. . -'lo~‘~ ‘ a uvfi‘flhmn-QNH°’.C -9 . s-ssmmzcfi 01: mag Mm gamma 9:91:49Pusmziasssms Ffimfi M3 fits-EAS- sJa'a- Pings“ he ¥?8§‘$§A 2}. ‘5’" " 5 an. - ~ 9?- 3‘5 ‘- mszs 103' $.23 333g?“ ca 2, 3 24* "w 4%; ?Q?P ",8: :4“ Wa #- .4 33:“; 7:533: ,4 AWL $22"; his 3 iii-Edd: 1978 :::: ‘q- a " H.155“! ABSTRACT SEPARATION OF FREE AND MEMBRANE-BOUND POLYRIBOSOMES FROM MINERAL OIL PLASMACYTOMA 21 BY Paul Jeffrey Freidlin Polysomes were obtained from the myeloma cell line MOPC-Zl which synthesizes and secretes an IgG-like molecule. Differential centrifugation of the total polysome population resulted in a supernatant which contained free polysomes and a pellet which con- tained membrane-bound polysomes. By this technique the membrane- bound polysomes were contaminated with free polysomes. Membrane— bound polysomes were separated from contaminating free polysomes by a combination of partition separation in a dextran-methylcellulose aqueous polymer two-phase system and differential centrifugation. The absorbance profile of the membrane-bound polysomes varied in a manner which appeared to be related to the physiological state of the cells. Free and membrane-associated polysomes were incubated in a cell-free system for protein synthesis. The polypeptide products were chromatographed on an anti-myeloma affinity column, but with the particular affinity chromatography system employed, the presence of newly synthesized myeloma polypeptide could not be unequivocally demonstrated. SEPARATION OF FREE AND MEMBRANE-BOUND POLYRIBOSOMES FROM MINERAL OIL PLASMACYTOMA 21 BY Paul Jeffrey Freidlin 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 1976 TO MY PARENTS, AND TO DEDICATED, RESPONSIBLE PEOPLE EVERYWHERE.. . ii ACKNOWLEDGEMENTS Throughout this study I was buoyed by the encouragement and aided by the patient counsel of Dr. Ronald J. Patterson. The Department of Microbiology and Public Health helpfully provided me with financial assistance. iii TABLE OF CONTENTS INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . Isolation and Description of Free and Membrane- Associated Polyribosomes . . . . . . . . . . . . . Different Modes of Attachment of Polyribosomes to Microsomal Membrane . . . . . . . . . . . . . . Segregation of Polyribosomes into Free and Membrane—Associated Fractions. . . . . . . . . . . i. Functional Differences . . . . . . . . ii. Affinity of Ribosomes for Membrane . . iii. Protein Composition of Subunits. . . . iv. Non-Ribosomal Proteins Associated with Ribosomes. . . . . . . . . . . . . . v. Relation Between Nascent Polypeptide and RER. . . . . . . . . . . . . . . vi. Kinetics and Composition of Free and Membrane-Associated RNA. . . . . . . vii. Proteins Bound to mRNA . . . . . . . . viii. RNA Other than mRNA and rRNA . . . . . ix. High Salt Wash "Factors" . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . RESULTS Cells. . . . . . . . . . . . . . . . . . . . . . . . Enzyme Assays. . . . . . . . . . . . . . . . . . . . Construction of the Aqueous Two-Phase Polymer System Isolation of Free and Membrane-Bound Polysomes . . . Mg++ Precipitation of Polysomes. . . . . . . . . . . System for Cell-Free Protein Synthesis . . . . . . . Affinity Chromatography Columns. . . . . . . . . . . Functional Assays. . . . . . . . . . . . . . . . . . Sucrose Gradient Analysis. . . . . . . . . . . . . . Cells. . . . . . . . . . . . . . . . . . . . . . . . Separation of Free and Membrane-Bound Polysomes by Differential Centrifugation. . . . . . . . . . . . Isolation of Membrane-Bound Polysomes by Partition Separation in an Aqueous Polymer Two-Phase System in Combination with Differential Centrifugation. . iv Page 12 12 13 14 16 16 17 18 19 20 22 22 23 25 28 32 32 33 34 35 36 36 38 52 Cell-Free System for Protein Synthesis . Assay for Myeloma Polypeptide Synthesized in the Cell-Free System for Protein Synthesis . DISCUSSION. . . . . SUMMARY . . . . . . LIST OF REFERENCES. O Page 66 68 72 9O 91 Table 10 ll 12 l3 14 LIST OF TABLES Generation times of MOPC-Zl tissue culture cells . . The effect of sample volume on the percentage of membrane—associated polysomes obtained by differential centrifugation at 27,000 x 9 (max) for 5 minutes . . The percentage of membrane-associated polysomes in various cell lines . . . . . . . . . . . . . . . . . Removal of microsomes and free polysomes by repeated differential centrifugation. . . . . . . . . . . . . The effect of RNase on the quantity of material pelleted by differential centrifugation. . . . . . . A test for unpelleted microsomal material by exten- Sive centrifugation. 0 O O O O O O O I O O O O O O O A test for unpelleted microsomal material by exten- sive centrifugation. . . . . . . . . . . . . . . . . The percentage of membrane material pelleted by repeated differential centrifugation . . . . . . . . The percentage of membrane-associated polysomes that will pellet after resuspension . . . . . . . . . . . Summary of enzyme marker data on polysome fractions. Differences between partitioned and crude membrane- associated material following treatment with RNase or high salt 0 I O 0 O O O O O O O O O O O O O O O 0 Separation of membrane-bound polysomes from added labeled free polysomes by a combination of differen— tial centrifugation and partition separation . . . . Affinity chromatography of polypeptides produced in a cell-free system for protein synthesis . . . . . . Generalizations about differential centrifugation. . vi Page 37 39 44 47 48 49 50 51 52 57 59 6O 71 74 Figure l 10 11 LIST OF FIGURES General schematic of the procedure used to separate membrane-bound from free polysomes . . . . . . . . . Variations in the percentage of membrane-associated polysomes isolated in the presence of RSB and RSB (suCh) O O O O O O O O I O O O O O I O O O O O O O Sucrose gradient profiles of membrane-associated polysomes pelleted by differential centrifugation at 27,000 x 9 (max) for 5 minutes (MP1). . . . . . . Sucrose gradient profiles of membrane-bound poly- somes partitioned in a dextran-methylcellulose aqueous polymer two-phase system . . . . . . . . . . Radioactivity profiles of the gradients shown in Figure 4 O O O O I O O O O O O O O O O O O O O O O 0 Separation of membrane-bound polysomes from added labeled free polysomes by a combination of differen- tial centrifugation and partition separation . . . . Sucrose gradient profiles of partitioned membrane- bound polysomes obtained from ascites cells freshly started in tissue culture. . . . . . . . . . . . . . Parameters of a cell-free system for protein syntheSiS O O O O O O O O O O O O O I O O l O O O O 0 Time needed to sediment from rmin to rmax by dif- ferential centrifugation at 27,000 x 9 (max) . . . . The minimum S value of particles pelleting from rmin to rmax in 5 minutes of centrifugation at 27,000 x 9 (max) . . . . . . . . . . . . . . . . . . Sucrose gradient profiles of MP1 . . . . . . . . . . vii Page 29 41 46 55 56 63 67 7O 76 78 82 INTRODUCTION Well defined free and membrane-associated fractions of poly- ribosomes (polysomes) are necessary for studies on the post- transcriptional control of immunoglobulin synthesis. The composition of these fractions may be examined to gain insight into the factors which cause some polysomes to be attached to membrane while others are not (for review see Rolleston, 1974). The fractions can also serve as a source of reactants for experiments that involve systems for cell-free protein synthesis (Uenoyama and Ono, 1972; Pryme, 1974; for review see Rolleston, 1974). It is generally accepted that membrane-associated polysomes in eukaryotic cells are responsible for the synthesis of proteins secreted from the cell and possibly for proteins incorporated into cell membrane, while free (unattached) polysomes produce intracellular proteins (for review see Rolleston, 1974). The myeloma cell line Mineral Oil Plasmacytoma 21 (MOPC-Zl) has been shown to secrete an immunoglobulin-like (IgG) gamma globulin and to produce excess light chains which are degraded intracellularly instead of being secreted (Baumal and Scharff, 1973). Cell lines which secrete immunoglobulin have been shown to contain more membrane-bound RNA than nonsecreting variants (Kimmel, 1969). Cioli and Lennox (1973) and Pryme (1973) reported that polysomes associated with membrane are perhaps the only polysomes involved in immunoglobulin biosynthesis. Thus a 1 reasonable approach to purifying myeloma messenger RNA would be to first isolate a well defined fraction of membrane-bound polysomes. The conventional technique used to separate membrane-bound from free ribosomes is that of zonal centrifugation in a discon- tinuous sucrose gradient (Bloemendal, 1974). With this technique free polysomes, shown to have a density of about 1.55 grams/cc (Dissous et al., 1974), have been defined as those which sediment through 2M sucrose of density 1.26 grams/cc (Bloemendal et al., 1974), while membrane-bound polysomes should float over 2M sucrose (Cioli and Lennox, 1973). There are many problems with the use of discontinuous sucrose gradients to isolate free and membrane-bound polysomes. Long periods of ultracentrifugation are required, and Blobel and Potter (1967) have demonstrated that the conditions of centrifugation (such as length of time centrifuged) must be critically monitored in order to effect complete separation of membrane-bound and free polysomes. Cioli and Lennox (1973) and Dissous et a1. (1974) found that the efficiency of the separation of membrane—associated from free polysomes depended on the physiological state of the cells used as the source of the polysomes. The pellet of free polysomes which results from the conventional discontinuous sucrose gradient tech- nique can be resuspended only with great difficulty (R. Patterson, personal communication). Another common method used to separate free from membrane- associated polysomes is differential centrifugation. Kimmel (1969) centrifuged a post-nuclear supernatant at 27,000 x g (max) for 5 minutes and used the pellet as a source of membrane-associated polysomes, while the polysomes in the supernatant were designated as free polysomes. Parameters such as the length of time a sample is centrifuged, and the 9 force employed, have been shown to affect the amount of membrane-associated polysomes which pellet in frac- tionation steps intended only for the removal of mitochondria (Blobel and Potter, 1967). Differential centrifugation has been described as an extension of classical Svedberg considerations, one important point being that complete separation of one class of polydisperse biological molecules from another such class is not possible by differential centrifugation alone (for review see de Duve, 1975). Consideration of the limitations of zonal and differential centrifugation led to a search for another method of separating membrane—bound from free polysomes. Albertsson (1959, 1960, pp. 177-182) used a dextran-methylcellulose aqueous two-phase system to partition free ribonucleoprotein particles from particles attached to membrane. The partition behavior of biological macro- molecules has been shown to depend on many complex factors such as size, surface area, and chemical nature of the macromolecule, and type of polymer used and ionic composition of the polymer (Albertsson, 1971, pp. 314-320). It was reasoned that a combination of (l) differential centrifugation and (2) partition separation in an aqueous non-ionic polymer two-phase system would