M sucrose and the 1.0 M sucrose. They were removed by puncturing the side of the tube about 3 mm below the interphase with an 18-gauge needle and withdrawing 0.8 to 1.0 ml in a 1 ml syringe. The sample was then dialyzed overnight at 0°C against polysome buffer to remove the sucrose. Sarkar and Moscona (1973) modified Schimke's technique for the SW 50.1 rotor which has smaller capacity tubes (5.5 ml). This technique uses 1.0 ml 2.5 M sucrose, 2.0 ml 1.0 M sucrose and 2.0 ml polysome preparation, centrifuging for 2 hours at 46,000 rpm 19 (250,000x 9 max.). We modified Sarkar's technique as follows. The 2.5 M sucrose pad was omitted, allowing the polysomes to pellet. The reasons for this were twofold. First, resuspending the pellet avoided the long dialysis with its subsequent loss of polysomes and, second, it was hoped that by pelleting through sucrose the polysomes would not be packed as firmly in the pellet, thus permit- ting easier resuspension. In another modification, Sarkar's technique was followed as reported except that dialysis was replaced by passage over a Sephadex G-25 column. The column was 3 ml packed volume of Sephadex in a 5 ml glass syringe. Approxi- mately 0.7 m1 of the polysome-sucrose solution was loaded onto the columns. The polysomes were eluted with polysome buffer in the void volume while the sucrose was retarded by the Sephadex. Sepharose Columns See Results, Section I. Analysis of Polysomes Polysomes were analyzed on 15-45% (w/v) linear sucrose gradients in either the SW 50.1 rotor (5.2 m1 sucrose per gradient) or the SW 41Ti rotor (12.4 ml sucrose per gradient). The former were centrifuged at 45,000 rpm (250,000 x 9 max.) for 35 minutes, while the latter were centrifuged at 38,000 rpm (246,000 x 9 max.) for 75 minutes. Gradients were collected from the top with continuous monitoring of absorbance at 254 nm. 20 Isolation of Specific Polysomes Specific Binding of Labeled Antiserum 1 Binding of 4C-labeled antibody to purified polysomes was done as described by Taylor and Schimke (1974). Varying amounts of polysomes (2-25 A2 units) were incubated with 10-15 pg 60 labeled antibody for 1 hour at 0°C. These were then layered onto sucrose gradients and centrifuged as described in Analysis of Polysomes. In competition studies, polysomes were incubated with 500 ug unlabeled antibody for 30 minutes followed by 10-15 ug labeled antibody for 30 minutes. Direct Precipitation of Polysomes This was done essentially as described by Delovitch et al. (1972, 1973b). Polysome fractions from sucrose gradients were incubated with antiserum (gamma globulin fraction) for 5 minutes at 37°C. Specific antigen was added, followed by incubation at 37°C for 5 minutes and 4°C for 2-4 hours (or overnight). Tubes were centrifuged for 15 minutes at 1000 x 9 max., the pellets washed once with polysome buffer, and centrifuged again. The pellets were dissolved in 1 N NaOH. All samples were dissolved in Bray's scintillation fluid (Bray, 1960) and counted in a Packard Tri-Carb liquid scintillation counter. Direct and indirect precipitation was also done on purified total polysomes. The procedure was modified from Shapiro et al. (1974). Polysomes were incubated with whole antibodies or pepsin treated antibodies for 45 minutes at 0°C. Then either specific 21 antigen or GARGG was added. This mixture was then incubated an additional 60 minutes at 0°C. Pellets were washed as above and dissolved with 5% SDS. All fractions were precipitated with 10% TCA, collected on Whatman GF/C filters and counted in toluene- Omnifluor. Volumes and concentrations of reagents used in specific experiments are reported in Results. Affinipy Chromatography of Polysomes The technique of affinity chromatography of polysomes was modified from the method reported by Palacios et al. (1973b) and Schimke et al. (1974). Briefly, it involves specific antigens or anti-antibodies bound to Sepharose, cross—linked to polysomes which have specific antibodies bound to them. Purified total polysomes were incubated with rabbit antibodies (specific or control) for 45 minutes at 0°C. Then Sepharose coupled with protein (specific or control antigens, GARGG) was added and the mixture incubated an additional 60 minutes at 0°C. The mixtures were then transferred to