Proc. Natl. Acad. Sci. USA Vol. 80, pp. 5510-5514, September 1983 Biochemistry

Nucleotide-stimulated proteolysis of histone H1 [protease/P',P4-di(-5') tetraphosphate/ATP/pyrophosphate/ lymphocytes] CAROL S. SUROWY AND NATHAN A. BERGER* Hematology/Oncology Division, Departments of Medicine and Biochemistry, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio 44106; and Hematology/Oncology Division of the Department of Medicine, Washington University School of Medicine and The Jewish Hospital of St. Louis, St. Louis, Missouri 63110 Communicated by Harland G. Wood, June 7, 1983 ABSTRACT We have identified a proteolytic system that se- triphosphate ([(3, y-CH2]ATP) were from P-L Biochemicals. Hi- lectively degrades histone HI in normal human lymphocytes. stone H1 (from calf thymus) was from Boehringer Mannheim. Treatment of permeabilized human lymphocytes with a series of Methods. Isolation of lymphocytes. Normal human lympho- nucleotides produced a marked decrease in their histone HI con- cytes (95% pure) were isolated by Ficoll/Hypaque gradient tent compared to untreated cells. The nucleotide-stimulated pro- centrifugation (1) and resuspended to 2 x 106 cells per ml in cess was selective for histone HI because gel electrophoresis showed a modified Eagle's medium supplemented with 10% heat-treated that almost all other lymphocyte protein bands remained constant fetal calf serum, penicillin at 50 units/ml, streptomycin at 50 while histone HI disappeared. The elimination of histone HI ap- mM Hepes buffer at a final pH of 7.2, as de- pears to be the result of proteolysis by a trypsin-like enzyme be- ,ug/ml, and 25 cause it was inhibited by phenylmethylsulfonyl fluoride, antipain, scribed (2). soybean trypsin inhibitor, and diisopropyl fluorophosphate. Pro- Cell "permeabilization.' Cells were made permeable to ex- teolysis was stimulated by P',P4-di(adenosine-5') tetraphosphate, ogenously supplied nucleotides as described (2), then resus- P',P3-di(adenosine-5') triphosphate, P',P5-di(adenosine-5') pen- pended to 1 x 108 cells per ml in a hypotonic buffer consisting taphosphate, adenosine 5'-tetraphosphate, ATP, adenosine 5'-[a43- of 10 mM Tris-HCI, pH 7.8/1 mM EDTA/4 mM MgCl2/30 methylene]triphosphate, adenosine 5'-[i,By-methylene]triphos- mM 2-mercaptoethanol at 0°C. Cells to be incubated with p- phate, ADP, CTP, GTP, UTP, dATP, or pyrophosphate, whereas (hydroxymercuri)benzoate, N-ethylmaleimide, or iodoacet- AMP, adenosine, adenosine diphosphoribose, NAD+, cAMP, or amide were resuspended before incubation in the same buffer sodium phosphate did not show this stimulation of proteolysis. ATP, containing no 2-mercaptoethanol. [a3,-methylene]ATP, [f3,y-methylene]ATP, and pyrophosphate Incubation of cells uith nucleotides. All nucleotides or their all stimulated proteolysis, suggesting that a pyrophosphate linkage components were prepared in 100 mM Tris, adjusted to a final was necessary for this process. Thus, resting human lymphocytes pH of 7.8 with HCI, and then added to the reaction system to contain a trypsin-like protease that is stimulated by nucleotides or give a final concentration of 0.67-10 mM. Fifty microliters of pyrophosphate to selectively degrade histone HI. the permeable cell suspension (5 x 106 cells) was added to tubes containing 25 ,ul of the nucleotide and the mixture was incu- During the course of our studies on the effect of P'1p4- bated at 37°C for 60 min. Control samples received an equal di(adenosine-5') tetraphosphate (Ap4A) on ADP-ribosylation in amount of buffer. human lymphocytes, we noticed that when cells were incu- Incubation of cells with Ap4A in the presence of protease bated with Ap4A the intensity of staining of histone Hi on poly- inhibitors. Protease inhibitors were prepared in 100 mM Tris, acrylamide gels was much less than when cells were incubated adjusted to a final pH of 7.8 with HC1, and then added to the in the absence of Ap4A. The