separate polysomes by their surface area in addition to the Svedberg parameters of density and molecular weight. Since a membrane-polysome complex has more surface area than a polysome alone, the combination of differential centrifugation and partition separation should provide 4 a more complete separation than that afforded by conventional techniques. This study documents the successful separation of free from membrane-bound polysomes by use of a combination of differential centrifugation and partition separation in a dextran 68- methylcellulose 4000 aqueous polymer two-phase system. Attempts were made to show the presence of myeloma messenger RNA by analysis of the product made in a cell-free system for protein synthesis, but the results were equivocal. LITERATURE REVIEW Isolation and Description of Free and Membrane-Associated Polyribosomes In 1955, Palade (1955) described small, 100 to 150 A, particles with high RNA content which had affinity for endoplasmic reticulum and the second (outer) nuclear membrane. These particles did not exhibit an affinity for other cellular membranes. The particles were preferentially associated with endoplasmic reticulum in cell types that seemed to have in common a high degree of differentiation, whereas in other cell types, characterized by rapid proliferation or a less differentiated state, the particles occurred more or less freely distributed in the cytoplasm (Palade, 1955). These obser- vations were extended to over forty different mammalian and avian cell types (Palade, 1955b). Palade and Siekevitz (1956) showed by electron microscopy and other means that membranous vesicles with attached RNA-rich particles, obtained by homogenization of rat liver cells and subsequent differential centrifugation, were derived from rough surfaced (studded with particles) endoplasmic reticulum. When cells are homogenized in sucrose containing buffers, fragments of rough endoplasmic reticulum with attached particles form vesicles termed microsomes. The term microsome was coined by Hanstein in 1880 and applied by Claude circa 1943 to a product rich in ribonucleic acid and phospholipid found during differential 6 centrifugation (for review see Bernhard, 1954; Palade, 1956; Hendler, pp. 154—201, 1968; Palade, 1975). Palade and Siekevitz (1956) observed that when microsomes were treated with the detergent deoxy— cholate (DOC), their membranes were disrupted and solubilized leaving the RNA-rich particulate components. Wettstein et al. (1963) treated a rat liver post-mitochondrial supernatant (PMS) with DOC and then centrifuged it through a dis- continuous sucrose gradient to separate the particulate components, by this time known as ribonucleoprotein particles or ribosomes, from other cellular material. They showed that protein synthetic activity resided mainly in ribonuclease-sensitive aggregates of ribosomes (polysomes). Webb et a1. (1964) observed that even without DOC treatment some polyribosomes would sediment through the 2M sucrose of a discontinuous gradient. This effect was markedly enhanced when homogenates from hepatomas were used in place of the usual rat liver homogenates. Webb et al. (1964) attributed this difference between cancerous and normal rat liver to differing amounts of polysomes associated with the RER, or to a difference in the type of association. Blobel and Potter (1967) demonstrated with rat liver homogenates that many factors must be taken into consideration when discon- tinuous sucrose gradients are used to obtain a quantitative estimate of free and membrane-associated polysomes in a cell type. They showed that the low centrifugal force used to remove nuclei from the homogenate also removed about 35% of the cellular RNA. Washing the nuclei could not reduce the loss below 25%. Since the nuclei contained about 5% of the RNA, 20% was reasoned to be RER either attached to the nuclei or large enough to co—sediment. Blobel and Potter (1967) found that when differential centrifugation was used to separate mitochondria from microsomes and free polysomes, a varying amount of microsomes pelleted with the mitochondria. Thus the PMS contained a variable amount of microsomes, and the quanti— tative estimate of the percentage of free and membrane-associated polysomes, obtained by centrifugation of the PMS through a discon— tinuous sucrose gradient, varied correspondingly. The amount of microsomal material co-sedimenting with the mitochondria depended on the degree of homogenization (with increased homogenization fewer microsomes pelleted with the mitochondria) and on the force used to effect separation (with increased force more microsomes pelleted with the mitochondria). Blobel and Potter (1967) also showed that depending on the force used, a certain minimum amount of centrifugation time was necessary to completely pellet the putative free polysomes in a discontinuous sucrose gradient. Cioli and Lennox (1973) tagged membrane with 14C-labeled choline. They showed that some membrane-associated polysomes from MOPC-Zl tissue culture cells would pellet through2.0 M but not 2,3basucrose. They suggested, based upon additional work, that this phenomenon might be common among rapidly proliferating cells. Dissous et a1. (1974) found that rat liver polysomes of two dif- ferent densities pelleted through 2.014sucrose. During starvation, the proportion of higher density polysomes increased. Polysomes which did not pellet through 2.0bdsucrose had a third characteristic density. Ribonuclease treatment of all three polysome fractions resulted in monoribosomes which had a unique fourth density. 8 Kimmel (1969), using a modification of the differential centri- fugation method of Attardi (1967), centrifuged a post-nuclear super- natant (PNS) at 27,000 x g for 5 minutes and used the pellet as a source of membrane—associated polysomes, while the polysomes in the supernatant were designated as free polysomes. Differential centri- fugation has been described as an extension of classical Svedberg considerations, one important point being that complete separation of one class of polydisperse biological molecules from another such class is not possible by differential centrifugation alone (for review see de Duve, 1975). Albertsson (1959; 1960, pp. 177-182) used a dextran—methyl— cellulose aqueous polymer two-phase system to partition free ribo- nucleoprotein particles from particles attached to membrane. The partition behavior of biological macromolecules has been shown to depend on many complex factors such as size, surface area, and chemical nature of the macromolecule, and type of polymer used and ionic composition of the polymer (for discussion see Albertsson, 1971, pp. 314-320). Aqueous polymer two-phase systems have recently been used to isolate plasma (cell surface) membranes (dextran- polyethylene glycol, Lesko et al., 1974; Brunette and Till, 1971) and chromosomal deoxyribonucleoprotein (dextran-polyethylene glycol, Turner et al., 1974), and to perform sensitive assays for antibody and different conformations of antigen with nanogram amounts of material (dextran-polyethylene glycol, Reese et al., 1973). In summary, free and membrane-associated polysomes have been operationally defined by differences in their behavior upon zonal centrifugation, differential centrifugation, and partition separation 9 in polymer two-phase systems. They have also been visualized by electron microscopy. Functional differences, that is, differences in the types of protein produced by each fraction, also exist as will be discussed later in this review. Different Modes of Attachment of Polyribo— somes to Microsomal Membrane Palade and Siekevitz (1956) treated microsomes obtained by differential centrifugation of rat liver homogenates with versene (ethylenediaminetetraacetate-EDTA) and ribonuclease (RNase). When the microsomes were reisolated by differential centrifugation, it was found that approximately 50% of the microsomal RNA had been released from the microsomes. Electron microscopy revealed that the loss of RNA was correlated with a loss of the particulate frac- tion (ribosomes) attached to the microsomal membrane. Washing and reisolation of untreated microsomes removed a smaller number of ribosomes from the membrane. The concentration of the divalent cation Mg++ is critically important for polysomal stability--at concentrations lower than 1.0 mM ribosomes begin to dissociate into their subunits, while polysomes begin to aggregate at concentrations higher than about 5 mM (Palade and Siekevitz, 1956; Hamilton and Petermann, 1958; Girard et al., 1965; Breillatt and Dickman, 1966). Palmiter (1974) found that approximately 10% of the total amount of ribosomes would precipitate at 5 mM Mg++, and he has used higher concentrations of Mg++ to quantitatively precipitate polysomes which were recovered in undegraded, biologically active form. Girard et al. (1965) found that EDTA treatment dissociated ribosomes and polysomal 10 associated ribosomes into their two subunits (presumably by chelating Mg++ to a concentration too low to maintain stability). These sub- units, however, sedimented at reduced 8 values. If the EDTA treated subunits were returned to a cytoplasmic extract at 1.5 mM Mg++, the subunits again sedimented at their higher S values. The cause of the shift in S value was not determined, but it seemed to require cytoplasmic extract and was not only a Mg++-induced effect. Sabatini et al. (1966) brought about the stepwise dissociation of ribosomes from guinea pig hepatic microsomal membrane with increasing concentrations of EDTA. Almost all of the small sub- units were released first, and then increasing amounts of large subunits were released. The initial release of small subunits sug- gested that ribosomes were attached to membrane by their large subunits. They supported this interpretation with electron microscopy findings. Approximately 30% of the RNA remained bound to microsomal membrane even after treatment with high concentrations of EDTA. This residual RNA was found to be mainly associated with large subunits. The particles resistant to detachment by EDTA contained approxi- mately 70% of newly synthesized protein. Rosbash and Penman (1971) used EDTA, puromycin and RNase to dissociate HeLa cell ribosomes and mRNA from membrane. The released material was called "loose", in contrast with the "tight" material which remained attached to membrane. Ribosomes resistant to the action of EDTA were also resistant to the action of puromycin and RNase. A combination of EDTA and puromycin released more mRNA than EDTA alone. The loose ribosomes had a higher density (1.55 gm/cc) than the tight ribosomes (heterogeneous, mean density 1.49 gm/cc) ll (Rosbash and Penman, 1971b). Bleiberg et al. (1972) incubated mouse myeloma cells with NaF, an inhibitor of protein synthesis, and found that approximately 25% of the membrane-associated ribosomes were released during the incubation. Addition of 1,4KC1 to an homogenate from NaF treated cells increased the amount of released ribosomes to nearly 100%, while KCl alone released about 50% of the ribosomes. EDTA treatment released all the small subunits and approximately 50% of the large subunits. EDTA and lbiKCl each released approximately 40% of the bound mRNA, while little or no mRNA was released by incubation with NaF. Incubation with NaF appeared to release a fraction of the same ribosomes released by EDTA. Sarma et al. (1972) treated