pasteur pipettes at 0°C and washed with column buffer (polysome buffer plus 0.5 M sucrose, 1% DOC, 1% Triton X-100, 100 ug/ml heparin) (Schimke et al., 1974). Columns were then washed with polysome buffer plus heparin, followed by elution buffer (10 mM Tris, pH 7.5, 50 mM EDTA). The EDTA causes dissociation of polysomes with subsequent release of ribosomal subunits and messenger RNA. Details of specific experiments are reported in Results. RESULTS Section I Article Polysome Isolation by Sepharose Column Chromatography submitted for publication to Biochimica et Biophysica Acta 22 POLYSOME ISOLATION BY SEPHAROSE COLUMN CHROMATOGRAPHY William H. Eschenfeldt and Ronald J. Patterson Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 (U.S.A.) 23 Running Title: POLYSOME ISOLATION 24 25 SUMMARY Polysomes from the mouse myeloma MOPC-21 were purified by gel filtration on Sepharose 6B, 4B and 2B columns. All three columns eliminated nearly all intracellular material smaller than 40 S subunits. In addition, passage through 4B and 2B columns substantially reduced the amount of subunits and monosomes in the preparations. Purified polysomes retained structural integrity when stored at -85°C for at least 9 weeks. INTRODUCTION In studies involving polysomes, it is often necessary that the polysomes be free from contaminating intracellular material. A common method for achieving this is by pelleting the polysomes by ultracentrifugation. This procedure requires several hours of centrifugation, and resuspension of the polysome pellets is often difficult due to their aggregation and degradation. A procedure using discontinuous sucrose gradients has been reported [1] in which the polysomes band at or just below the interphase between 1.0 and 2.5 M sucrose. This method requires dialysis of the polysomes to remove the sucrose. Recently, Palmiter [2] has reported the use of magnesium precipitation for the isolation of polysomes. Sephadex <3-100 columns have been used to isolate ribosomal subunits [3], and 'the purification of polysomes on hydroxyapatite has been reported [4,5]. Tangen et al. [6] have reported the use of Sepharose gel fjnltration for the isolation of microsomes, and Darnbrough et al. [7:] have reported purification of polysomes using Sepharose column chznamatography. We have extended the original observations of 26 Darnbrough et al. [7] and have rapidly purified polysomes on columns of Sepharose 6B, 4B and 2B. MATERIALS AND METHODS Preparation of Polysomes: The mouse myeloma line MOPC-Zl (kindly provided by The Cell Distribution Center, Salk Institute) was carried as an ascites tumor in female BALB/c mice. For polysome isolation, the mice were sacrificed by cervical dislocation and ascites fluid was withdrawn with a sterile syringe. Cells were pelleted at 500 x g for 10 minutes, then resuspended to a density of 5-6 x 106 cells/ml in Dulbecco's Modified Eagle Medium (GIBCO) supplemented with 10% fetal calf serum (Flow Laboratories). After 30—60 minutes incubation at 37°C in an atmosphere of 95% air, 5% CO2 [5,6-3H]uridine (New England Nuclear, specific activity 45 Ci/ mmole) was added to a concentration of 1—2 uCi/ml. Incubation was continued for 2-3 hours. The cells were then rapidly cooled by pouring over crushed frozen saline. All subsequent procedures were performed at 0-4°C. The cells were pelleted by centrifugation for 10 minutes at 500 x g, washed twice with RSB (10 mM Tris, pH 7.4, 10 mM NaCl 3 mM MgClZ), resuspended in RSB and lysed by the addi— tion of one-tenth volume of 5% (v/v) Nonidet P—40 (Shell). After 5 minutes, nuclei were removed by centrifugation for 5 minutes at 900 x g and the supernatant fluid was treated with one-tenth volume of a solution of 5% (w/v) sodium desoxycholate (Nutritional Biochemicals), 5% (v/v) Triton X-100 (Packard). This preparation is referred to as crude polysomes. 