Proc. Natl. Acad. Sci. USA Vol. 85, pp. 2464-2468, April 1988 Biochemistry On the of y-glutamylcysteine synthetase: Relationships to , inhibition, and regulation (/cystamine//kidney/ inactivation) CHIN-SHIou HUANG, WILLIAM R. MOORE, AND ALTON MEISTER Cornell University Medical College, Department of Biochemistry, 1300 York Avenue, New York, NY 10021 Contributed by Alton Meister, December 4, 1987

ABSTRACT y-Glutamylcysteine synthetase (glutamate- dithiothreitol, suggesting that cystamine forms a mixed ; EC 6.3.2.2) was isolated from an Escherichia between cysteamine and an enzyme thiol (15). coli strain enriched in the gene for this enzyme by recombinant Inactivation of the enzyme by the L- and D-isomers of DNA techniques. The purified enzyme has a specific activity of 3-amino-1-chloro-2-pentanone, as well as that by cystamine, 1860 units/mg and a molecular weight of 56,000. Comparison is prevented by L-glutamate (14). Treatment of the enzyme of the E. coli enzyme with the well-characterized rat kidney with cystamine prevents its interaction with the sulfoxi- enzyme showed that these have similar catalytic prop- mines. Titration of the enzyme with 5,5'-dithiobis(2- erties (apparent Km values, specificities, turnover nitrobenzoate) reveals that the enzyme has a single exposed numbers). Both enzymes are feedback-inhibited by glutathione thiol that reacts with this reagent without affecting activity but not by y-glutamyl-a-aminobutyrylglycine; the data indicate (16). 5,5'-Dithiobis(2-nitrobenzoate) does not interact with that glutathione binds not only at the glutamate but the thiol that reacts with cystamine. Evidence has also been also at a second site on the enzyme that interacts with the thiol obtained that y-methylene-D-glutamate, which inactivates moiety of glutathione but not with a . Both the enzyme, binds in the glutamate binding site of the active enzymes are inactivated by buthionine sulfoximine in the pres- center and forms a covalent linkage with the active site thiol ence of ATP, suggesting a common y-glutamyl through a Michael-type addition reaction (17). The enzyme is intermediate. However, unlike the rat kidney enzyme that has also inactivated by S-sulfocysteine and S-sulfohomocys- an active center thiol, the bacterial enzyme is insensitive to teine; these inactivators are tightly but noncovalently bound cystamine, r-methylene glutamate, and S-sulfo amino acids, and their interaction with the enzyme is postulated to indicating that it does not have an active site thiol. Thus, the rat involve effects of the active site thiol (18). These and earlier kidney and E. coli enzymes share several catalytic features but (14) observations have led to the suggestion that the active differ in active site structure. If the active site thiol of the rat center thiol may play a role in the mechanism of action of is in this enzyme. kidney enzyme involved catalysis, which seems likely, there In contrast to the results summarized above obtained in would appear to be differences in the mechanisms of action of studies on rat kidney y-glutamylcysteine synthetase, the the two y-glutamylcysteine synthetases. findings on the y-glutamylcysteine synthetase ofEscherichia coli, reported here, indicate that the bacterial enzyme has The first step in the synthesis of glutathione is catalyzed by properties that do not reflect the presence ofan active center y-glutamylcysteine synthetase (glutamate-cysteine ligase; thiol. Nevertheless, the bacterial enzyme exhibits a number EC 6.3.2.2) [Reaction 1 (ref. 1, pp. 671-697)]. This reaction, of characteristics that closely resemble those of the mam- usually the rate-limiting step in glutathione synthesis, is malian enzyme, including, for example, high turnover num- feedback-inhibited by glutathione (2). y-Glutamylcysteine ber, feedback inhibition by glutathione, and inactivation by synthetase, like synthetase (ref. 1, pp 699-754, incubation with buthionine sulfoximine in the presence of and refs. 3 and 4), is inactivated by sulfoximine ATP. A preliminary account of this work has been presented