rats with etionine or CC14. Hepatic microsomes isolated from these rats released a larger amount of ribosomes on exposure to 0.411KC1 than microsomes from saline treated rats. Sarma et al. (1972) also studied ribosomal attachment to mouse liver microsomal membrane. High salt plus puromycin was more effective than high salt alone in detaching ribosomes from membrane. Neither RNase nor puromycin alone could effect release of ribosomes; moreover, high salt plus RNase was not more effective than high salt alone. High salt treatment with other monovalent cations, Li+, Na+, and Ca+, had the same effect as treatment with high K+ concentration. Rosbash and Penman (1971a), Bleiberg et al. (1972), and Sarma et al. (1972) reported that ribosomes were released from membrane in the form of monomers or subunits, but not polyribosomes. Adelman et al. (1973a) used a combination of differential cen- trifugation and zonal centrifugation to obtain a pure but degraded 1n 12 fraction of rat liver microsomes. In a companion paper (Adelman et al., 1973b), they reported that up to 40% of ribosomes (in the form of subunits) could be released by high KCl concentrations, with an additional 40% released when puromycin was included with the KCl. Treatment with puromycin at low salt concentration did not result in ribosome detachment. A fraction, approximately 15%, of the ribosomes could only be released from membrane under conditions which caused extensive unfolding of ribosomal subunits. Segregation of Polyribosomes into Free and Membrane-Associated Fractions The preceding sections of this review illustrate that it has been fairly well documented and accepted that some polyribosomes are bound to endoplasmic reticulum while others are not, and that there are at least two classes of membrane-associated polysomes-—a tightly and a loosely bound class as defined by various criteria (for review see Rolleston, 1974). The molecular events which are necessary and sufficient to cause a selected fraction of polysomes to become membrane-associated have not yet been elucidated. The remainder of this review documents other approaches taken to gain insight into the molecular events affecting polysomal segregation. i. Functional Differences It is generally accepted that membrane-associated polyribo- somes are responsible for polypeptides destined to be secreted or incorporated into cell membrane, while free polyribosomes produce polypeptides for intracellular use (except incorporation into intra- cellular membrane). The data gathered so far support the above generality (for comprehensive review see Rolleston, 1974). 13 Whether tight or loose membrane-associated polysomes produce the same polypeptides remains an unresolved question. Tanaka and Ogata (1972) found that a distinct amount of ribosomes, approxi- mately 25%, was released by RNase treatment of rat liver microsomes. Nascent serum albumin was found almost exclusively on the tight (RNase resistant) ribosomes. Nascent immunoglobulin has also been reported to be almost exclusively on tight (KCl resistant) ribo- somes (Zauderer and Baglioni, 1972). Harrison et al. (1974a,b) found that both loose and tight (KCl released, unreleased, respec- tively) fractions contained immunoglobulin mRNA. Zauderer et al. (1973) showed that high salt treatment, EDTA exposure, and repeated washing through 15% to 30% sucrose gradients released approximately equivalent fractions of ribosomal RNA. In contrast to other investi- gators, they found a significant amount of loose RNA to be polysomal instead of monosomal or in the form of subunits. Histone mRNA (7-9S RNA) was found in the free and loose fractions, but was essentially excluded from the tight fraction. Rosbash (1972) reported that inhibition of protein synthesis with cycloheximide prevented attachment of 35% to 50% of newly synthesized mRNA. ii. Affinity of Ribosomes for Membrane A great deal of research has been directed towards finding differences between the affinities of free and membrane-associated ribosomes for membrane. Baglioni et al. (1971) found that 608 sub- units of mouse myeloma cells attached to membrane even when protein synthesis was inhibited by cycloheximide, and that these 608 subunits were not in equilibrium with free 608 subunits. Borgose et a1. 14 (1973) demonstrated that membrane-bound rat liver ribosomes would exchange small subunits but not large subunits with free ribosomes. Ekren et al. (1973) and Rolleston (1972) determined that the binding capacity of stripped RER was greater for large ribosomal subunits than for small subunits (ribosomes must be removed from RER before new ribosomes can be added, Pitot and Shires, 1973). Rolleston (1972) found no difference in binding to stripped RER between free and membrane-associated ribosomes. Rolleston and Mak (1973) reported no difference in binding to stripped RER between free and membrane-associated polyribosomes. Binding to smooth endoplasmic reticulum (SER) was sensitive to ionic strength, moderate binding occurring at 25 mM KCl and very low binding at 100 mM KCl. Pitot and Shires (1973) also found no difference between the binding of free or membrane-associated polyribosomes to membrane. They clas- sified binding reactions into two types, a temperature-independent type observed only on derivatives of RER, and a temperature- dependent type which requires incubation of the polysomes with membrane at 25-37°C. They also advanced the concept of a membron, a "functioning, regulatable, translating polyribosome complex with a specific surface area of membrane." Rolleston and Lam (1974) reported that smooth endoplasmic reticulum binds ribosomes with the same affinity as RER, but has fewer binding sites. iii. Protein Composition of Subunits Another line of investigation has been directed towards finding differences in the protein make-up of ribosomal subunits from free and membrane-associated ribosomes. Borgese et a1. (1973) found that 15 the protein complement of subunits from rat liver free and membrane— associated ribosomes was similar except for one band in polyacrylamide gel electrophoresis (PAGE) which was more intense in free large subunits. Hanna et al. (1973) found an additional protein in rat liver free monosomes analyzed by two—dimensional PAGE; however, the protein pattern for free and membrane—associated monosomes was iden- tical when both were treated with DOC. Fehlmann et al. (1975) used two—dimensional PAGE to analyze rabbit reticulocyte ribosomal proteins. They found two proteins which seemed unique to the free monosomes and four proteins which seemed to belong only to the membrane-associated monosomes. Kaulenas and Unsworth (1974) reported a difference in the protein complement of the large sub- units of ribosomes from adult mouse liver and kidney. The difference became apparent from 11 days to 14 days of gestation, coinciding with the time of initiation of kidney differentiation. Starvation (Hanna and Godin, 1974) and hepatectomy with accompanying regenera- tion (Sheinbuks et al., 1974) resulted in a change in the protein complement of both subunits of rat liver ribosomes. Hanna and Godin (1974) reported that the free and membrane-associated ribo- somes underwent an identical change when rats were starved. Differences have been sought in the pattern of phosphorylation of free and membrane-associated ribosomal proteins. Egly et al. (1972) found that newly phosphorylated protein was mainly associated with DNA-like RNA, but could often be co-isolated with polysomes due to the sedimentation characteristics of the ribonucleoprotein particle. Pierre et al. (1974) reported that rat liver ribosomal phosphoproteins occurred primarily in the small subunits. They found 16 a difference in the pattern of phosphorylation of free and membrane— associated polysomes. iv. Non-Ribosomal Proteins Associated with Ribosomes Non-ribosomal proteins associated with ribosomes have been studied only with great difficulty, due to suspected artifacts of non-specific adsorption inherent in isolation procedures. Olsnes (1970) observed the adsorption of solubilized RER proteins to polysomes. The adsorbed proteins could be removed by DOC alone, or by a combination of Triton X-100 and high salt concentration. Henshaw et a1. (1973) and Hirsch et a1. (1973) reported the isola- tion of two types of Ehrlich ascites small ribosomal subunits, those found free in the cytoplasm and those derived from polyribosomes. The small subunits found free in the cytoplasm were shown to be combined with substantial amounts of non-ribosomal protein, and would not join with large subunits until this protein had been removed by treatment with 0,5quC1. Shires et a1. (1971) found that trypsin treatment under mild conditions destroyed the ribosome binding capacity of stripped RER. Kreibich and Sabatini (1974) reported that smooth and rough endoplasmic reticulum had similar sets of proteins with the exception of the ribosomal-associated proteins on RER. v. Relation Between Nascent Poly- peptide and RER Since functional differences have been found between free and membrane-associated polysomes, it is reasonable to consider the relationship between nascent chain and RER as a possible factor in 17 the attachment of polysomes to RER. Relevant studies which employed puromycin to release nascent chain have already been presented in the section on different modes of attachment of polyribosomes to microsomal membrane. Other studies have examined the fate of polypeptides synthe— sized by membrane-associated polysomes. Vectorial discharge, through membrane into a cisternal space, of polypeptides synthesized on ribosomes attached to RER or microsomal membrane has been fairly well documented and accepted (Redman and Sabatini, 1966; Redman et al., 1966; for review see Palade, 1975). Sabatini and Blobel (1970) reported that controlled proteolysis of microsomes resulted in two broad categories of nascent chain fragments: smaller carboxy- terminal segments which were protected by the large subunit and larger membrane-associated fragments which depended on intact membrane for their protection. Kreibich et al. (1973) showed that the vesicu- lar content of microsomes could be released by low levels of deter- gent insufficient to produce an extensive change in microsomes other than the reversible formation of openings in membrane which may allow leakage of contents. vi. Kinetics and Composition of Free and Membrane-Associated RNA Free and membrane-associated ribosomal and messenger ribo- nucleic acids have been examined for differences in kinetic behavior or base composition. Loeb et a1. (1967) reported that the base composition and rate of synthesis of ribosomal RNA was the same for both free and membrane-associated ribosomes in rat liver. Mishra et al. (1972) found the same decay pattern and half-life for free 18 and membrane—associated rat liver ribosomal RNA. Murty and Sidransky (1972) reported that for hepatic mRNA, free polysomal mRNA appears to have a short half—life as compared with mRNA of membrane— associated polysomes. Storb (1973) examined the half-life of mRNA (poly(A%+ RNA) in myeloma cells and found that microsomal poly(A) + RNA had a longer (by a factor of about 4) half-life than free poly(A)+ RNA. Attardi and Attardi (1967) showed that HeLa cell membrane- associated mRNA had a base composition very different from that of free polysomal mRNA. The membrane-associated mRNA was especially high in adenylic