derived from liver. A subsequent paper will report the cell-free synthesis, by

Transcription

1 STUDIES ON CELL-FREE SYNTHESIS OF RAT IMMUNOGLOBULINS, I. A CELL-FREE SYSTEM FOR PROTEIN SYNTHESIS PREPARED FROM LYAMPH-NODE MIICROSOMAL VESICLES BY PIERRE VASSALLI DEPARTMENT OF PATHOLOGY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE Communicated by Michael Heidelberger, September 29, 1967 The heterogeneity of the cell population of antibody-producing tissues poses a potentially serious problem in attempts to devise a cell-free system in which the primary interest is to study immunoglobulin synthesis. Since it is believed that proteins intended for excretion are synthesized mainly, if not exclusively, on the membrane-bound ribosomes of the endoplasmic reticulum (ER), and since this reticulum is much better developed in plasma cells than in most other lymphoid cells, it was decided to select the ER membranes of lymph nodes as a protein-synthesizing fraction. This paper shows that it is indeed possible to prepare from lymph nodes a cell fraction consisting almost exclusively of ER microsomal vesicles, which is, in optimal conditions, as active in cell-free protein synthesis as systems derived from liver. A subsequent paper will report the cell-free synthesis, by this cell fraction, of immunoglobulin with antibody activity.' Materials and Methods.-Sprague-Dawley rats of gm were injected with 2 X 109 killed H. pertussis (Eli Lilly Co.) in each footpad. ATP, GTP, phosphoenolpyruvate (PEP), pyruvate-kinase, nonradioactive amino acids, and puromycin dihydrochloride were obtained from Sigma Co., H3-L-leucine (5 c/mm) from New England Nuclear Corp., polyvinylsulfate from Eastman Organic Chemicals, and ribonuclease from Worthington Biochemical Corp. Fractionation of tissue: Six to eight days after immunization the rats were killed, and popliteal, axillary, and cervical lymph nodes excised and minced in small fragments with fine scissors in chilled Hanks' solution. Nodes from up to 15 rats were pooled. The fragments were centrifuged, resuspended in 3 vol of medium A (0.05 M Tris-HCl, ph 7.7; M KCl, and M MgC12) containing M 2-mercaptoethanol, 4 pg/ml polyvinylsulfate, and 0.88 M sucrose, and homogenized under ice by 10 strokes of a loose-fitting Teflon-glass, motor-driven homogenizer, followed by 3 strokes of a tight-fitting Dounce homogenizer. The homogenates were centrifuged 20 min at 20,000 g, and the supernatant was further centrifuged 20 min at 59,300 ga,. in a Spinco L2-65 ultracentrifuge (30,000 rpm in a type-40 rotor) to obtain a microsomal fraction. The last supernatant was centrifuged 90 min at 105,000 ga., or 60 min at 226,400 a in a 50 Ti rotor to obtain cell sap. All centrifugations were performed at -40C. To obtain liver microsomes, livers were homogenized in the same medium but centrifugation of the 20,000 g supernatant was for 60 min at 105,000 g.mz (34,000 rpm with rotor 40), which sediments the rough membranes of the liver.2 Liver sap was prepared as lymph node sap, except that when only cell sap, and no microsomes, was needed, homogenization was in 0.25 M sucrose. "ph 5 Fractions" were obtained from diluted cell sap by precipitation with 1 M acetic acid at ph 5.2 at 0C, and resuspension of the precipitate in the incubation medium described below. 'When necessary to remove free amino acids from cell sap, ph 5 fraction, or the supernatant of ph 5 fraction, 10 ml were passed through a 20 X 1.5- cm column of Sephadex G25 equilibrated with medium A and the first 10 ml of the eluted protein solution were collected. Analyses of free and membrane-bound ribosomes were performed by centrifugation over 2 M sucrose as described by Loeb et al.3 Cell-free incubation: Incubations were carried out at 370C in a volume of 0.5 ml of incubation medium (Tris-HCl, M, ph 7.7; MgCl2, MU; KCl, 0.07 M; 2-mercaptoethanol, M; polyvinylsulfate, 4,ug/ml; sucrose, 0.1 M), containing: ATP, M; GTP, 0.4 mm; PEP, 2117