27 Sepharose Chromatography: A11 columns were poured and maintained at 4°C. Columns (1.5 x 15 cm) of Sepharose 6B, 4B and 2B (Pharmacia) were poured and washed with at least ten bed volumes of polysome buffer plus heparin [8] [25 mM Tris, pH 7.6, 25 mM NaCl, 5 mM MgCl 100 ug/ml sodium heparin (Sigma)]. Crude polysomes were 2: applied to the columns in volumes up to 2.5 ml and eluted at a flow rate of approximately 10 ml/hr with polysome buffer. Poly- somes were eluted in the void volume and were visible as cloudy white fractions. Following thorough washing with polysome buffer, columns could be reused. One of our columns has been in use for over three months. The columns were periodically treated with a solution of 0.1% diethylpyrocarbonate (Calbiochem) in polysome buffer. Sucrose Gradients: Polysomes were analyzed on 15-45% (w/v) linear sucrose gradients in polysome buffer plus heparin. The gradients were centrifuged at 250,000 x g for 40 minutes at 4°C in a Beckman SW 50.1 rotor in the Beckman Model L5-50 ultracentrifuge. Frac- tions of 0.2 ml were collected from the top using an ISCO Model 640 density gradient fractionator. Absorbance at 254 nm was moni- tored continuously with the ISCO Model UA-4 absorbance monitor and recorded on a Gilford Model 2400-S recorder. Fractions were collected directly into scintillation vials and counted in 5 m1 of Bray's scintillation fluid [9] at 4°C. RESULTS Aliquots from a single preparation of crude MOPC-21 polysomes were purified on Sepharose 2B, 4B and 6B columns. The absorbance 28 and radioactivity profiles from sucrose gradients are shown in Figure l. A large peak of material smaller than 40 S subunits is evident in the crude polysomes which is absent in the purified polysomes. Sepharose 6B, with an exclusion limit of approximately 4 x 106 Daltons for globular proteins, excludes polysomes and subunits, while retarding the remainder of the intracellular material. Sepharose 4B and 2B, with approximate exclusion limits for globular proteins of 20 x 106 Daltons and 40 x 106 Daltons, respectively, retard subunits and monosomes. Sepharose 2B also retards the smaller polysomes. Thus the void volume is enriched for the larger polysomes. Table 1 compares the percentage of radioactivity in the poly- some region and the nonpolysome region of the gradients. The poly- some region is defined as that region larger than monosomes. The percentage of nonpolysomal radioactivity in the crude fraction is high due to the presence of unincorporated [3H]uridine. The data from both 4B and 2B Sepharose fractions show a substantial increase in the percentage of polysomal radioactivity over that in the 6B fraction. Purified polysomes from the columns were stored frozen without further preparation. Figure 2 shows the absorbance profiles of MOPC-21 polysomes purified over a 6B column. Figure 2A is a sucrose gradient profile of the polysomes analyzed on the day of isolation. Figure 2B is a profile of the same preparation after storage at -85°C for 9 weeks. Slight degradation is evident. The 40 S and 60 S subunit peaks have increased with corresponding decrease in the 80 S monosome peak. The percentage of subunits and monosomes has 29 increased from 26.6% on the day of isolation to 32.9% after 9 weeks storage. DISCUSSION Passage of crude polysome preparations over any of the Sepharose columns substantially reduces the contamination by intracellular material smaller than the 40 S subunits. Columns of Sepharose 4B and 2B enrich for the polysomal fraction, retarding subunits and monosomes, while Sepharose 2B also retards the smaller polysomes, yielding a population enriched for the largest polysomes. Furthermore, the polysomes remained structurally intact. The gel filtration technique yields polysomes that are diluted during puri- fication. Crude preparations applied to the Sepharose 6B column at about 100 A units/ml are eluted as purified polysomes at about 260 25-30 A260 units/m1. For our purposes this dilution of purified polysomes is acceptable. Preliminary results indicate that the polysomes are active in an endogenous cell-free protein synthesizing system. We have also used the columns to purify polysomes from the post—nuclear supernatant from cells which have been lysed without the use of detergents. Under these conditions, the eluted fraction contains intact microsomes, as well as free polysomes permitting subsequent separation of membrane-bound and free polysomes. This procedure offers a relatively simple and rapid technique for the purification of total polysomes. Fractions from the columns need not be monitored continuously, as the fractions containing polysomes are readily