acid. Baglioni et a1. (1972) found that myeloma cell free and membrane-associated mRNA contained similar poly(A) sequences. Stevens and Williamson (1972) isolated poly(A)-containing immunoglobulin mRNA from the cytoplasm and nucleus (hnRNA) of myeloma cells. This mRNA contained messengers for both heavy and light chains. Poly(A) sequences may be involved in the attachment of polysomes to microsomal membrane (Milcarek and Penman, 1974; Lande 8t al., 1975). vii. Proteins Bound to mRNA Messenger RNA, independent of ribosomal subunits, has been found associated with protein. The relationship of this protein to RER or to ribosomes has not yet been clearly established. Whether membrane-associated ribonucleoprotein (RNP) complexes contain the same or different protein complements as free RNP complexes is still unresolved. Baltimore and Huang (1970) reported that a heterogeneous group of HeLa cell soluble cytoplasmic proteins could bind to all types 19 of RNA. The bond was unstable at high ionic strength. Lee et al. (1971) released mRNA from polysomes by starvation of mouse sarcoma 180 ascites cells and showed that the mRNA was complexed with spe— cifically bound protein--the protein could not be removed by high salt concentration, nor would it significantly bind to nuclear RNA. Milcarek and Penman (1974) reported an association of HeLa cell microsomal membrane and poly(A). Lande et al. (1975) found an association between human diploid fibroblast microsomal membrane and mRNA at or near the 3' poly(A) end. Kwan and Brawerman (1972) found a particle of apparent protein composition attached to the poly(A) segment of mouse sarcoma 180 mRNA. Two proteins, a larger one of about 78,000 Daltons and a smaller one of about 50,000 Daltons, have been reported bound to ribosome-free polysomal mRNA of mouse L cells and rat hepatocytes (Blobel, 1973) and chick cerebra (Bryan and Hayashi, 1973). Blobel (1973) found that the larger protein was bound to the poly(A) sequence of mRNA. Gander et al. (1973) prepared a post—ribosomal supernatant from which mRNA—protein complexes were isolated. They demonstrated that none of the complexed proteins was identical to any of the proteins associated with polysomal mRNA, and that some of the proteins were phosphorylated and contained phosphoserine. Immunoglobulin has been reported to bind to mRNA (for review see Stevens and Williamson, 1974), and to ribosomes (Moav and Harris, 1970; Eschenfeldt and Patterson, 1975). viii. RNA Other than mRNA and rRNA Little work has been done on the role of transfer RNA (tRNA) in the segregation of free and membrane—associated polysomes. Jones 20 and Mach (1973) reported that met-tRNAF provides N—terminal methionine while met-tRNAM provides internal methionine in mouse myeloma cells. Darnbrough et al. (1973) showed that in reticulocyte cell lysates, met—tRNAF can combine with the small ribosomal subunit independently of mRNA. Gerlinger et al. (1975) examined the translation of ovi- duct and reticulocyte polysomal RNA in a tRNA dependent Krebs—II ascites cell-free system. They found that translation proceeded better when both tRNA and polysomes were from the same tissue than when the tRNA came from a heterologous source. Shafritz (l973a,b) demonstrated that a significant amount of mRNA in polysomes is not translated in rabbit liver cell-free systems. A low molecular weight RNA, termed translational control RNA (tcRNA), has been found which affects the binding of mRNA to small subunits, and met-tRNA to polysomes, in a rabbit reticulocyte cell- free system (Kennedy et al., 1974). ix. High Salt Wash "Factors" A heterogeneous mix of initiation factors, tRNA, diverse pro- teins and other unassigned material is obtained by exposing free and membrane-bound fractions to high concentrations of salt (usually KCl). After exposure to high salt, the ribosomes are pelleted and the supernatant dialyzed to a salt concentration compatible with polysomal stability. This dialysate is termed a high salt wash. Membrane-associated polysomes (derived from detergent solu- bilized microsomes) from rabbit liver (Shafritz and Isselbacher, 1972) and myeloma cells (Abraham et al., 1974) have been reported to show a marked preference for homologous high salt wash (that is, 21 a high salt wash from microsomes as opposed to free polysomes) as assayed by increased translation in cell-free systems for protein synthesis. A corresponding preference by free polysomes for homologous wash was not observed by Abraham et al. (1974). Uenoyama and Ono (1972) mixed free and membrane-associated ribosomes, mRNA, and high salt wash in different combinations in a cell-free system for protein synthesis. When the components of the cell—free system were homologous, better translation resulted than when the components were not all from the same source. Abraham et al. (1974) showed that chain initiation on membrane—associated polysomes was stimu- lated only by a microsomal high salt wash, whereas initiation on free polysomes was stimulated by either free or microsomal wash. Pryme (1974) reported that membrane-associated polysomes from myeloma cells (MPC-ll) in the G1 phase could be stimulated in a cell-free system for protein synthesis by either 61 or 62 high salt wash, but that G2 membrane-associated polysomes could only be stimulated by G2 wash. MATERIALS AND METHODS _Ce_11_s; Mouse plasmacytoma cells maintained in tissue culture were used in this study. The myeloma cell line Mineral Oil Plasmacytoma 21 (MOPC-Zl) secretes an immunoglobulin-like (IgG) gamma globulin and produces excess light chains (of the k class) which are degraded intracellularly instead of being secreted (Baumal and Scharff, 1973). Three other cell lines were employed. The XC.1 cell line neither secretes nor contains intracellular immunoglobulin. The 849.1 cell line consists of lymphoma cells derived from thymus (T) cells which do not produce detectable amounts of gamma globulin. Cells of the 8180 line are not part of the immune system and do not produce gamma globulin. All lymphoid cell lines used in this study were kind gifts of the Cell Distribution Center, Salk Institute. The S180 cell line was maintained in ascites form in Swiss Webster mice. MOPC-Zl cells, in addition to being grown in tissue culture, were also maintained in ascites form in BALB/c mice. The MOPC-Zl cells were passaged in the peritoneum at approximately 7- to lO-day intervals. The MOPC-21, XC.1, and 849.1 cell lines were maintained in vitro in tissue culture. The culture medium consisted of Dulbecco's Modified Eagle Medium (GIBCO) supplemented with antibiotics (75 micrograms/m1 streptomycin, 100 units/ml penicillin, 40 units/ml 22 23 mycostatin) and 10% fetal calf serum. Tissue culture cells were maintained in roller bottles which were flushed with 95% air, 5% C0 sealed and incubated at 37°C at a rotation speed of one—half 2: revolution per minute. Typically, MOPC—2l cells were grown to a density of 4 to 8 x 105 cells/ml and diluted to 2 to 4 x 105 cells/ ml with fresh medium. The MOPC-21 cell line was periodically checked for antibody (myeloma protein) production. MOPC—21 cells were grown in tissue culture and pelleted by centrifugation. The supernatant medium received ammonium sulfate to 50% saturation in order to precipitate the gamma globulin fraction. The precipitate was centrifuged (3500 x 9 max for 30 min) resuspended in distilled water, and reprecipitated with 40% saturated ammonium sulfate. This precipi- tate was centrifuged, resuspended in distilled water, and reprecipi- tated with one volume of saturated ammonium sulfate added dropwise by burette. The final precipitate was centrifuged, resuspended in distilled water and dialyzed against buffer (20 mM Tris, pH 7.4, 150 mM NaCl). Ouchterlony immunodiffusion revealed one line against specific antisera. The MOPC-21 cell line was routinely checked for mycoplasma contamination. The method used, observation for cytoplasmic incor- poration of radioactive thymidine, yielded results which were con- sistently negative (that is, indicative of no contamination). Enzyme Assays Assay parameters (proper reagent concentrations, temperatures, etc.) for the assay of 5'-mononucleotidase and glucose-6—phosphatase were generously provided by Dr. Walter J. Esselman. 24 Glucose-6-phosphatase is a conventional marker for microsomal membrane. To assay for it a stock solution was prepared of EDTA (pH 6.5), histidine (pH 6.5), glucose-6—phosphate (adjusted to pH 6.5 with]_bJHCl), and deionized 1X glass distilled water. When combined with the proper volume of enzyme fraction, this stock solution gave the following final concentrations of reagents: 1 mM EDTA, 7 mM histidine, and 40 mM glucose-6—phosphate. The total volume of the reaction mixture was 0.5 ml. The reaction mixture was incubated at 37°C for 20 minutes, at which time the reaction was stopped by the addition of 2.5 m1 of 10% TCA. The mixture was then cooled on ice for 15 minutes. Any precipitate was removed from the mixture by filtration (with a Swinney adapter and a 10 ml syringe) through Whatman GF/C glass fiber filters (2.4 cm). Alternatively, the precipitate was removed by centrifugation at 1,500 x 9 (max) for 15 minutes in an International refrigerated centrifuge. Phosphate was determined by the method of Fiske and SubbaRow by using a Fisher Gram-Pac (Fisher A974 ANS) pre-mix of l-amino-Z- naphthol-4-sulfonic acid-~dry mixture. One and eight-tenths milli- liter of the precipitate—free enzyme reaction mixture was added to 2.5 m1 of deionized 1X glass distilled water. To this 0.5 m1 of 25 gm/l ammonium molybdate in 5N H2804 was added followed by 0.2 ml of Fisher A974 ANS. After 10 minutes the spectral absorbance at 660 nm was read. The blank was an identically treated sample which had received buffer in place of buffer plus enzyme fraction. The assay for 5'-mononucleotidase, a conventional marker for plasma membrane, depended on a different stock solution but other- wise followed the same procedure as the glucose-6-phosphatase assay. 