2 2118 BIOCHEMISTRY: P. VASSALLI PROC. N. A. S M.k; pyruvate-kinase, 40 lug/ml; H3-leucine, 6-1() uc/ml. The amounts of inicrosonmes, (ell sap, ph 5 fraction, or other cell fractions are indicated under Results. Incubations were stopped by precipitation in 0.5 M HCl04; tubes were left 10 min in ice and 20 min at 90'C, centrifuged, and the sediments were washed 3 times with 0.25 M HC104, once with ethanol-ether (3:1), and once with ether. The pellets, dissolved in 0.5 ml of formic acid, were added to 4 ml of ethanol and 15 ml of toluene containing Omnifluor (4 gm/i) (New England Nuclear), and counted in a Packard liquid scintillation counter. Formation of aminoacyl-trna was determined similarly with the omission of the 900 treatment, all steps being performed at 4VC. All assays were done in duplicate. Radioactivity measured in control, nonincubated tubes was subtracted from all results. Proteins were measured by the method of Lowry et al.4 and RNA by the method of Fleck and Munro.5 Results and Discussion.-Characteristics of the tissue used: Lymph nodes of rats injected in the footpads with H. pertussis appeared especially suitable for establishing a cell-free system for immunoglobulin synthesis, since, in this species, the injection results in a 10- to 20-fold increase in size of the draining lymph nodes associated with massive accumulations of plasma cells. Except for plasma cell tumors, such tissue probably contains the largest available concentration of plasma cells. Electron microscopic study of lymph nodes one week after immunization with H. pertussis shows that only two types of cells are rich in ER: cells of the plasma cell series and large cells with imprecise outlines, present in the medulla between plasma cells, whose exact nature is unknown, but which can tentatively be classified as macrophages or reticular cells (Fig. 1). Thus, any microsomal fraction selected from such lymph nodes by differential centrifugation would contain mainly the membrane-bound ribosomes of these two types of cells. Characteristics of the protein-synthesizing cell fractions used: A medium containing 0.88M sucrose was selected for homogenization, since in high molarity of sucrose, lysosomes appear to be more stable6 and selective separation of rough membranes by differential centrifugation can be achieved more easily. In order to reduce possible contamination of the microsomes by heavier particles, such as lysosomes, the supernatant of a centrif- 4.. ~ ~ ~ ~ ~ ~ / ugation of 20 minutes at 20,000 g was. used for further steps, although 50 per cent of the ribosomes are found on * microsomes in the 20,000 g pellet. In order to separate microsomes from free ribosomes this supernatant and FIG. 1pellets obtained from it after various node ay ate imuiztin it r-centrifugation schedules were centritussis.fuged on sucrose gradients to analyze ththeir content for microsomes and free on the right. pribosomes. In these experiments the -ell ~~> s' *~~~~~~~'~~~ anim~~~~als were pulse-labeled withhleucine so that the distribution of FIG. 1.-Electron micrograph of a rat lymph nascent, ribosome-bound polypeptide node 7 days after immunization with H. per- chains could also be studied. The tussis. High magnification of a field, showing the flattened ER of a mature plasma cell at the degradative action of tissue ribonulower left, contrasting with the round shape of clease on polyribosome structure was the rough vesicles of a macrophage or reticularprvndalesprtlyb th cell on the right. rvnea es atalb h

3 VOL. 58, 1967 BIOCHEMISTRY: P. VASSALLI 2119 addition of fresh liver sap to the homogenizing mediunm and to the cell fractions analyzed; liver sap contains powerful inhibitors of rihonuclease, and its use allows good preservation of the polyrihosome profile on the sucrose gradients.7 8 Sucrose-gradient analysis of the whole 20,000 g supernatant shows microsomes, free polyribosomes, and free single ribosomes (Fig. 2A). Centrifugation for 20 minutes at 59,300 gav. gives a pellet which appears to be made selectively of microsomes or fast-sedimenting particles without significant contamination by free ribosomes (Fig. 2B). After solubilization of the