disappearance of histone Hi sug- reaction system to give a final concentration of 5 mM, except gested that it may have been modified or proteolytically de- STI, which had a final concentration of 50 ,uM. Phenylmethyl- graded. The present study was conducted to more fully deter- sulfonyl fluoride and pepstatin A were dissolved in 1/20 vol of mine the nature of this decrease in histone HI and to characterize 95% (vol/vol) ethanol, before addition of 100 mM Tris and ad- the system that induced it. Our results show that Ap4A, other justment to pH 7.8 with HC1. The final concentration of ethanol nucleotides (including ATP), and pyrophosphate all stimulate present was shown to have no effect on the reaction. Fifty mi- proteolysis of histone Hi by a trypsin-like protease. croliters of the permeable cell suspension (5 x 106 cells) was added to tubes containing 25 ,ul of the protease inhibitor and MATERIALS AND METHODS 10 p.1 of Ap4A (final concentration of 10 mM) and incubated at 37°C for 60 min. Control samples received an equal amount of Materials. Pl,P3-Di(adenosine-5') triphosphate (Ap3A), Ap4A, buffer. P1,P5-di(adenosine-5') pentaphosphate (Ap5A), adenosine 5'- NaDodSO4/Polyacrylamide gel electrophoresis. After incu- tetraphosphate (p4A), 5'-AMP, ADP, ATP, NAD+, adenosine bation, reactions were stopped by addition of an equal volume diphosphoribose (ADP-), 3',5'-cAMP, adenosine, phe- of sample solution composed of 4% (wt/vol) NaDodSO4, 0.2 M nylmethylsulfonyl fluoride, p-(hydroxymercuri)benzoate, iodo- dithiothreitol, 20 mM potassium phosphate, 40% (vol/vol) acetamide, N-ethylmaleimide, antipain, leupeptin, soybean tryp- glycerol, and 0.006% bromophenol blue, pH 7.0 (3), and the sin inhibitor (STI), pepstatin A, o-phenanthroline, N-a-rham- mixture was immediately boiled for 2 min. Electrophoresis was nopyranosyloxyhydroxyphosphinylleucyltryptophan (phos- carried out in. 7.5% polyacrylamide gels, using a stacking gel of phoramidon), and diisopropyl fluorophosphate were from Sig- ma. GTP, UTP, CTP, dATP, adenosine 5'-[a,4-methylene]tri- Abbreviations: Ap4A, P',P4-di(adenosine-5') tetraphosphate (the tri- phosphate ([a,4-CH21ATP) and adenosine 5'-[3, y-methylene]- phosphate and pentaphosphate homologs are abbreviated, similarly); p4A, adenosine 5'-tetraphosphate; [a,,-CH2]ATP, adenosine 5'-[a,,4meth- The publication costs of this article were defrayed in part by page charge ylene]triphosphate (the 83,.y analog is abbreviated similarly); STI, soy- payment. This article must therefore be hereby marked "advertise- bean trypsin inhibitor. ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 5510 Downloaded by guest on September 30, 2021 Biochemistry: Surowy and Berger Proc. Natl. Acad. Sci. USA 80 (1983) 5511 5% polyacrylamide (4). The buffer system of the stacking gel histone Hi that decreased on incubation with Ap4A. Fig. LB was 62.5 mM Tris HCI/2 mM EDTA, pH 6.8, and that of the shows the concentration dependence of the effect of Ap4A on separating gel was 0.238 M Tris HCI/2 mM EDTA, pH 8.8. histone Hi from lymphocytes from another donor. When cells The electrode buffer consisted of 50 mM Tris.HCI/0.39 M gly- were incubated with 0.67 mM Ap4A (lane b) there was a slight cine/1.89 mM EDTA, pH 8.3. Both gel and electrode buffer decrease in the histone Hi band relative to control (lane a). systems contained NaDodSO4 at a final concentration of 0.1%. Further increases in the concentration of Ap4A caused pro- Gels were stained with 0.25% R-250 gressively greater decreases in histone Hi such that cells in- in water/methanol/acetic acid, 5:5:1 (vol/vol), and then de- cubated with 3.33 mM (lane c), 6.7 mM (lane d), and 10 mM stained with water/methanol/acetic acid, 16:2:2 (vol/vol). For Ap4A (lane e) showed progressively less histone Hi relative to quantitation of histone H1, gels were dried and photographed, control. Thus, the disappearance of histone was directly de- and negatives