25 The stock solution for the 5'-mononucleotidase assay was prepared with AMP (adjusted to pH 7.0 with 1.0 N NaOH), glycine (pH 9.1), MgClZ, and deionized 1X glass distilled water. When combined with the proper volume of enzyme fraction this stock solution gave the following final concentrations of reagents: 5 mM AMP, 100 mM glycine, and 10 mM MgC12. A standard curve was constructed using NaZHPO4 in buffer (10 mM NaCl, 3 mM MgCl 10 mM Tris, pH 7.4, 0.25 M sucrose, 100 ug/ml 2’ heparin; this buffer is abbreviated as RSB[such]). Two standard concentrations of NaZHPO4 were included with each experiment. .Construction of the Aqueous Two-Phase Polymer System A 10% (w/w) solution of dextran 68 (D68) (Sigma Type 2000) was made according to the method of Albertsson (1960, pp. 28-29). The undried dextran was first wetted and mixed to a paste with a small amount of sterile, deionized 1X glass distilled water. The rest of the water was then added and the dextran dissolved by stirring and slowly heating the mixture to boiling. The flask was then covered and the solution allowed to cool. Typically this step involved 5 gm of D68 plus 45 gm of water. A 1% (w/w) solution of methyl- cellulose 4000 (MC4000) (a kind gift of Dow Chemical Company) was also prepared according to the method of Albertsson (1960, pp. 31-32). The methylcellulose was dried at 110°C for 16 to 24 hours. Two hundred grams of a 1% (w/w) solution of MC4000 was prepared in the following way: 2.0 gm of dry MC4000 were weighed into an Erlenmeyer flask and 100 gm hot (80-90°C) sterile, deionized 1X glass distilled water added. The flask was closed and shaken 26 vigorously for a few minutes in order to wet the powder. Ninety- eight grams of cold water were then added, the flask shaken and allowed to stand with occasional stirring until it reached room temperature. The powder then swelled and dissolved slowly. Care was taken not to allow material to sediment to the bottom of the flask. The solution was then cooled to 4°C and kept at this temperature. To make the stock solution the following constituents were added in the given order to an approximately 200 m1 capacity bottle with rectangular sides: (1) 13.6 gm of a 10% (w/w) solution of D68; (2) 72.0 gm of a 1% (w/w) solution of MC4000; (3) 0.5 gm of 0.2 M MgCl (4) 0.5 gm of 0.2 M K HPO ; and (5) 13.4 gm of water 2’ 2 4 (after Albertsson, 1959). The bottle was then placed almost hori- zontal and parallel to the body of the person making the stock solu- tion, at which time the bottle was rotated 21 rotations toward the person and 21 rotations away from the person, at a speed of about 1 rotation/sec, to mix the components. Directions for mixing were worked out during the course of this study and should be followed because formation of the phases is a highly empirical process. The stock solution, routinely stored at 4°C in a standard refrigerator, kept well for at least 4 months with only minimal attention given to sterile technique. This stock solution, when diluted 1:1 with a polysome fraction, gave the desired phase system. To construct the phase system, the stock solution was mixed as described above and 2.5 gm were withdrawn with a 10 ml pipette and deposited in a 15 ml Corex centrifuge tube. The following components were added in the order given to the 2.5 gm of stock: (1) 0.05 ml 27 of heparin at 10 mg/ml; (2) 0.8 ml of 15% (w/v) RNase-free sucrose in water; (3) 0.01 ml of 0.2 M KZHPO4; (4) 0.64 ml of RSB made up with 100 ug/ml of heparin [RSB(HEP)]; and after mixing the Corex tube 10 rotations in a manner similar to that described in mixing the stock solution, (5) 1.0 ml of crude 27,000 x g membrane pellet (MP1) suspended in RSB(such). The Corex tube was then rotated in a manner similar to that described above. The tube was then lowered to an approximately 45° angle with an axis perpendicular to the floor, and subsequently rotated around the axis 10 times clockwise and 10 times counter- clockwise. Finally, the tube was rocked from almost perpendicular to almost horizontal position 10 times. This mixing algorithm was important to follow, since other mixing procedures did not allow consistent formation of the two phases. After mixing, the aqueous polymer system was centrifuged for 10 min at 1,400 x g max in an International refrigerated centri- fuge to separate the phases. It should be mentioned that all pro- cedures were done at 4°C in order to minimize RNase degradation of polysomal material. This low speed centrifugation consistently allowed formation of two phases from a properly mixed aqueous polymer system. The final weight of the aqueous polymer two-phase system was 5.0 gm, which was equivalent to 5.0 ml since the density of the system was very near that of water. The bottom phase was of course denser than the top phase but, curiously, it was much less viscous. The final concentration of each component in the system was 0.68% (w/w) D68, 0.36% (w/w) MC4000, 133 ug/ml of heparin/ml, 1.4 mM 28 ++ Mg , 0.9 mM K HPO 2 4, 3.2 mM Tris, pH 7.4, 3.2 mM NaCl, and 0.12 M RNase—free sucrose. Isolation of Free and Membrane- Bound Polysomes Figure 1 presents a general schematic of the procedure used to isolate free and membrane-bound polysomes. Cells were grown to a density of usually 5 to 9 x 105 cells/ml. If labeled polysomes were desired, 0.5 or 1.0 uC/ml of 3H-uridine was added 10 to 20 hours before termination of the incubation period. At the end of incuba- tion, cells were poured over crushed frozen saline and pelleted for 8 minutes at 500 x 9 (max) in an International refrigerated centri- fuge. Unless otherwise indicated, all procedures were done at 4°C. The cells were resuspended in RSB(hep) and allowed to swell for 15 minutes. They were then recentrifuged and resuspended in approxi- mately one volume of RSB(such) in a 7 m1 Kontes Dounce homogenizer. Immediately after resuspension in the sucrose buffer, the cells were Dounced 10 times with a tight pestle. This resulted in greater than 90% lysis of cells; however, if the cells were allowed to equili— brate in the sucrose buffer for about 10 minutes, the efficiency of lysis was reduced to 50 to 70%. The nuclei were then pelleted at 900 x 9 (max) for 5 min and washed once with RSB(such). The wash was added to the first supernatant and the total supernatant was coded PNSW (post-nuclear supernatant plus wash). The PNSW was then spun at 27,000 x 9 (max) for 5 minutes in a Sorval RC2-B centrifuge. The supernatant (PMSl--first post-mitochondrial supernatant) was decanted. It contained crude free polysomes. The pellet (MPl--first pellet), which contained the crude microsomal material, was resuspended 29 .meOmmaom moum Scum ocsonumcmunfime mumwmmwm ou pom: mnsvmooum on» mo UHumegom Hmwmcmu .H musqfim AUCDOQImQMHQEmE u mxmmzv Ameom>Hom =mmooH=v umaamm umaamm AcflE m MOM . ixmav m x ooo.nmv .ucmu [nanosmvmmm an“; :mmz EE 3 now ixmé m xoooémv 323$ch Mao: a wow SE mmv mumuflmflomum ++mz . I uma mm ucmumcwwmsm wmmnm momma wmmnm momma . (47 AI Aces m Hon ixmev m x ooo.>mv mmsufluucmo icosmcmmm snag musaflo umaamm + mmmnm HwBOA ACHE OH now AmeV 0 x oovav wmsmfluucwu Megan Eonm wmmcm Momma nuHB xwz uwaamm + mmmnm umBOq AmmEOmmaom mmwm u mmzmv ucmumcummsm ‘7 . ACHE OH mom Axmev m x oovav mmzmwwucmo ACME m “Om AmeV AOOOVUZImmov m x ooo.hmv xm madmwuucmu umsxfiom wmmcmlozu msomsvm nufi3 tz Aamzmv ucmumcwwmdm HmEOmoonEIumom Hm uwaamm HmEOmouon .. A so _ laws m How Axmev m x ooo.emv mmsmwuucmu Azmzm u Hwaosc mo cmmz mco +V ucmumcuwmzm Hmwaoscumom HmWMsz ACHE m wow me m x oomv mmSMAwucmoawocsoo_ mHHwU OHDHHSU 05mmHB HNIUQOE 30 in RSB(such) and frozen at —80°C for later use. The supernatant was then centrifuged two more times at 27,000 x g (max) for 5 minutes. This supernatant (PMS3) was used as a source of free polysomes. For early experiments, including those in which enzyme assays were performed, the PMSl was chromatographed on Sepharose 6B (Pharmacia). The procedure was similar to that of Eschenfeldt (thesis, 1975, p. 27), except that a different elution buffer was used, namely RSB(such). A column (1.5 x 15 cm) was poured and washed with at least 10 bed volumes of RSB(such). PMSl was applied to the column in a volume of 2.5 ml or less. The polysomes were eluted at a flow rate of approximately 10 ml/hr with RSB(such). Polysomes were eluted in the void volume. The cloudy, white frac- tions were combined and designated PMS,Sl (the extra S indicated that the PMSl had been chromatographed on Sepharose 6B). The PMS,Sl was then centrifuged two more times (27,000 x 9 (max) for 5 minutes), labeled PMS,S3, and used as a source of free polysomes. Storage was at -80°C. After thorough washing with RSB(such), the column could be reused. The column was periodically treated with a solution of 0.1% diethylpyrocarbonate (Calbiochem) in RSB(such). The previously frozen MP1 was routinely processed within a few days of freezing. MP1 was partitioned as described in the section "Construction of the Aqueous Two-Phase Polymer System." The bottom phase was consistently cloudy and contained the membrane-bound polysomes.‘ Some material would always pellet. After the first partition separation, the clear top phase was withdrawn with a 10 m1 pipette and saved. To the bottom phase was added the top phase of a blank made with RSB(such) in place of MP1. Without resuspending 31 the pellet, the new system was mixed and centrifuged as described previously. The clear top phase was again withdrawn and saved. The bottom phase was then diluted with 5 ml of RSB(such), and the contents mixed with a 10 ml pipette, resuspending the pellet in the process. The contents of the tube were then distributed evenly to other 15 m1 Corex tubes so that no one tube would hold more than 3 ml. The tubes were then centrifuged for 5 minutes at 27,000 x 9 (max) in a Sorvall RC2—B centrifuge. The supernatants were decanted and combined with the previous two top phases. The pellets were combined and resuspended in 2 m1 of RSB(such) and recentrifuged at 27,000 x 9 (max) for 5 minutes. The supernatant was decanted and added to the other supernatant plus top phases. This mixture was distributed evenly among Corex tubes (15 m1 size) so that no tube would hold more than 6 ml. The supernatant polysomes were then Mg++ precipitated as described in the section "Mg++ Precipitation of Polysomes.“ These supernatant polysomes were termed “loose"; they were found in the crude 27,000 x 9 (max) pellet but they did not stay with the microsomes upon partitioning and further dif— ferential centrifugation. The pellet was resuspended in approxi- mately 1 ml of RSB(such). Resuspension of membranous material was commonly accomplished with the aid of a small (approx. 2 ml) homogenizer. The polysomes in this final pellet were termed "tight" and were coded MP2XP for membrane-bound polysomes, twice partitioned. The MP2XP fraction was either frozen immediately or ++ Mg precipitated and then stored at -80°C. 32 ++ , Mg Precipitation of Polysomes The polysome fraction to be precipitated was made 25 mM with MgCl2 and allowed to sit for one hour at 4°C. If no membrane was desired, the fraction was first given 100 ug/ml heparin, then treated for 5 minutes with 0.5% TX-100 (v/v), and then precipitated as above. The precipitate was centrifuged for 10 minutes at 27,000 x 9 (max), then resuspended in 20 mM HEPES (Sigma) buffer, pH 7.4. The polysomes were then stored at -80°C until needed. The magnesium precipitation procedure is a variant of a technique presented by Palmiter (1974). System for Cell-Free Protein Synthesis The system was a modification of a system developed by Marcu and Dudoch (1974). Reactions were done in a total volume of 0.5 ml or 1.0 m1 unless otherwise noted. Final concentrations of reactants were as follows: (a) 100 mM KCl, 7 mM Mg acetate, 20 mM HEPES (pH 7.4), 6 mM 2-mercaptoethanol; (b) 1 mM ATP, 0.02 mM GTP, 8 mM creatine phosphate; (c) 2.325 or 4.65 units/m1 creatine phospho- kinase; (d) 25 x 10'.6 M amino acids other than those in the 3H-L- amino acid mix which was added to 4 uC/ml; (e) high speed super- natant at approximately a final concentration of 0.1 mg protein/ml and 0.006 mg nucleic acid/m1; and (f) varying amounts of polysomes generally ranging from 1 to 5 A units/ml. Reactant groups (a) 260 and (b) were stored as pre-mixed stock solutions at -20°C. All reactant groups were lO—fold concentrated and thus were diluted 1:10 for the reaction. For reactant groups (b) and (d), the pH was adjusted to about neutrality (7-7.5). Creatine phosphokinase was usually diluted in RSB(such) and stored at -20°C. 33 High speed supernatant was prepared by centrifugation of a PNSW, or a PMS, at 250,000 x 9 (max) for 90 minutes at 4°C in a Beckman SW 50.1 rotor in the Beckman Model L5-50 ultracentrifuge. The supernatant was dialyzed against two changes of 100 volumes of buffer adjusted to about pH 7.4 (20 mM HEPES, pH 