membranes by deoxycholate, the ribosomes in this microsomal fraction are mostly polyribosomes of very heterogeneous size, which are broken down into single ribosomes by mild treatment with ribonuclease (Fig. 2C). It was not possible to detect polyribosome peaks corresponding to the synthesis of H and L chains of immunoglobulin. For further characterization, the microsomal pellet was resuspended and submitted to prolonged centrifugation over a layer of medium containing 2 M sucrose; the free ribosomes sediment through high density sucrose, but not the bound ribosomes, which are attached to membranes of low density.9 A small degree of contamination by free polyribosomes, which are undetectable on sucrose gradients probably because they sediment with the microsomal peak, thus becomes apparent, but these free ribosomes do not represent more than per cent of the total ribosomal content of the pellet. Electron microscopic study of the pellet shows that it consists mainly of smooth and rough membranes, forming vesicles of variable size (Fig. 3). The surface layer of the pellet has the same composition, but the bottom contains cell debris, mostly nuclear, representing no more than 2-3 per cent of the total volume CPM OD 260 CPM OD 260 CPM,'. OD- 05 OD 00 CPM--_ 20- CPM,. CPM- OD AFT_- 0,4 zoo0 10-2oo00~ t ': 0 500o I FRACTION NUMBER (A) (B) (C) FIG. 2.-Sucrose density-gradient analysis of cell fractions. Rats were killed 5 min after ,c H3-leucine I.V. Pooled lymph nodes were homogenized and centrifuged as in Methods but in a medium containing fresh liver sap. Cell fractions in 0.25 M sucrose (1 ml vol) were placed on top of linear 10-34% sucrose gradients in med. A. In (A) and (B), gradients consisted of 25 ml on top of a bottom layer of 5 ml of 2 M sucrose in med. A.; in (C) of 30 ml without bottom layer. Gradients were centrifuged in a Spinco SW25.1 rotor at 25,000 rpm for 150 min at -4C. Fractions were collected through a 20-gauge needle and absorbancy at 260 mya (1-cm light path) read at appropriate dilutions (corrections for ferritin were found unnecessary). One mg of bovine serum albumin was then pipetted into each fraction, which was precipitated in 0.5 M HC104 and processed for determination of radioactivity as under Methods. (A) Whole supernatant of a 20,000 g centrifugation. The homogenizing medium contained 0.25 M sucrose instead of 0.88 M; microsomes peak at tube 8, and free, single ribosomes at tube 33. (B) Pellets obtained after a 20-min centrifugation at 59,300 g., of the 20,000 g supernatant. (C) Pellet similar to (B), but resuspended in medium containing 1% deoxycholate with either liver sap to protect polyribosomes, or ribonuclease (1 pg 5 min at 250C) to break these structures.

4 2120 BIOCHEMISTRY: P. VASSALLI PROC. N. A. S. Characteristics of the cell-free system: After preliminary trials, it became apparent that both the concentration of microsomes and the nature of the cell sap or ph 5 fractions are critical factors in cell-free systems derived from lymph nodes (LN). (1) Effect of microsomal concentration and presence of inhibitors of cell-free protein synthesis on microsomal membranes: Under otherwise optimal conditions, as described later, incorporation of H3-leucine into polypeptide chains is strikingly dependent upon the concentration of LN microsomes: it increases linearly up to LIVER 0 x *vhi Or*1 0 /LYMPH NODES.: i MG MICROSOMAL PROTEIN/ML FIG. 3. Electron micrograph of the FIG. 4.-Effect of microsomal concenmicrosomal pellet used for lymph node tration on H8-leucine incorporation in cell-free synthesis. Rough and smooth cell-free systems containing lymph node vesicles, and small circular clusters of or liver microsomes and 2 mg/ml of liver membrane-bound ribosomes are seen. ph 5 fraction. Incubation, 20 min at 370C. microsomal concentrations of 1 mg/ml, then decreases, and is very low at 4 mg/ml. In contrast, when liver microsomes are used, the incorporation increases almost linearly with microsomal concentrations up to 4 mg/ml (Fig. 4). Protein synthesis proceeds for 20 minutes or more