were scanned with an LKB Zeineh laser scanning pendent on the amount of Ap4A present. Similar results were densitometer. obtained with lymphocytes from 30 different normal donors. Effect of Other Compounds on Histone HI Disappearance. RESULTS To determine whether other nucleotides or their components Effect of Ap4A on Histone HI. Fig. 1A shows the effect of could cause the elimination of histone Hi, we incubated cells Ap4A on histone Hi in human lymphocytes. Cells were per- for 60 min with a number of compounds and compared their meabilized and incubated with Ap4A for 60 min, and then the effects with those of Ap4A. Each compound was present at a proteins were analyzed by NaDodSO4/polyacrylamide gel final concentration of 10 mM. In each case the decrease in his- electrophoresis. Fig. 1A, lane a, shows the proteins from cells tone HI observed on NaDodSO4/polyacrylamide gels was de- that were incubated in the absence of Ap4A. Histone H1 was termined by densitometry. The results of this analysis are shown seen as a heavily stained band migrating at a position equivalent in Table 1. A number of the compounds tested were found to to a molecular weight of 32,000. Fig. 1A, lane b, shows the ef- cause a drastic decrease in histone Hi. These were Ap4A, Ap3A, fect of incubating cells with 10 mM Ap4A. Under these con- Ap5A, p4A, ATP, [a,,BCH2]ATP, [3,8y-CH2]ATP, ADP, CTP, ditions the amount of histone Hi was substantially less than GTP, UTP, dATP, and pyrophosphate. In contrast, AMP, cAMP, that in the control; Ap4A caused a drastic decrease in the amount ADP-ribose, NAD+, and sodium phosphate had little effect, of histone Hi in lymphocytes. Acid extraction of histones fol- and adenosine had no effect on histone Hi. Thus, compounds lowed by acid/urea gel electrophoresis confirmed that it was containing pyrophosphate or pyrophosphate itself are capable

A a b

I. B a b c d kzII. i2 id[ a4 I,I!I w s^

Histone Hi -

Histone HI -

10 mM 0 0.67 3.33 6.7 10 Ap4A mM Ap4A FIG. 1. Protein profiles ofpermeabilized human lymphocytes. (A) Effect ofAp4A on histone H1. The permeabilized lymphocytes were incubated (5 x 106 cells per reaction system) for 60 min in the absence or presence of 10 mM Ap4A. The protein profile ofeach reaction was analyzedby NaDodSO4/ polyacrylamide gel electrophoresis followed by Coomassie brilliant blue staining. Molecular weight standards were as follows: myosin, 200,000; (- galactosidase, 116,500; phosphorylase b, 94,000; bovine serum albumin, 68,000; ovalbumin, 43,000; carbonic anhydrase, 30,000; STI, 21,000; ly- sozyme, 14,300. Histone H1 from calf thymus was also used as a standard. (B) Concentration dependence of effect of Ap4A on histone H1. The per- meabilized lymphocytes were incubated (5 x 106 cells per reaction system) for 60 min in the absence or presence of Ap4A at the indicated concen- trations. The protein profile of each reaction was analyzed as described for A. Downloaded by guest on September 30, 2021 5512 Biochemistry: Surowy and Berger Proc. Natl. Acad. Sci. USA 80 (1983) Table 1. Effect of nucleotides, pyrophosphate, and other compounds on histone H1 100 Compound (10 mM) % histone Hi Untreated control 100 Ap4A 5 80 Ap3A 8 Ap5A 6 p4A 8 ATP 7 60 [a,3-CH2]ATP 9 [/,y-CH2]ATP 6 ADP 9 AMP 65 40 cAMP 67 ADP-ribose 55 NAD+ 56 Adenosine 97 CTP 5 20 GTP 9 UTP 6 dATP 6 PPi 9 0 10 20 30 40 50 60 P04 60 Time, min Permeabilized human lymphocytes were incubated (5 x 106 cells per reaction system) for 60 min. The percentage of histone H1 remaining, FIG. 2. Time course of effect of Ap4A, ATP, or pyrophosphate on relative to control, was determined by densitometry of Coomassie bril- histone Hi. Permeabilized human lymphocytes were incubated (5 x liant blue stained histone H1 on NaDodSO4/polyacrylamide gels ofto- 106 cells perreaction system) inthe absence orpresence of 10mM Ap4A tal cellular proteins. ATP, or pyrophosphate for the indicated time periods. The protein