7.4, 2 mM 2- mercaptoethanol, 2.5 mM Mg acetate, and 100 mM KCl). The super- natant was then stored at -80°C. It retained activity (stimulated the synthesis of polypeptide) for about 5 weeks. Reactions were incubated at 37°C for 40 minutes in a New Brunswick Shaker Bath (model G76). After incubation, the tubes were removed to ice, aliquots were taken for TCA precipitation, and the remainder was saved to use in affinity column studies. TCA precipitation was followed by heating at 90°C for 15 minutes. The tubes were then cooled on ice for 15 minutes, and the precipitate collected by vacuum filtration on Whatman GF/C filters which were counted in 5 ml of toluene plus omnifluor. Mg++ and K+ optimums were obtained by varying the concentra- tions of the respective salts in group (a). pH optimums were obtained by varying the pH of the buffer in group (a). As such, the pH optimum was empirically an optimum for the pH of group (a), but might not have been an accurate value for the pH of the whole reaction. Affinity Chromatography Columns Protein was coupled to Sepharose 6B (Pharmacia) by the method of Cuatrecasas (1970). The method in detail can also be found in the master's thesis of Eschenfeldt (1975, p. 15). Briefly stated, 34 the method allows protein to couple to cyanogen bromide activated Sepharose. Sepharose 6B coupled with normal rabbit gamma globulin and Sepharose 68 coupled with anti-myeloma protein were used. Columns were poured (at room temperature) in disposable Pasteur pipettes to a final packed volume of 0.3-0.5 m1 Sepharose. They were washed twice with buffer (25 mM Tris, pH 7.6, at 4°C, 175 mM NaCl, 5 mM MgC12, and 100 ug/ml sodium heparin). The columns were washed three times with a total of about 4.0 ml of 10% fetal calf serum to saturate nonspecific binding sites. They were then rewashed three times with buffer. Sample was applied in 0.4 m1 aliquots. Unbound sample was eluted with about 4.0 ml of buffer. Eluate was precipitated with 10% trichloroacetic acid (TCA). The precipitate was collected by vacuum filtration on Whatman GF/C glass fiber filters (2.4 cm) and counted in 5 ml or 10 ml of toluene— Omnifluor (New England Nuclear) scintillation fluid. Functional Assays Polypeptides were prepared from polysome fractions in a system for cell-free protein synthesis by methods described elsewhere in . . . . . . 3 this theSis. The reaction mixture containing the H-labeled poly- peptides was brought to 33 mM EDTA with 100 mM Na EDTA in water, pH 2 7.0, to dissociate ribosomes into subunits and release nascent chains. After 10 minutes on ice, 1 M KCl in RSB was added to a final concentration of 250 mM. Sample was then applied to an anti- myeloma protein column and a control column of normal rabbit gamma- globulin (NRGG). The method of application and preparation of eluate (unbound material) for counting has already been described. 35 For each experiment control columns were prepared which were tested against purified 14C-labeled myeloma protein. Sucrose Gradient Analysis Polysomes were analyzed on 15-45% (w/v) linear sucrose gradients intimeSW 50.1 rotor (5.2 ml sucrose per gradient). Centrifugation was at 45,000 rpm (250,000 x g [max]) for 35 minutes (occasionally this was changed to 40 minutes). Gradients were collected from the top with continuous monitoring of absorbance at 254 nm. For some experiments, 0.2 ml fractions were counted in toluene-Omnifluor plus TX-100 plus water (6:3:1). RESULTS 2.1.1: As mentioned in the literature review, the physiological state of a cell affects its polysomes. One measure of the physiological state is the generation or doubling time of the cell. Throughout this study records were kept of MOPC-Zl cell density and time of measurement. Conventional reasoning about generation time was translated into a simple FORTRAN program and measurements represent- ing the span of this study were converted into generation times, a running calculation of mean generation times, and a corresponding running calculation of standard deviations as shown in Table 1. It is apparent there was variation in the generation times of the cells. It is doubtful that the variation is solely due to counting inac- curacies. Some of the variation reflects differences in the length of time cells were incubated before being counted (that is, cells may have been in stationary phase for different time intervals). Other variation may reflect the fact that toward the latter part of this study, tissue cultures were started from slower growing MOPC-21 ascites cells which probably took time to become adapted to the culture conditions. Finally, diverse factors such as variation in incubation room temperature and in partial pressure of 0 (due to 2 frequently leaky roller bottle caps and to larger volumes of media per bottle toward the latter part of this study) may have contributed 36 37 Generation times of MOPC-21 tissue culture cells Table l . standard deviation generation me an time (hrs) 300.00000033000000009000303000000050000 06000.05030.0GOUUUOAU‘UCODODOC‘U55.000.000.000 P000f+ff+f.76....9}ffff+§+§+++fif§GO+§+ff++ 6:222.:C.C:_::C::CC:C.L:r:._22:3.:L_r=.._f:..C...C::.::CC__..::.:.:CCLCL.C 09.141.1.51.3.09.036733693032220523c1nv6391.9591 Sal“£538.3557L‘6h914551126“QC)262.£L~005J260;56 “2.070.51.141097.01754432714369361431.09.972.927... 91555337921909593150299003957393205357 5180.387C527759153382Q,29362907.u31217689 25C).«rOQnUSIOJrOCJZTO3336“35125985307“v4“ 1.635 093739205865QQQQ3QQ33222212222211198793 .00.0.09.0000...OOOOOOCOOOCOOOOOOOOOOOO ““33333222222222222222222222229.2223333 11111.11111111.11111111111111111111111111 000000000000009030050000000005000000030 +O+++++O++++++++§O§§§+§++++f6+++9§f§t§+ E.F:LE_EEEEEEEEEr.EEEEEEEEEEEEEEEEEEEEEEEEE nu“c.145115003359355300.138766614552.423313052 125.».979200951965'9[4552.92616320142661.336606. 7&5}le.530.5%.b5h901720283284405140121410.5.4773 05181453D7..7_33.u§2894.3702r01h3r).72h3h.u.47.:h37)1 01.19“3577Q145512.528.3735165175739409.9653w7 h947h7hk6Q555h6552332232334556565683355 17655hhhh9§1wQQQQQQQQ“HQ“QHQQQQQBQQQSSSCJ 000000000000no.0000.00.00.00.00000000000 211111111111111111111111111111111111111 11111111.1111111111111111.111111111111111. OOCCCOUOOOOCQOOOCCGCCGUOCCOCCour-«00000000 +++f++++++++o++++++++§+++++++++++++++§+ I...:C_CCCE..C::::=L_C..L_:..:L.C—L.CC2;_:C=C.C.L::CCCF:—:GEES—£62.32.» €030?#3753577381.Q33991.9Q6653Q11697232150 1372-4.OQ:29§8537793982995“2.080019182135167 7176:..731h0.5255239413140231463.»?32836573314 CC3597326593C59993661h5227686613CLC3677 030523.00.2?..H72615.0299786900h2QQOg392815“ “h109317533566.“ng.926556596Q57OQBQ1796535 1.43314129625“Q273305Q2352633656Q3h612h35 COOOOOOOIOOOOOIOOOOOOOOI00000000000000. 21111111111111111111111111111111112312... nu000000000000000000033000000000.000000300030000 03.900350300003000000400000055300003303.«000033.00 +§+++§+§++ff+f++++§+++f§§f+OOOf§f§§§+§+Of++§OL E:2.3253232...L:=C_s::::r_.—:C.L..:C.:Eczlntgpl..2CrCPLC:L..:......:.:Z_C_EEC;”1EZS.» 133145715960169.3507OSZPDURQ10770.3013599975163736 291539ufl73.0“577_67:4775:49]0.71“1.§c39.3“““07771.0020601 3146956569134412.6535157564299235906973055595037 7:4“03655765011811637.052614313995679h515.602.196.630)... 254277068752.0000L777.0,756701CQ0L5.UQ.371H7r)1.7r.447118q17 727663230631514Ufi?09r)0625h?0.3557h602652853136an 88890.99593890332221110099090000301.433322221111 OOCOOOOOCOOOOOOOOOOOO.OCOOOOOOOOOOOOOOOOOOOO~OQ 333333333333gulflhgssssr§5hIQ5“CI—IJFJSSSSSSSC/rJSFJSSS.95 111111.11.11.111111111111111111111111111114.111111 0000000000000000000000UOCOUCUOOOO‘UGOOOCQU00.000. .v.t++++++++f++++++++++++++6+++++++f++++++++§++o .LEEEC5E2CEEEEEE—LC:CEC.EE:_EESLFCEEFELFCF_ENCEC:CCC::CEEEC:.:ZE 0..0.15931550.9700655663663797050157763530. 2374931135 659113207522859.6048170.525233101013.0317013995352685 1435:46.47190293550201H663325061017.56332_J.b3.0q815qH.114“ 179.91913255014237993.5.51479.....9175709114920,.“37.15:»3110 56955939992625“14670209980410.3703h97709232hd53952 6670103.95?7891333377776666539098.090323333333222 S555655555556666666666666666766667777777777777 o09.000000000000000000000coo.cocoooooooooooo.Oo 11111111111111111111111111111111111111111111T1 11.1111111111111111111111111.1.111111111111111111 0CCUQCCCUCGUOOCCF.CUCCGOOOUCUCUUGOUCUUOUGOCCGCO ++6+++++++++O+f++++++++++f+§++++++++++++++6+§t p:7.3:.:C£3.22:$56.32»...Cr.C_C_.»:.=C..:.:C_.2C::.=..C.E..._L1C_»._._C_.p..25:16£252.22”... U3607840.“..737656671981438“962““1.530;.Iw9taz7“6506573“ O;(00056,0In0583.039390Q73SZFJ30'51“Skid? qu31'00491..146.07 h91003923h90395279191§35h895573612277725525868 b21C9235579B16pL6C656165950,72587.901R8723517 1:493 7020338321458709109752901597?“0735h07hqr0693Q751 73251781PJ9I45h28c/67335197514229781866010Q7890396 8612032B1572985679275665757681002h3798680“633% O0.0000000000000000000OOOOOOOOOOOOOIOOOOOOOOOO 11.222111111222111.31111111212111222311111211111. 38 to variations in the observed cell generation time. The mean of all analyzed MOPC-Zl cell generation times was 17 hours, and the standard deviation was 5 hours. The data which follow were relatively consistent and made a coherent description of certain properties of MOPC—21 cells, even with the apparent variation in cell generation time, and thus should be pertinent to the isolation of free and membrane-bound polysomes from MOPC-21 cells regardless of their physiological state. Separation of Free and Membrane-Bound Polysomes by Differential Centrifugation Cells were lysed by Dounce homogenization and a PNSW (post- nuclear supernatant plus wash) was prepared as described in Materials and Methods and centrifuged at 27,000 x 9 (max) for 5 minutes. Unless otherwise noted, the percentage of membrane-associated poly- somes was calculated as . * . A260 units in pellet ' ' + A260 units in supernatant A 100 x . . 260 units in pellet or if the polysomes were labeled, CPM in pellet CPM in supernatant + CPM in pellet 100 x The pellet contained membrane-associated polysomes and the supernatant free polysomes (Kimmel, 1969). However, a major concern was how efficient a separation of membrane-bound from free polysomes had been achieved. * A260 units = absorbance at 260 nm minus absorbance at 320 nm. 3 ”VJ SC S 39 Figure 2 shows two sets of data. The first set of data was gathered early in this study and is summarized by the regression lines. A greater concentration of A units/m1 of sample centri- 260 fuged resulted in a greater amount of A units found in the pellet. 