in the presence of ATP (5). and (*; ref. 19). certain other sulfoximines, such as buthionine sulfoximine [which inhibits y-glutamylcysteine synthetase but not gluta- EXPERIMENTAL PROCEDURES mine synthetase (6-9)], are phosphorylated by ATP on the enzymes; the phosphorylated sulfoximines bind tightly, but Materials. Agarose-hexane-ATP (Al6; type 2), CM-Seph- noncovalently, to the enzymes, thus producing inhibition. adex C-50, Sephadex G-100, and Sephadex G-150 were purchased from Pharmacia. DE-52 was obtained from What- L-Glutamate + ATP + L-cysteine man. L-Buthionine-(SR)-sulfoximine (6-8), S-sulfocysteine L-y-glutamyl-L-cysteine + ADP + Pi [1] (18), S-sulfohomocysteine (18), and y-methylene glutamate (17) were synthesized as described. Other compounds were The presence of a thiol at the active site of y-glutamyl- obtained from Sigma. cysteine synthetase was suggested by the finding (10) that Microorganisms. Previously a strain of E. coli enriched by highly purified 'y-glutamylcysteine synthetase from rat kid- recombinant DNA techniques in its content of the two ney is inactivated by low concentrations of L-2-amino-4-oxo- synthetases required for glutathione synthesis was described 5-chloropentanoate (11), that such inactivation is associated (20, 21). The genes for the synthetases were isolated and with covalent binding of the inactivator, and that glutamate inserted into a vector that was used to transform the original protects against inactivation. Subsequently it was found that strain. The transformed strain, in an immobilized form, has the enzyme is strongly inactivated by cystamine (12-14) and been used for the synthesis of isotopically labeled gluta- that inactivation by cystamine is reversed by treatment with thione (22) and for the synthesis of glutathione analogs (23). In the present work a bacterial strain enriched in its content The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" *Huang, C.-S., Moore, W. R. & Meister, A., American Society of in accordance with 18 U.S.C. §1734 Alely to indicate this fact. Biological Chemists Meeting, June 6-10, 1987, Philadelphia, PA. 2464 Downloaded by guest on October 4, 2021 COST AND EFFICIENCY IN HEALTH AND EMPHYSEMA 497 z 0 Z 0 - 7s I/ . / II100 200. O 0 0E NO1/ d)/ P1 80 10V CLJ 0 2 150 x 4p,, x > -a Zn 60 - C) u0 -~ 100 -w°X 1. r0 40 IZ I z I . . ol 0~. :-0c LA 20 0 10 20 30 4I0 0 6 3 'D VENTILATION A z (L/min. B.T.P.S.) 0 2.0 4.0 6.0 8.0 FIG. 3. THE CHANGE IN OXYGEN CONSUMPTION As- ADDED MECHANICAL WORK SOCIATED WITH CHANGE IN MINUTE VENTILATION IN 22 (kgm.m./min.) PATIENTS WITH EMPHYSEMA FIG. 4. THE EXTRA OXYGEN CONSUMPTION REQUIRED Also shown are isopleths representing the oxygen TO HANDLE ADDED RESPIRATORY WORK LOADS IN NINE cost of per liter of ventilation. breathing NORMAL AND SEVEN EMPHYSEMATOUS SUBJECTS Also shown are isopleths representing the efficiency of tion of the respiratory muscles per liter of ventila- the respiratory muscles for handling added work loads. tion was considerably higher in the patients with 0 represents normal subjects; X represents emphysema- emphysema than in the normal subjects. The tous subjects. cost ranged between 0.45 to 1.87 ml. per L. of ventilation (mean 1.16) in the normal subjects, was high for even slight increases in ventilation while it ranged between 1.68 and 18.50 ml. per L. and rose still further when ventilation was in- of ventilation (mean 5.96) in the emphysematous creased. subjects. The extra oxygen consumption associated with In Figures 2 and 3 the increments in oxygen added work loads in nine normal subjects and consumption associated with changes in ventilation seven patients with emphysema is shown in Fig- in the normal subjects and patients with emphy- ure 4. It can be seen that the extra oxygen sema are shown. It can be seen that in the normal consumption per unit added work load was greater subjects the oxygen cost of an increase in ventila- in the patients with emphysema than in the tion of 30 L. per minute above the resting ventila- normals. tion was less than 2 ml. per L. On the other hand, In Table III the calculated efficiencies of the in the subjects with emphysema the oxygen cost respiratory muscles of the nine normal and seven