with 0.5 or 1 mg/ml of LN microsomes, but usually stops after 10 minutes with 2 mg and after 5 minutes with 4 mg/ml (Fig. 5). Decrease in the efficiency of the system with increase in concentration of microsomes suggested the presence of inhibitors of protein synthesis in the microsomal fraction used. Assays for ribonuclease and acid phosphatase ruled out lysosomal contamination, since the concentration of these enzymes was almost as low as in the liver microsomal fraction, which does not show an inhibitory effect at high concentrations (Table 1). Inhibiting factors seemed therefore associated with the microsomes themselves, and each of the two steps required for incorporation of amino acids into polypeptides was investigated as a possible target for their action. (a) Inhibition of binding of amino acids to trna by microsomal A TPase: When the formation of H3-leucyl-tRNA is measured in a ph 5 fraction from liver incubated for five minutes in the presence or absence of microsomes, the microsomes have an inhibitory effect which is more marked with LN than with liver microsomes, and is maximal with spleen microsomes (Fig. 6). Spleen microsomes

5 VOL. 58, 1967 BIOCHEMISTRY: P. VASSALLI 2121 were used because spleen cell-free systems are even more inhibited by increasing microsome concentration than LN systems.10 When the deacylation of preformed H3-leucyl-tRNA is tested during similar incubations performed in the presence or absence of microsomes, LN and spleen microsomes considerably enhance the speed of deacylation, an effect which can be duplicated, in the absence of microsomes, by adding AMP to the system. This effect of the microsomes of 20-1 L 20 2IG/K ML 4 04 X, 0.5 mg/ml_ CL MG/ML II I I 1 1, I I I TIME (MIN.) FIG. 5.-Effect of concentration of lymph node microsomes on duration of H3-leucine incorporation in cell-free system. lymphoid organs on the amino acid-trna linkage could be explained by an excess of ATPase in these fractions, since a poor aminoacylation of trna can result from a rapid hydrolysis of the ATP necessary for the reaction, and accelerated deacylation from accumulation of AMP, the reaction being reversible.1" ATPase assays on microsomal fractions of lymphoid organs showed an activity 7 times (LN) to times (spleen) higher than in liver microsomes (Table 1). That a relative lack in aminoacyl-trna, due to an excess of ATPase, is indeed an inhibitory factor in cell-free systems using high concentrations of LN and spleen TABLE 1 ENZYME ASSAYS ON MICROSOMAL FRACTIONS ATPaset RNase' Acid Phosphatas t (micromoles of Pi (cpm of HZ-labeled RNA (micromoles of p-nitrotihenol liberated/mg micros. rendered acid-soluble) liberated/mg micros. prot./min) prot./min) Micr. liver 180 d 10 Micr. liver 0.13 Micr. liver 0.14 Micr. LN 240 i 20 Micr. LN 0.25 Micr. LN gsg RNase 5300 ± 400 Micr. LN "washed" 0.22 Micr. spleen 2.7 * Acid-soluble radioactivity present in an aliquot of the 30-min incubation of 0.5 mg of RNA containing H3- uridine (1.5 X 105 cpm) with 0.5 mg microsomal protein in 1 ml of inol~bation mixture ph 7.7. t Assay performed with p-nitrophenylphosphato, 0.02 M at ph 5.6" with 0.05 and 0.1 mg microsomal prot./ml. At ph 7.7, values were times lower. "Washed" microsomes Otre obtained by resuspension of the microsomal pellet in med. A with 1.53 M sucrose followed by centrifugatio4,eliminating from the pellet any contaminating lysosomes, since in this density of sucrose (1.2), the lysosomal Articles of lymphoid tissue (dens and 1.19) do not sediment.'6 "Washing" has been used successfully to rsihove the lysosomes contaminating a microsomal fraction of the liver, thus markedly reducing the acid phospihatase content and enhancing the proteinsynthesizing ability of this fraction." "Washed" LN microsomes have no improved efficiency in protein synthesis. t Assay performed in 1 ml of incubation medium, ph 7.7, containing ATP, M, and microsomes (12.5, 25, and 50 ;&g protein/ml), incubated 10 min at 370C. Assay for Pi ivas according to."7 This ATPase activity is resistant to ouabain and is Mg++-dependent.