260 Regression was done by translating conventional thoughts about least squares analysis (as discussed in Kreyszig, 1970, sections 17.1-17.5) into a FORTRAN program to analyze the data points. Early in the study a buffer lacking sucrose (RSB) was used to lyse the cells, while shortly thereafter and for the remainder of the study, 0.25 M sucrose was included with the RSB isolation buffer. Since this may have made a difference, two lines were calculated, one for all points and one for those measurements taken when 0.25 M sucrose was included with the buffer. The second set of data shown in Figure 2 and Table 2 shows the mean values for percentage membrane-associated polysomes versus sample volume, bracketed by their corresponding standard deviations, Table 2. The effect of sample volume on the percentage of membrane- associated polysomes obtained by differential centrifuga- tion at 27,000 x 9 (max) for 5 minutes volume of mean percent membrane— standard number of sample (ml) associated polysomeS' deviation measurements 0 - 1.5 32.8 5.4 16 1.5 - 2.5 31.1 3.64 16 2.5 - 3.5 29.1 3.9 11 3.5 - 4.5 29.5 4.1 6 40 Figure 2. Variations in the percentage of membrane- associated polysomes isolated in the presence of RSB (O) and RSB(such) (O). The regression lines summarize variations which accompany changes in A260 units/ml of sample for all points (—--) and for polysomes isolated in the presence of RSB(such) (-——9. The connected points (+-—+) summarize data presented in Table 2 on the average variation which accom- panies a change in sample volume. N PELLET 260 /\ PERCENT 45 PELLET | N PE T RCEN A250 41 VOLUME OF SAMPL E 0.0- 1.5- 2-5- 3.5- 4.5- 1.5 2.5 3.5 4.5 5.5 45 M O 6‘: (0 o 6 C O) 1O 20 30 4O 50 60 7O 80 A260 PER ML Figure 2 42 with the total number of points used for the calculation in paren- theses. Measurements were made throughout the latter part of this study. It is apparent that as the sample volume increased, the percentage of membrane-associated polysomes decreased! A t test was performed to determine whether the results obtained with the largest sample volume (4.5 to 5.5 ml) differed significantly from the results obtained with the smallest sample volume (0.0 to 1.5 ml). According to the t test, the possibility that there was no difference could be rejected at a significance level of 5%. Explanations were sought for the variations in the percentages of membrane-associated polysomes obtained by differential centri- fugation at 27,000 x g for 5 minutes. These variations were not reported by other investigators, although many did wash the crude 27,000 x g pellet before using it as a source of membrane-associated polysomes. RSB(such) was the buffer routinely used in the isolation of membrane-associated polysomes. The low salt concentration of the buffer presented the possibility that some fonm of ionic interaction resulting in aggregation could occur at higher sample concentrations. To test this possibility membrane-associated polysomes were isolated in the presence of different concentrations of cations: 25 mM KCl, 160 mM NaCl, 2 mM lysine (pH 7.3), 20 mM lysine (pH 7.1), 6 mM Mg++, and a dialyzed 0.5 M KCl wash fraction (which may or may not have been cationic). No appreciable effect was observed, although 160 mM NaCl and 20 mM lysine did slightly reduce the concentration of membrane-associated polysomes obtained from concentrated samples. 43 When 3H-labeled free polysomes (PMS,S3 as prepared in Materials and Methods) were added to the PNSW before centrifugation, 160 mM NaCl did prevent sedimentation of some of the polysomes as compared with a control performed with low salt. Nevertheless, this study was completed using standard RSB(such) with its low salt concentra- tion, primarily because the sucrose gradient profile of non— detergent treated MP1 showed evidence of being contaminated with free polysomes even when MP1 was isolated in the presence of 160 mM NaCl (profiles not shown). In summary, ionic interactions may be a factor, but they are not the sole or even major source of the variations in the amount of polysomes found in the pellet. To test whether differences among cell types could be detected with differential centrifugation, PNSW fractions and membrane- associated polysomes from three different cell types were prepared as previously described. The results are shown in Table 3. The results indicate that differential centrifugation for 5 minutes at 27,000 x 9 (max), even with all its variability, was sufficient to detect differences among cell lines. As compared to the MOPC-Zl cell line (data summarized in Figure 2), the 849.1 cell line had a generally greater percentage of membrane-associated polysomes, the XC.1 cell line had approximately the same percentage of membrane- associated polysomes, and the 5180 cell line contained fewer membrane-associated polysomes. What is the source of the variations observed in percentages of membrane-associated polysomes? What can be done to obtain a well defined, pure fraction of membrane—associated polysomes? These 44 Table 3. The percentage of membrane—associated polysomes in various cell lines sample A260 per m1 percent membrane-associated cell line volume of sample polysomes 849.1 2.0 5.36 58.8 1.0 44.1 47.5 1.0 24.5 50.0 1.0 9.8 45.2 1.0 4.9 42.8 XC.l 1.0 25.0 34.9 5.0 5.0 25 0 1.0 26.6 26.7 1.0 13.3 25.8 1.0 5.3 13.5 5.0 5.3 17.9 8180 2.0 29.8 18.0 2.0 14.9 17.8 2.0 5.1 15.6 1.0 66.8 19.2 1.0 26.7 17.8 1.0 6.7 18.3 5.0 6.7 11.6 5.0 26.1 11.9 45 questions are answered in the discussion with the help of the following pertinent data. A PNSW was prepared as described in Materials and Methods. After centrifugation for 5 minutes at 27,000 x 9 (max) the pellet, MP1, was resuspended in RSB(such). Equal aliquots were taken and one aliquot was treated for 5 minutes with 0.5% DOC TX—lOO to solu- bilize membranes. Each aliquot was analyzed by linear sucrose gradient centrifugation. Figure 3A is a representative profile of an MP1 fraction which has not been treated with detergent, and Figure 3B is the profile of the detergent treated replicate MP1 fraction. It is evident that a polysome profile appears in the non-detergent treated sample with a large peak of putative membrane— aggregate (non-microsomal) at the bottom of the gradient. A frac— tion of the sample also pelleted. The polysome profile apparently reflects the presence of free polysomes. The large peak disappears on exposure to DOC TX-100. Table 4 shows the results of six differential centrifugations of a non-detergent treated PNSW which had first been column chromato- graphed on Sepharose 68 as described in Materials and Methods. The polysomes had been labeled for 22 hours with 1.0 uC of 3H-uridine per ml. Notice that material, albeit less material, continues to pellet after the first spin which supposedly removed the membrane— bound polysomes. Similar percentages of pelleted material on second and third centrifugations were observed in other experiments, even in one in which the Sepharose chromatographed PNSW had been treated with either 0.5 or 1.0% DOC TX-100. This indicates that a fraction of free polysomes has pelleted. 46 .Amomv mmsomocos mmumoflccfl zouum .AIIIS «mma .ooH-xe coo wm.o cuHB Umummwe .m .pmummuu ucmmnmumbucoz .4 .Aamzv mmuscHE m How AmeV q x ooo.nm um coflummSMAuu Icmo HmflucmumMMHp >Q Uwumaamm mmsom>aom cwumHUOmmMIwcmwQEmE mo mmHflmoum ucwflpmwm wmouosm .m musmam ZO;Ooa oom.vvo.H umsm m.m o.m oom.em mm6.mmm umaamm mcoc o.Hm m.mH snm.omfl oov.mvm.m umsm 4.4 k.m ohm.qm wma.oov umaamm mmmzm He m.om m.mH mmm.kaa ooo.mmn.a umsm o.mm o.H mem.~oa Nk~.kmn umaamm mac: 4.4m m.Hm smm.kaa omm.mam.a umsm o.mm o.H mmh.mm 4mm.mm umaflmm mmmzm Ha m.m~ H.0H mmw.vm mom.oo~ umsm v.m o.H vam.qo mm~.mv umaamm mcoc H.- m.ka www.mm mom.0m~ umsm v.m o.H mv~.em oma.moa “madam mmmzm He 6.6H m.o mmH.me om~.>mv.a umsm o.m o.m N»~.Hm Ham.hmfl panama macs m.oH H.0H mee.om mam.aek.a umsm o.m o.m www.mm Hmm.mmH “madam .«mmmzm He m.mH m.ma mmm.mm eHv.omH.H umsm m.vm o.H nma.kv omm.kom umaamm mcoc «.mm H.Hm www.mo omn.~ma.a .umsm m.vm o.H ucmEumwuu umaamm uwaamm coma Emu mHQEMm HE wEsHo> cw comm w ca zmo w “mm zmo umm comm cofiummsmwwucmo Hmflucmwmmmwp an Uwumaamm Hwflumumfi mo muwucmsv mcu c0 mmmzm mo uommmm one .m wanna 49 percentage of membrane-associated polysomes differs between experi- ments. This is one case in which the physiology of the cells, as discussed in the first part of the Results section, might have made a difference in observed percentage of membrane-associated polysomes. It is possible that not all membrane-bound polysomes are pelleted during differential centrifugation of a larger sample volume. If this is so, then one would expect a second extensive application of differential centrifugation (for example, 30 minutes instead of 5 minutes) to pellet more membrane-associated polysomes from the supernatant of a previously centrifuged 5 m1 sample than from the supernatant of a previously centrifuged 1 m1 sample. The results of Table 6 were consistent with this possibility, but the results Table 6. A test for unpelleted microsomal material by extensive centrifugation sample volume of sample (m1) A260 per ml % A260 pelleted Initial isolation (27,000 x g for 5 min) A 1.0 25.0 37.2 B 1.0 50.0 36.4 C 5.0 5.0 24.7 D 5.0 10.0 25.7 Second centrifugation (27,000 x g for 30 min) A 4.8 2.7 12.4 B 4.8 5.4 13.5 C 4.6 3.5 15.6 50 were not unequivocal so a second experiment was performed with labeled polysomes. The results of the second experiment are presented in Table 7. Only a very slight difference is seen in the percentage of radio- activity pelleting in the second differential centrifugation step. This indicates that not many, if any, membrane-associated polysomes are left in the sample with the larger volume after the first dif- ferential centrifugation, or at least not many more than are left in the sample with the smaller volume. There is, however, a marked difference in the quantity of absorbance units pelleted in the second differential centrifugation in contrast to the results noted with the radioactivity. The supernatant of the sample with the large volume Table 7. A test for unpelleted microsomal material by extensive centrifugation volume of A260 per CPM in % CPM in % A260 in sample sample (ml) ml pellet pellet pellet Initial isolation of 27K x g pellet 27K x g for 5 minutes A 1.0 25.0 300,038 38.5 35.8 B 5.0 5.0 200,390 18.1 27.2 C 1.0 10.3 134,461 29.5 32.3 Second 27K xjg centrifugation 27K x g for 30 minutes A 4.5 2.6 72,281 11.8 8.9 B 4.5 3.8 103,286 12.5 13.0 C 4.5 1.1 28,411 10.7 8.9 51 in the first differential centrifugation pelleted the most absorbance units in the second differential centrifugation. Since the extra absorbance was not associated with the polysomal radioactivity, it represented miscellaneous non—microsomal membrane fragments. The data in Table 8 support this interpretation. Cells were incubated for 29 hours with 0.5 uC of 3H-choline/ml to label membrane. A PNSW Table 8. The percentage of membrane material pelleted by repeated differential centrifugation centrifu- CPM per % CPM in A260 in gation fraction CPM* 3260 pellet pellet initial supt 119,248 9,755 47.0 16.6 pellet 105,777 43,422 second supt 70,920 -** 20.1 - pellet 17,851 - third supt 54,581 6,606 8.3 - pellet 4,951 - * cells labeled with 3H-methyl choline for 29 hours ** not determined was prepared, chromatographed on Sepharose 6B, and then subjected to three consecutive differential centrifugations at 27,000 x 9 (max). The sample volume was about 3.0 m1. It is apparent that a consider— able amoiunt of membrane which did not pellet in the first differen- tial centrifugation was able to pellet in the second differential centrifugation and, further, a significant amount of membrane was available to be pelleted in the third differential centrifugation. 