pro- file of each reaction was examined by NaDodSO4/polyacrylamide gel electrophoresis followed by Coomassie brilliant blue staining. The per- of stimulating a drastic decrease in the histone Hi content of centage of histone H1 relative to control at zero time on the stained lymphocytes. However, the finding that NAD' and ADP-ri- gels was determined by densitometry. e, Control lymphocytes; o, lym- bose are not very effective in causing a decrease in histone Hi phocytes incubated with 10 mM Ap4A; *, lymphocytes incubated with indicates that not all pyrophosphate groups are capable of stim- 10 mM ATP; A, lymphocytes incubated with 10 mM pyrophosphate. ulating this process. For NAD and ADP-ribose, the failure to stimulate may be attributable to steric hinderance of the py- suggested that each compound produced direct effects on his- rophosphate group or to the fact that, in these molecules, both tone H1 degradation. ends of the pyrophosphate are blocked. Those compounds with Effect of Protease Inhibitors on Histone HI From Cells In- only one phosphate or no phosphate had little or no effect on cubated with Ap4A. One of the causes for the disappearance of the histone. histone H1 could be proteolytic digestion. To evaluate this pos- Nucleoside triphosphates can be cleaved in permeabilized sibility, we incubated cells with 10 mM Ap4A for 60 min in the and nucleoside presence of various protease inhibitors. Inhibition of the de- cells to nucleoside diphosphates monophos- crease in histone H1, observed on NaDodSO4/polyacrylamide phates (5). It has also been shown that Ap4A can be converted gels, was quantitated by densitometry. The results of this anal- to ATP by an Ap4A hydrolase (6). Because Ap4A, ATP, and py- ysis are shown in Table 2. As previously observed, incubation rophosphate were all capable of causing a decrease in histone with Ap4A caused a drastic decrease in histone Hi. Incubation Hi, we considered the possibility that this decrease might be in the presence of various protease inhibitors showed that a caused only by pyrophosphate and that the nucleotides had to number of these agents were capable of significantly inhibiting be processed to yield pyrophosphate before they could stim- this Ap4A-induced decrease in histone Hi. Phenylmethylsul- ulate histone Hi disappearance. To investigate this possibility fonyl fluoride (7), antipain (8), STI (9), and diisopropyl fluo- we compared the time courses of histone Hi disappearance in rophosphate, all of which inhibit proteolysis by trypsin or tryp- the presence of Ap4A, ATP, and pyrophosphate. Cells were in- sin-like enzymes, inhibited the Ap4A-induced decrease in histone cubated in the absence or presence of each compound for var- Hi. Neither leupeptin (10), which inhibits plasmin, trypsin, ious periods of time and then subjected to NaDodSO4 gel elec- and related enzymes, nor pepstatin A, which inhibits acid pro- trophoresis followed by densitometry to determine the amount teases (11), had any effect on the decrease in histone Hi. p- of histone Hi. If pyrophosphate were the only stimulating agent (Hydroxymercuri)benzoate, N-ethylmaleimide, and iodoacet- then cells incubated with pyrophosphate should show the most amide, which all inhibit proteases requiring a sulfhydryl group rapid disappearance of histone Hi. In contrast, Ap4A-treated for activity, and phosphoramidon (12), o-phenanthroline, and cells should show the slowest rate of histone HI disappearance. EDTA, which inhibit various metalloproteases, were also found The results shown in Fig. 2 indicate that all three compounds to have no effect. The inhibitors showed very similar specifici- produced approximately the same rate of disappearance of his- ties toward the histone H1 proteolysis induced by ATP or py- tone Hi. In fact, incubation with pyrophosphate caused the rophosphate. These results suggest that the decrease in histone slowest rate of histone Hi