6 2122 BIOCHEMISTRY: P. VASSALLI PROC. N. A. S. microsomes is indicated by the enhancing effect (50 to several hundred per cent) in such systems of "preincubating" the ph 5 fraction, i.e., allowing the formation of aminoacyl-trna in the absence of microsomes, and also of increasing the efficiency of the ATP-generating system by using 0.03 N1t PEP (increasing the concentration of ATP itself is inhibitory). Such procedures have no effect with low concentrations of LN microsomes or with liver microsomes. (b) Inhibition of the transfer reaction by microsomal inhibitors: The microsomal membranes also contain inhibitors of protein synthesis whose activity can be demonstrated in the solubilized membrane fraction after dissolution of the mem- 10. LYMPV NDE 0 5- MG MICROS INHIB./m& SPLEEN FIG. 7.-Effect of microsomal inhibitors ff W ; on the transfer of H3-leucine from H leucyl-trna to nascent polypeptides on MG MICROSOMAL PROTEIN/ML liver polyribosomes during incubation for.5 min. Inhibitors were obtained from microsomes washed by recentrifugation, FIG. 6.-Effect of microsomes of lymph resuspended in 1% deoxycholate, and nodes, liver, and spleen on formation of centrifuged 120 min at gay.; the Hs-leucyl-tRNA in cell-free system incu- supernatant was dialyzed against bated 5 min. To reduce the incorporation M Tris-HCl, ph 7.7, and lyophilized. of H3-leucine into protein to a minimum, H3-leucyl-tRNA was provided by a liver microsomes with low incorporating ac- ph 5 fraction incubated 5 min in presence tivity (long storage at -20'C) were incu- of H3-leucine and 19 cold amino acids, bated in the presence of 100,gg/ml of reprecipitated at ph 5 and resuspended puromycin, which does not affect the in an incubation medium containing excess formation of aminoacyl-trna but pre- cold leucine. Liver polyribosomes (300 vents polypeptide synthesis. The radio- jug RNA/ml) were obtained from the activity in tubes washed after hydrolysis pellet from centrifugation of deoxycholateat 90'C ( cpm) was subtracted treated microsomes through 0.5 and 2 M from the radioactivity in tubes washed at sucrose for 4 hr at ga... This 4VC. The difference, indicated in the system is dependent on transfer enzyme, ordinate, represents leucyl-trna formed supplied by LN ph 5 fraction supernatant during the incubation. (3 mg/ml). branes in deoxycholate followed by dialysis to remove the detergent (this destroys the ATPase activity). Addition of increasing amounts of these membrane extracts to a cell-free system progressively decreases its efficiency, but the inhibitory effect is two to three times more marked with membrane extracts from LN than from liver. This inhibition results, at least in part, from an impairment of the "transfer reaction," as can be shown when a system is used (Fig. 7) for studying specifically the transfer of H3-leucyl-tRNA to peptide linkage on the ribosomes. Thus, it is likely that an excess of microsomal "transfer inhibitors" is in part responsible for the poor efficiency of systems with high concentration of LN microsomes.

7 VOL. 58, 1967 BIOCHEMISTRY: P. VASSALLI 2123 As further evidence, addition of a TABLE 2 fraction rich in transfer enzymes SOME CHARACTERISTICS OF SYSTEMS USING LN MICROSOMES CELL-FREE (supernatant of the ph 5 fraction, or System: Efficiency (%) fraction of the supernatant precipitat- -GTP ing between 40 and 70 per cent am- -ATP-generating system 1 - Microsomes 0 monium sulfate saturation) enhances -ph 5 Fraction 4 the efficiency of these systems RNase (1 img/ml) 2-3 +Puromyin (looug/ml)2-3 +Puromycin (100,~g/ml) per cent; in contrast, cell-free systems with low concentrations of micro- set at 100% efficiency. somes are not improved. It is likely * Incorporation in the complete system was arbitrarily that these transfer inhibitors, which increase the requirements for transfer enzymes, are related to the inhibitors described by Hoagland et al. in liver microsomal membranes.'12 13 Similar and still more powerful inhibitors are found on spleen microsomes.'0 (2) Effect of cell sap or its ph 5 fraction: Liver sap gives consistently better results than LN sap (20-> 100% higher). The lower efficiency of LN sap does not appear to be due to a larger amino acid pool, lack of transfer enzyme activity, or excess of lyzosomal enzymes. ph 5 Fractions are more effective than plain cell saps, and liver ph 5 fraction gives optimal results at a concentration of 2 mg/ml. LN ph 5 fractions are markedly less effective, probably because of an insufficient precipitation at ph 5 of the transfer enzymes of LN sap, since addition to the ph 5 fraction of ph 5 supernatant gives per cent enhancement, an effect which is not seen when ph 5 supernatant (liver or LN) is added to liver ph 5 fraction. Another procedure which leads to marked enhancement when LN ph 5 fraction is used is its preincubation for five minutes at 370C before addition of the microsome fractions. The reason is unclear, but Scornik et al. have reported that "aging" may increase the transfer enzyme activity of fresh cell sap with poor activity.'3 With these procedures it is possible to obtain cell-free systems derived exclusively from lymph nodes which function almost as well as systems using liver ph 5 fraction, although the results are less consistent. Characteristics of the system under optimal conditions: With respect to both the amount and duration of amino acid incorporation, LN cell-free systems function optimally with concentrations of mg/ml of microsomal protein and 2 mg/ml of liver ph 5 fractions. Under these conditions preincubation of the ph 5 fractions, addition of fractions rich in transfer enzymes, or increase in the concentration of PEP have no beneficial effect. Incubations at various ph and Mg++ concentrations show best results between ph 7.4 and 7.8, with M Mg++. When the ph 5 fraction is passed through Sephadex G25 to remove free amino acids, addition of cold amino acids enhances the system: in this case, concentrations of 0.02,mole/ml of cold amino acid and Aumole/ml of H3-leucine give optimal results, allowing incorporation of as much as 1.4 X 106 cpm or 80 /A/Amoles of leucine per milligram of microsomal protein. Other characteristics are indicated in Table 2. It has been reported that cell-free systems derived from spleen are relatively resistant to puromycin and ribonuclease, and this unusual observation was considered to reflect special mechanisms of synthesis for immunoglobulin molecules.'4 This is not borne out by the high sensitivity to these agents observed in the present experiments.