visible. Also, stability of the purified 3O polysomes when stored at —85°C is excellent, probably due to the reduction of intracellular nucleases achieved through the gel filtration procedure. ACKNOWLEDGMENTS We wish to thank William Chaney for helpful discussions during the initial stages of this study. This investigation was supported by USPHS grant AI-11493. This is journal article no. 7073 from the Michigan Agricultural Experiment Station. REFERENCES Palacios, R., Palmiter, R. D., and Schimke, R. T. (1972) J. Biol. Chem. 242, 2316-2321 Palmiter, R. D. (1974) Biochemistry 13, 3606-3615 Brown, G. E., Kolb, A. J., and Stanley, W. M., Jr. (1974) in Methods in Enzymology (Moldave, K., and Grossman, L., eds), Vol. 30, Nucleic Acids and Protein Synthesis, Part F, pp. 368-387, Academic Press, New York Hoffman, W. L., and Ilan, J. (1974) Biochim. Biophys. Acta 366, 199-214 Hoffman, W. L., and Ilan, J. (1974) Preparative Biochem. 4, 367-380 Tangen, O., Jonsson, J., and Orrenius, S. (1973) Analyt. Biochem. 54! 597-603 Darnbrough, C., Legon, 8., Hunt, T., and Jackson, R. J. (1973) J. Mol. Biol. 1g, 379-403 31 Schimke, R. T., Palacios, R., Sullivan, D., Kiely, M. L.,. Gonzales, C., and Taylor, J. M. (1974) in Methods in Enzymology (Moldave, K., and Grossman, L., eds), Vol. 30, Nucleic Acids and Protein Synthesis, Part F, pp. 631-648, Academic Press, New York Bray, G. A. (1960) Analyt. Biochem. 11 279-285 .mcflcflwommma pmumuomuoochs ou moo ma coflmmu HmEOmwaomlcoc .mpsno on» CH oomucoo tumm 30H: >Hm>ammwoxo map was» .mufl>fluum0Homu mandaomcfl one mandaom swan mosaocfl mucmsouammmz 32 n -.wcfloflusmm L cuflz mcflamnma capes cw musoc o.m woumm maaoo mmufiomm Hmnomoz Eoum cmumHomfl moEommHomm m o.am ooo.moa o.m oon.oa H5.H mm v.6m ooa.m~a o.ma 00m.mm mn.a mo o.mm oom.mva v.0v oo~.>m H5.H mm 0.0m oom.vma 0.0m oov.nmm mo.a guano ommucooumm mono HMEOmhaom wmmucmouom mono HQBOmMHomncoc omnd\oom< Mmmsomwaom on neuw>fiuomowomm an omen>fluomowomm moEOmmaom coAMHusm omoumnmom can mmEOmhaom mpsuo ca mufl>flu080flomu mo coaudnfluumwo .H magma 33 Figure l. Sucrose gradient profiles of crude and Sepharose purified polysomes prepared as in Materials and Methods. Arrow indicates monosomes (80 S). A254 ( ); [3H]uridine (O——-—O). A254 34 § 9 CRUDE 68 0.8 ‘ 4 0.6 ‘ 3 0.4 1 2 0.2 I] 0.6 0.4 0.2 T FRACTION Figure l 35 Figure 2. Sucrose gradient profiles of Sepharose 6B purified polysomes. The crude polysomes were prepared from MOPC-Zl tissue culture cells maintained in Vitro in Dulbecco's Modified Eagle Medium, supplemented with 10% fetal calf serum, in an atmosphere of 85% air, 15% C02. Cells were labeled with [3H]uridine and harvested at 5-8 x 105 cells/ml. The remainder of the isolation procedure was identical to that described in Materials and Methods. A. Analyzed on day of isolation. B. After storage at -85°C for 9 weeks. Arrow indicates monosomes (80 S). 36 N ousmflm 203.014”; 11 l a Nd v.0 6.0 $6 vszv RESULTS Section II Isolation of Free and Membrane-Bound Polysomes Tissue culture cells were lysed without the use of detergent and the postnuclear supernatant fraction was applied to a Sepharose 6B column. The purified polysomes were pooled and centrifuged at 30,000 x 9 max. for 5 minutes to fractionate free and membrane-bound polysomes. The pellet was resuspended in polysome buffer plus 0.5% (w/v) DOC, 0.5% (v/v) Triton X-100, which released the bound poly- somes from the membrane. Figure 3A is the sucrose gradient profile of the free polysomes; 3B is the profile of the membrane-bound polysomes. To test the effect of the 30,000 x g centrifugation step, crude polysomes prepared with the use of detergents to release all membrane- bound polysomes were centrifuged at 30,000 x g for 5 minutes. Sucrose gradient profiles of the supernatant fraction and the pellet are shown in Figure 4A and 4B, respectively. The absorbance profile in 4A decreases rapidly in the region of the larger polysomes, while the profile of the pelleted material shows a significant peak in the region of the largest polysomes. This latter peak is probably due to aggregation of some of the polysomes. It should be noted that in Figure 4B the absorbance profile falls below the baseline in the 37 38 Figure 3. Sucrose gradient profiles of free and membrane- bound polysomes. A. Free polysomes. B. Membrane-bound polysomes. A254 (, ). 