TABLE III Total mechanical work and efficiency of the respiratory muscles in normal and emphysematous subjects

Normal Emphysematous Total mechanical work Total mechanical work Subj. Efficiency at rest Subj. Efficiency at rest % Kg. M./L. Kg. M./min. % Kg. M./L. Kg. M./min. 1 10.80 0.173 1.52 17 2.74 0.141 1.39 2 7.45 0.172 1.38 18 1.45 0.203 1.77 3 7.60 0.123 0.82 19 2.30 0.159 1.39 4 8.20 0.280 2.69 20 2.04 0.120 0.83 5 11.10 0.326 3.00 21 1.24 0.188 1.20 6 8.04 0.204 2.74 22 1.47 0.284 2.68 7 7.20 0.203 1.83 23 1.60 0.123 1.22 8 7.43 0.215 1.89 9 9.35 0.349 2.27 Mean 8.58 0.227 2.02 1.83 0.174 1.49 Downloaded by guest on October 4, 2021 2466 Biochemistry: Huang et al. Proc. Natl. Acad. Sci. USA 85 (1988) 1.0, and 0.3 mM. The relative rates of reaction found with Table 2. Effects of several compounds on E. coli and rat kidney the enzyme isolated from E. coli strain KM when L-cysteine y-glutamylcysteine synthetases was replaced by L-a-aminobutyrate, /3-chloro-L-, S- % activity remaining methyl-L-cysteine, and L- were 0.85, 0.79, 0.70, and 0.18, respectively. The corresponding values for the enzyme E. coli isolated from E. coli strain W were 0.81, 0.99, 0.72, and 0.13. Inhibitor Strain KM Strain W Kidney Less than 9% of the activity obtained with t-glutamate and L-cysteine was found with both enzyme preparations when 4-Methylene glutamate 98 100 41 L-glutamate was replaced with D-glutamate or L-aspartate S-Sulfocysteine 100 109 5 and when L-cysteine was replaced by L-norvaline or L- S-Sulfohomocysteine 99 100 10 methionine. Buthionine sulfoximine* 16 7.6 16 Inhibition Studies. The enzyme was incubated with cysta- For details see text. mine (0.1-10 mM) in Tris HCl buffer (50 mM; pH 8.2) at 37°C. *No inhibition was observed when ATP was omitted from the A portion (10 ,ul) ofthe mixture was diluted into 0.99 ml ofthe preincubation mixture. standard assay solution. Inactivation by 4-methylene-DL- glutamate was carried out by preincubation of the enzyme isolated kidney enzyme exhibits a specific activity of about with this (10 mM) in a buffer containing Tris HCI 1600 units/mg of , whereas the enzyme isolated from (50 mM; pH 8.2) and 0.25 mM MnCl2 for 1 hr at 37°C. After E. coli strain KM has a specific activity of about 1900 incubation, a 2O-,u portion of the solution was diluted into units/mg. The turnover numbers of the E. coli and kidney 0.98 ml of the assay solution. Studies with S-sulfo-DL- enzymes are calculated to be 2400 and 3130 min-1, respec- homocysteine and S-sulfo-DL-cysteine were carried out in the tively. same manner. In the experiments with buthionine sulfoxi- As shown in Fig. 1, the E. coli strain KM enzyme was not mine, the enzyme was preincubated with 10 mM L-buthi- inactivated by cystamine; indeed, this enzyme, as compared onine-(SR)-sulfoximine in Tris HCl buffer (10 mM; pH 8.2) to the kidney enzyme, is highly resistant to inactivation by containing 20 mM MgCl2 and 5 mM Na2ATP at 370C for 10 cystamine. Thus, the E. coli strain KM enzyme was not min. Then a 20-' 1 portion ofthe solution was diluted into 0.98 inactivated after incubation with 10 mM cystamine for 1 hr, ml of assay solution. In the studies on inhibition by gluta- whereas the kidney enzyme was completely inactivated by thione and y-glutamyl-a-aminobutyrylglycine, the enzyme incubation with 0.1 mM cystamine for 10 min. When the E. was added to the standard assay mixture containing 2-10 mM coli enzyme was incubated with 10 mM cystamine for 4 hr, glutathione or y-glutamyl a-aminobutyrylglycine. there was about 25% inactivation; this was not reversed by treatment with 10 mM dithiothreitol. The E. coli strain W enzyme preparation was also resistant to inactivation by RESULTS cystamine.t The enzyme isolated from E. coli strain KM (molecular 4-Methylene glutamate, which inactivates the kidney en- weight, 56,000) was found to be homogeneous on polyacryl- zyme by reaction with the active center thiol (17), did not