8 2124 BIOCHEMISTRY: P. VASSALLI PROC. N. A. S. Summary.-A cell-free system has been established from lymph nodes of immunized rats, using as the protein-synthesizing fraction microsomal vesicles with little contamination by other cell particles. The concentration of mierosomes plays a critical role in the efficiency of the system because of the presence on microsomal membranes of high amounts of ATPase and inhibitors of the transfer reaction. The system functions best with ph 5 fractions from liver, but ph 5 fractions from lymph node cell sap are also effective if supplemented by fractions rich in transfer enzymes. The expert technical assistance of Mr. Reynaldo Fernandez is gratefully acknowledged. The author is indebted to Dr. M. Lieberman and Dr. B. Goldberg for the electron micrographs. This research was supported by grant AI0765I-02 from the USPHS. Abbreviations: trna, transfer or soluble RNA; LN, lymph nodes; Pi, inorganic phosphorus; ER, endoplasmic reticulum; PEP, phosphoenolpyruvate; ATP, adenosine triphosphate; AMP, adenosine monophosphate; GTP, guanosine 5'-triphosphate. l Vassalli, P., B. Lisowska-Bernstein, M. E. Lamm, and B. Benacerraf, these PROCEEDINGS, in press. 2 Chauveau, J., J. Moul6, C. Rouiller, and J. Schneebeli, J. Cell. Biol., 12, 17 (1962). 3 Loeb, J. N., R. R. Howell, and G. M. Tomkins, J. Biol. Chem., 242, 2069 (1967). 4 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 5 Fleck, A., and H. N. Munro, Biochim. Biophys. Acta., 55, 571 (1962). 6 Sawant, P. L., S. Shibko, V. S. Kumta, and A. L. Tappel, Biochim, Biophys. Acta., 85, 82 (1964). 7 Lawford, G. R., P. Langford, and H. Schachter, J. Biol. Chem., 241, 1835 (1966). 8 Blobel, G., and V. R. Potter, these PROCEEDINGS, 55, 1283 (1966). Bloemendal, H., W. S. Bont, and E. L. Benedetti, Biochim. Biophys. Acta., 87, 177 (1964). '0Vassalli, P., unpublished observations. '1 Hoagland, M. B., M. L. Stephenson, J. F. Scott, L. J. Hecht, and P. C. Zamecnik, J. Biol. Chem., 231, 241 (1958). 12 Hoagland, M. B., 0. A. Scornik, and L. C. Pfefferkorn, these PROCEEDINGS, 51, 1184 (1964W. 13Scornik, 0. A., M. B. Hoagland, L. C. Pfefferkorn, and E. A. Bishop, J. Biol. Chem., 242, 131 (1967). 14 Stenzel, K. H., and A. L. Rubin, Science, 153, 537 (1966). '6 Shibko, S., and A. L. Tappel, Biochim. Biophys. Acta., 73, 76 (1963). "I Bowers, W. E., and C. de Duve, J. Cell. Biol., 32, 339 (1967). 17 Weil-Malherbe, H., and R. H. Green, Biochem. J., 49, 286 (1951).