39 m ouomfim ZO_._.OHom Hanna meOmhaom mEOmAHOQ CH Iwaom Ca Emu wEOm>H0mICOC ICOC CH Emu Emu quouwm CH Emu quonm mCECHoo mpHCHmmm um>o mommmmm mo mmsommaom Co uomumm .NH magma 74 separation on the basis of size. These results would indicate, then, that monosomes and subunits may bind nonspecifically to the immuno— adsorbent. Binding of antibody to the polysomes does not appear to have been affected by passage over the columns, although it is interesting to note that there appears to be antibody binding to the monosomes and subunits. In Figure 11B, with the reduced monosome-subunits peak, the antibody levels decreaseafour fractions earlier than in 11A. There is a small peak of antibody coinciding with the monosome—subunits peak. A sample of the same polysomes used in the previous experiment was treated with EDTA, final concentration 33 mM, for 10 minutes at 0°C. One-tenth milliliter of this solution was applied to a Sepharose-NRGG column (0.25 ml packed volume) and incubated for 30 minutes at 0°C. The column was then washed with 2 ml polysome buffer and the eluted radioactivity was determined by TCA precipi- tation. Of 101,700 cpm applied, 22,800 (22.5%) were eluted. Thus, 77.5% of the radioactivity was bound to the column. A second sample of the EDTA treated polysomes was incubated with 2.5 pg l4C-anti- MOPC for 1 hour at 0°C, then analyzed on a 10-30% (w/v) linear sucrose gradient (10 mM Tris, pH 7.4, 10 mM NaCl, 10 mM EDTA) by centrifu- gation at 50,000 rpm for 105 minutes at 4°C in an SW 50.1 rotor. The profile is shown in Figure 12. The EDTA treatment has reduced the polysomes to subunits. Thus, the sample which was passed over the column had been completely converted to 40 S and 60 S subunits. A significant amount of the labeled antibody has sedimented with the subunits. The two peaks of antibody fall just to the heavy side of 75 Figure 12. Binding of l4C-anti-MOPC to ribosomal subunits. EDTA treated MOPC-21 polysomes labeled with 3H-uridine (O——-O) were incubated with l C-anti-MOPC (O——-—O) and analyzed on 10-30% linear sucrose gradients. PERCENT CPM 76 121 ‘ 10w. i 603 408 - 81 6+ 1 4h. 2'1 1'0 2?) FRACTION Figure 12 77 the subunit peaks. The shoulders on the heavy sides of the subunit peaks may be due to cross—linking by the antibody. With the sub- units region of the gradient defined as fractions 6-28, 60.9% of the labeled antibody is in the subunits region. The above experiment was repeated, incubating the EDTA-treated' polysomes with 125 pg unlabeled anti-MOPC or unlabeled NRGG for 30 minutes. The samples were then incubated with 2.5 pg l4C-anti- MOPC for 30 minutes. A third sample was incubated for 1 hour with labeled anti-MOPC. All samples were analyzed on sucrose gradients as above. Figure 13A shows the profile of the sample which was not pre-incubated with competing protein. The antibody profile is similar to that seen in Figure 12. There are peaks of antibody coin- ciding with the subunits peaks, as well as a large shoulder of anti- body in the region larger than 60 S. The subunits region contains 54.7% of the antibody. Figure 13C is the profile of the sample pre-incubated with NRGG. The results are nearly identical to 13A. The subunits region contains 50.0% of the antibody. The profile of the sample pre-incubated with unlabeled anti-MOPC is shown in Figure 13B. Although there are antibody peaks coinciding with the subunits peaks, there is little antibody in the region larger than the 60 S subunits. Overall, only 25.4% of the antibody is in the subunits region. The addition of the unlabeled antibody to the sub- units caused visible precipitation. Only 50% of the 3H-uridine radioactivity of the subunits was recovered on the gradients. Pre- sumably, the other 50% was pelleted as a precipitate. Recovery of the l4C-antibody on the gradient was 100%. 78 l . 4C-anti-MOPC to ribosomal subunits: Figure 13. Binding of l4c-anti- specificity. A. EDTA treated polysomes incubated with MOPC. B. EDTA treated polysomes incubated with unlabeled anti- MOPC prior to incubation with l4C-anti-MOPC. c. EDTA treated polysomes incubated with unlabeled NRGG prior to incubation with 14c-anti-M0pc. 3H—uridine (0—0); l4c-anti-M0Pc (O——O). 79 ma musmflm ZO_._.O