52 Free polysomes were detected in the 27,000 x g pellet by wash- ing MP1 fractions which had been isolated from samples with large and small volumes. The results are shown in Table 9. Supernatants from the washes of pellets from smaller volume samples contained considerably more A units than supernatants from the washes of 260 pellets from larger volume samples. In relation to polysomal Table 9. The percentage of membrane—associated polysomes that will pellet after resuspension volume of A260 per % A260 in % A260* sample sample ml pellet repelleted A 1.0 47.9 38.8 65.7 B 1.0 25.0 - 38.5 62.0 C 5.0 5.0 29.7 75.9 D 5.0 5.0 29.9 80.3 * repelleted = resuspended pellet in 1.5 ml and centrifuged for 5 minutes at 27,000 x 9 (max) material, the interpretation given is that more free polysomes pelleted in the smaller volume than in the larger volume during the initial centrifugation. Isolation of Membrane-Bound Polysomes by Partition Separation in an Aqueous Polymer Two-Phase System in Combination with Differential Centrifugation The data from the previous section indicated that a single dif— ferential centrifugation of a PNSW would not adequately separate membrane-bound polysomes from free polysomes. Free polysomes were apparently contaminated with negligible amounts of membrane—bound 53 polysomes after one differential centrifugation; thus following two additional differential centrifugations, the supernatant (PMSB) was used as a source of free polysomes. Membrane-associated polysomes obtained after one differential centrifugation needed to be further processed in order to separate membrane-bound from contaminating free polysomes. The approach taken was to combine differential centrifugation with partition separation. Albertsson (1959) reported that the partition behavior of a particle in a dextran—methylcellulose aqueous two-phase polymer system depended primarily on the surface area of the particle. The partition coefficient in a liquid two-phase system is defined as: concentration of substance in top phase concentration of substance in bottom phase Polysome fractions labeled with 3H—uridine were prepared and then partitioned once in a D68-MC4000 aqueous two-phase polymer system as described in Materials and Methods. The partition coef- ficient for the crude pellet of membrane-associated polysomes MP1 was 0.043. The partition coefficient for the free polysomes PMS,SB was 0.179. The partition coefficient for an unlabeled PMS was 0.353. These values agree fairly well with expected values put forth by Albertsson (1960, p. 178). The values support what is readily ascertained visually--that membrane-bound polysomes partition into the bottom phase. Membrane-associated polysomes (MP1) labeled with 3H-uridine were processed by a combination of differential centrifugation and partition separation. The final pellet (MP2XP) was resuspended in 54 RSB(such) and analyzed on linear sucrose gradients. Figure 4 shows a profile of non-detergent treated MP2XP. The polysome profile which could be seen in non-detergent treated MP1 (Figure 3A) is not evident. Figure 4 also shows the profile of a replicate aliquot of MP2XP treated with 0.5% DOC TX-100 (the profile is labeled " + DOC"). Figure 58 is a plot of CPM per fraction and Figure 5A is a similar plot of percent radioactivity for both the non-detergent and detergent treated MP2XP. Notice that the large peak at the bottom of the gradient shown in Figure 4 does not have a correspondingly large amount of radioisotope. The exact nature of this peak remains something of an enigma. Further characterization of polysome fractions was sought by following enzyme markers throughout the isolation procedure. Free and membrane-bound polysomes were isolated as described in Materials and Methods with two modifications: (1) the PMS was chromatographed on Sepharoxe 6B before further differential centrifugation, and (2) membrane-bound polysomes were not partitioned a second time with the top phase of a blank; instead they were recovered by dif- ferential centrifugation and partitioned a second time in a com— pletely fresh system as at first. Various parameters of the isolation procedure are presented in Table 10. The final pelleted microsomal fraction has been designated MP6. The membrane fractions have typi- cally lower A / ratios. The Sepharose chromatographed PMS 260 A280 has a typically higher A / ratio. The yields shown for 260 A280 recovery of A units from the Sepharose column are representative; 260 that is, when this column was used an average of about 47% of the 55 + DOC A254 \ T 8 Figure 4. Sucrose gradient profiles of membrane-bound poly- somes partitioned in a dextran-methylcellulose aqueous polymer two- phase system. Arrow indicates monosomes (805). A254 (--). 56 1 ”T I 10+ CPM °/o 103 CPM 4; T 16 20 B FRACTION Figure 5. Radioactivity profiles of the gradients shown in Figure 4. A. Percent CPM per fraction. B. Absolute CPM per fraction. Non-detergent treated (O-——O). + detergent (O———O). 57 Table 10. Summary of enzyme marker data on polysome fractions parameter PNSW PMS,Sl MP6 A26O/A280 1.648 1.765 1.466 ' * % yield A260 46.7 32.2 units G-6-Pase** 22.7 7.9 (76.7)* 7.0 (111.0) 33.6 3.3 (13.3) 3.2 (52.5) units 5'-mononuc 130.7 16.4 (26.3) 15.1 (30.2) 218.7 54.9 (63.9) 12.4 (20.8) 212.4 95.4 (77.6) 8.4 (17.7) * PMS,Sl . MP6 . —_ = % I — = % P PMS x 100 yield PMS 81, MP1 x 100 yield M 6 ** one unit = one microgram phosphate released per minute A260 units (PMS,Sl) of a PMS could be recovered. The final method adopted for partition separation (using the top phase of a blank) gave slightly higher yields, 40 to 50%, of membrane-bound polysomes (MP6) than the yields of about 30 to 36% recorded in Table 10. One microgram of phosphate released per minute was defined as a unit. Table 10 also presents a summary of the glucose-6- phosphatase activity found in the polysome fractions. It appears that the reaction was not optimized (data not shown), and therefore was not sensitive enough to serve as a probe for microsomal membrane. Nevertheless, the results suggest a slight enrichment for micro- somal membrane in the partitioned fraction. It also seems that some enzymic activity remains in the PMS after the first differential 58 centrifugation. This activity is probably associated with small fragments of membrane not associated with polysomes. Table 10 further shows the activity of 5'-mononucleotidase found in the polysome fractions. An adequate amount of activity was obtained with this enzyme. Again, some enzyme activity is still retained by the PMS after one differential centrifugation. This is consistent with the data presented in Table 8 and could be due to small frag— ments of plasma membrane which will not pellet under the conditions employed. The activity was reduced in the partitioned fraction. This may indicate that some of the plasma membrane fragments do not partition with the microsomes during the isolation procedure. Alterna- tive reasons for reduction in activity such as competing enzyme reac- tions or inactivation due to harsh treatment were not investigated. The sucrose gradient profiles of non-detergent treated MP1 and MP2XP (partitioned MP1) differed, as has already been mentioned. To examine the difference between these fractions from another perspec- tive, MP1 and MP2XP were either exposed to RNase or to 0.5 M KCl and then pelleted by differential centrifugation. Untreated frac- tions served as controls. The results are shown in Table 11. Con- sistently more A units remained in the supernatants of crude 260 (MP1) fractions than in the supernatants of partitioned (MP2XP) fractions. This indicated that the partitioned fractions had been separated from free polysomes, which presumably contaminated the crude fractions. Another approach to determine whether free polysomes contami- 3 nated the MP2XP fraction was to add H-uridine labeled free polysomes to a PNSW and to proceed through the fractionation scheme and 59 Table 11. Differences between partitioned and crude membrane- associated material following treatment with RNase or high salt isolation A260 per m1 procedure fraction % A260 treatment 3.75 27,000 x g supt 22.4 none** pellet 77.6 3.41 partition supt 2.4 none pellet 97.6 3.75 27,000 x g supt 36.6 RNase A*** pellet 63.4 3.41 partition supt 20.2 RNase A pellet 79.8 3.75 27,000 x g supt 30.8 0.5 M KC1** pellet 69.2 3.41 partition supt 9.6 0.5 M KCl pellet 90.4 2.94 partition supt 12.9 none pellet 87.1 2.94 partition supt 16.6 RNase A pellet 83.4 2.94 partition supt 17.6 T1 RNase**** pellet 82.4 2.94 partition supt 12.6 0.5 M KCl pellet 87.4 * all samples 1.0 ml volume ** 5 min at 4°C *** RNase A 1 min at 37°C, **** Tl RNase l min at 37°C, 1 microgram/ml 10 units/m1 60 determine radioactivity at each step. The free polysomes (PMS,SB) were highly labeled making it possible to add only two A260 units (less than 2% of the total). Thus, the environment of the PNSW remained essentially the same after the addition of the labeled free polysomes. Table 12 gives the percentage counts found with each fraction. The volume for the first differential centrifugation was 2.9 ml, there were 46.1 A units/ml, and the percentage membrane- 260 associated polysomes was 23%. Only 0.4% of the counts remained Table 12. Separation of membrane-bound polysomes from added labeled free polysomes by a combination of differential centrifugation and partition separation Fraction: PNSW PMS MP1 MP2XP Loose CPM in 333,790 282,003 30,195 1,246 11,676 fraction: Percent CPM: 100 84.5 9.1 0.4 3.5 associated with the membrane-bound polysomes. The expected percentage based on the yield of MP2XP from MP1 was 2.3%. The percentage CPM of MP2XP plus Loose do not add up to that found in the MP1 since MP2XP and the loose fraction were isolated from only a fraction of the total MP1. The percentage of CPM in this fraction was 6.0%. A combination of differential centrifugation and partition separation was sufficient to allow only a negligible quantity of free polysomes to contaminate the membrane-bound polysomes. 61 Figure 6 shows profiles of the various fractions. The profiles of MP1 are shown in Figures 3A and 38. The percentage CPM of labeled free polysomes per fraction has been plotted with certain absorbance profiles. It is obvious that a labeled free polysome profile is not present in the MP2XP fraction, but it is present in all the other fractions. Notice also that the large peak at the bottom of the gradient of non-detergent treated MP2XP is not enriched for labeled free polysomes, thus tending to preclude it from being implicated as an aggregate involving free polysomes. It is interesting to note that the detergent treated MP2XP fraction seems to be somewhat degraded (enriched for smaller poly- somes, monosomes, and subunits) in contrast with all the other frac- tions. This same occurrence has been noted in other experiments. This implies that membrane—bound polysomes can be degraded (or appear degraded) while other polysome populations in the cell are not. An artifact, if it exists, would have to be one that could exclusively cause degradation of membrane-bound polysomes. The detergent or the aqueous polymer could be the source of such an artifact. Nuclease action of the polymer (that is, the polymer being a nuclease or containing a nuclease) is ruled out because the loose polysomes have an undegraded profile and were also extensively exposed to the polymer. The possibility that Mg++ precipitation artifically produced a good profile was ruled out by precipitating polysomes in the presence of a small amount of RNase. The profile of the precipitated polysomes showed complete degradation (profile not shown). The possibility that the detergent contained a nuclease was ruled out because other detergent treated fractions were not 62 Figure 6. Separation of membrane-bound polysomes from added labeled free polysomes by a combination of differential centrifugation and partition separation. A. A. PNSW. B. PNSW + detergent. C. PMS B. A. MP2XP. B. MP2XP + detergent C. Loose (Figure 3 shows the MP1 profiles) A254 (-—). Percent CPM (O——O) 64 mm mwswwm ZO_._.U