disappearance. These observations H1 caused by Ap4A, ATP, pyrophosphate, or other nucleotides suggest that conversion of nucleotides to pyrophosphate is not is a result of stimulation of proteolysis of the histone. The en- required for stimulating histone Hi disappearance. Moreover, zyme that catalyzes this proteolysis appears to be a trypsin-like because the time courses of histone HI disappearance were protease that does not require sulfhydryl groups or metal ions similar upon incubation with Ap4A, ATP, or pyrophosphate, this for activity. Downloaded by guest on September 30, 2021 Biochemistry: Surowy and Berger Proc. Natl. Acad. Sci. USA 80 (1983) 5513 Table 2. Effect of protease inhibitors on the Ap4A-induced tin system in that the latter requires both sulfhydryl groups and decrease in histone H1 Mg2+ for activity (25, 26). Histone H1 A proteolytic system that is activated by both nucleotides and pyrophosphate has been detected in rat liver (27). This system % of % inhibition is Treatment control of proteolysis similar to that which we describe in human lymphocytes; however, it differs in that the liver enzyme appears to be a Untreated control 100 sulfhydryl-dependent protease. In the rat liver protease study Ap4A 13 - the physiological substrate was not identified. In this study, + phenylmethylsulfonyl fluoride 88 86 histone Hi appeared to be the natural substrate of the human + antipain 64 57 lymphocyte nucleotide- and pyrophosphate-stimulated en- + diisopropyl fluorophosphate 96 95 zyme. Further studies will be required to determine whether + STI 90 89 the liver can + leupeptin 18 6 enzyme cleave histone Hi. + pepstatin A 19 7 It is interesting that both liver cells and human lymphocytes + phosphoramidon 14 0 are intermitotic Go cells. Our preliminary experiments have + o-phenanthroline 17 2 shown that stimulation of lymphocytes to undergo a transition + EDTA 15 1 from resting to proliferating cells is accompanied by a decrease + p-(hydroxymercuri)benzoate 23 10 ini histone Hi relative to other cellular proteins. In fact, a cor- + N-ethylmaleimide 15 0 relation between proliferative rate and histone proteolysis has + iodoacetamide 20 6 been reported (13, 23). The expression of protease and pro- tease-inhibiting activity has also been found to vary between Permeabilized human lymphocytes were incubated (5 x 106 cells per resting human mononuclear leukocyte cultures and those stim- reaction system) for 60 min with 10 mM Ap4A in the absence or pres- to ence ofthe various agents listed. The agents were each present at 5 mM ulated proliferate by concanavalin A (28). Furthermore, his- except for STI, which was 50 kLM. The amount of histone H1 and in- tone Hi complements of dividing and nondividing mouse cells hibition ofdecrease in histone H1 were determined by densitometry of have been shown to differ (29). Thus, it is possible that the ac- Coomassie brilliant blue stain on NaDodSO4/polyacrylamide gels of tivity of this proteolytic enzyme system is somehow involved total cellular proteins. Percent inhibition ofhistone proteolysis in each in removing and replacing histone Hi as cells shift from the Go case was calculated by subtracting the percent of histone H1 observed state to that present in actively growing cells. Further in the presence ofAp4A from that observed in the presence ofAp4A and support the protease inhibitor and then dividing this figure by the percent de- for this possibility comes from the observation that Ap4A, one crease in histone H1 caused by Ap4A alone. of the stimulators of histone Hi proteolysis, has been shown to increase markedly during the transition from resting to pro- DISCUSSION liferating cells (30). The stimulation by nucleotides may be the result of a direct or indirect effect on the protease, histone Hi, Our studies have revealed a proteolytic system in human lym- or some other aspect of the proteolytic system. Further in- phocytes that is activated by nucleotides and pyrophosphate to vestigations into this intriguing proteolysis of histone HI will selectively degrade histone H1. This proteolytic system has be required to determine its biological significance and the trypsin-like properties, because it is inhibited by phenylmeth- manner in which it may affect nuclear events. ylsulfonyl fluoride, antipain, STI, and diisopropyl fluorophos- phate. While a number of studies have described the occur- These studies were supported by National Institutes of Health Grant rence of histone proteolysis during subcellular fractionation (13- GM 32647. Some of the studies were performed in facilitites supported 17), we know of no other reports by the Sohio Foundation. A travel grant to C. S.S. from the Wellcome of histone HI proteolysis that Trust, England, is acknowledged. N.A.B. is a Leukemia Society of is activated by nucleotides and pyrophosphate. The protease America Scholar. that we describe also differs from other histone proteases (18) in that it does not appear to be dependent on sulfhydryl groups 1. Mendelsohn, J., Skinner, S. A. & Kornfeld, S. (1971)J. Clin. In- for activity. In addition, the histone H1 protease is different vest. 50, 818-826. from the protease that cleaves protein A24 to give 2. Berger, N. A., Adams, J. W., Sikorski, G. W., Petzold, S. J. & ubiquitin and Shearer, W. T. (1978) J. Clin. Invest. 62, 111-118. histone H2A, because the latter is not sensitive to phenylmeth- 3. Ito, S., Shizuta, Y. & Hayaishi, 0. (1979)J. Biol. Chem. 254, 3647- ylsulfonyl fluoride (19). Thus, this nucleotide-stimulated his- 3651. tone H1 proteolytic system appears to be unique and also spe- 4. Studier, F. W. (1973)J. Mol. Biol. 79, 237-248. cific for histone H1. A proteolytic activity with unique specificity 5. Berger, N. A. & Johnson, E. S. (1976) Biochim. Biophys. Acta 425, for histone H2A has also been described (14, 20). 1-17. Nucleotide-activated proteolytic systems have been previ- 6. Hohn, M., Albert, W. & Grummt, F. (1982) J. Biol. Chem. 257, ously described but none 3003-3006. with the specificity of the present 7. Fahrney, D. E. & Gold, A. M. (1963) J. Am. Chem. Soc. 85, 997- system. For example, reticulocytes contain an ATP-activated 1000. proteolytic system that selectively degrades abnormal proteins. 8. Suda, H., Aoyagi, T., Hamada, M., Takeuchi, T. & Umezawa, H. This system is composed of proteases (21), ubiquitin (22), and (1972)1. Antibiot. 25, 263-267. a ubiquitin-activating enzyme (23). This ubiquitin system does 9. Kassel, B. (1970) Methods Enzymol. 19, 853-862. not appear to be involved in 10. Suda, H., Aoyagi, T., Takeuchi, T. & Umezawa, H. (1973) J. the histone H1 proteolysis that we Antibiot. 26, 621-623. observe because it is restricted to stimulation by adenine nu- 11. Aoyagi, T., Takeuchi, T., Matsuzaki, A., Kawamura, K., Kondo, cleotides (24) whereas, in contrast, the histone H1 proteolysis S., Hamada, M., Maeda, K. & Umezawa, H. (1969) J. Antibiot. that we observe occurs on incubation of lymphocytes with a 22, 283-286. number of nucleotides, including ATP, CTP, GTP, UTP, and 12. Umezawa, H., Aoyagi, T., Morishima, H., Matsuzaki, M., Ap4A and also with pyrophosphate. In addition, the ubiquitin Hamada, M. & Takeuchi, T. (1970)J. Antibiot. 23, 259-262. system has been reported to be on ATP whereas 13. Bartley, B. & Chalkley, R. (1970)J. Biol. Chem. 245, 4286-4292. dependent (25), 14. Eickbush, T. H., Watson, D. K. & Moudrianakis, E. N. (1976) the protease in our system is stimulated by both [a,,8-CH2]ATP Cell 9, 785-792. and [P3,y-CH2]ATP and also by pyrophosphate. The histone H1 15. Fornells, M. & Subirana, J. A. (1977) Biochem. Biophys. Res. proteolytic system of lymphocytes also differs from the ubiqui- Commun. 78, 217-221. Downloaded by guest on September 30, 2021 5514 Biochemistry: Surowy and Berger Proc. Nati. Acad. Sci. USA 80 (1983)

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