gel electrophoresis in the presence and absence of inactivate the E. coli enzymes (Table 2). S-Sulfohomocys- NaDodSO4. The catalytic properties (apparent Km values, teine and S-sulfocysteine did not affect the activity of the E. substrate specificity) ofthis enzyme and those of the homog- coli enzyme under conditions in which the kidney enzyme enous enzyme obtained from rat kidney (1, 24) are similar as was substantially inactivated. On the other hand, buthionine noted above. The kidney enzyme (molecular weight, sulfoximine in the presence of ATP inactivated the enzyme 104,000) is composed of two subunits (molecular weights, preparations obtained from E. coli as well as the kidney 73,000 and 27,700); the catalytic and feedback properties of enzyme (Table 2). the enzyme are associated with its heavy subunit. The tFollowing our initial report (19), we became aware of the paper by (D 100 Watanabe et al. (30), who reported purification of E. coli y- glutamylcysteine synthetase and its inhibition by cystamine. Wa- Z KEcoli (I hour) z~~~~~~80 tanabe et al. obtained a protein that was homogeneous on poly- 4 hours acrylamide gel electrophoresis, but the specific activity of this UJ 60 enzyme calculated from their data is 21.6 units/mg (compared to 1860 units/mg found in the present work). The apparent discrep- ancy in specific activity may be ascribed to differences in the assay F- 40 procedures used. Watanabe et al. assayed the enzyme in a mixture containing ATP, glutamate, cysteine, and relatively low levels of and enzymes. used 7 of 0.98 20 zKIDNEY (10 MgCl2 coupling They umol MgCl2, min) unit of lactate , and 0.90 unit of pyruvate in a &1- 0- total volume of 0.7 ml; in the present work, the respective concentrations were 20 ,mol, 10 units, and 5 units per ml. We have 0 2 4 6 8 10 observed that although cystamine activates mark- CYSTAMINE (mM) edly, it inhibits ; however, at the concentra- tion of lactate dehydrogenase used in our assay, cystamine does FIG. 1. Effect of cystamine on y-glutamylcysteine synthetases. not affect coupling and therefore does not prevent efficient mea- Purified isolated from E. coli strain surement of the ADP formed in the y-glutamylcysteine synthetase- y-glutamylcysteine synthetase catalyzed reaction. In our assay, we substituted L-a-aminobutyrate KM (6 units) and from rat kidney (4 units) was incubated at 37°C in for L-cysteine. This substitution, as noted elsewhere (1, 24), is 100 ,ul containing Tris HCl buffer (50 mM; pH 8.2) and cystamine. desirable because of the marked tendency of cysteine to undergo A portion (0.01 ml) ofeach reaction mixture was removed and added oxidation and to form mixed . Cystamine would be to 0.99 ml of the standard enzyme assay solution. E. coli enzyme expected to be highly active in forming a mixed disulfide with was assayed after treatment with cystamine for 1 hr or 4 hr; kidney cysteine under assay conditions. On the basis of our studies on the enzyme was assayed after treatment with cystamine for 10 min. properties of the coupled assay system, we conclude that the Inactivation of the kidney enzyme was completely reversed by reported (30) apparent inhibition of E. coli y-glutamylcysteine treatment with 10 mM dithiothreitol, whereas the 25% inactivation synthetase by cystamine is a function of the assay system used observed after incubating the E. coli enzyme with 10 mM cystamine rather than an effect of cystamine on -glutamylcysteine synthe- for 4 hr was not reversed by dithiothreitol. tase. (See Note Added in Proof i.) Downloaded by guest on October 4, 2021 Biochemistry: Huang et al. Proc. Natl. Acad. Sci. USA 85 (1988) 2467

0 thiol found at or close to the active center of the kidney z 100 Y-GLU-aABA-GLY enzyme does not play a role in catalysis. Accordingly, the z observed reactions of the thiol of the kidney enzyme may 90 simply serve to block the glutamate binding site. However, it - GSH LUI is difficult to conceive that such a reactive thiol group that is 80 located so close to the y-carboxyl moiety of glutamate is not involved in catalysis. That a moiety is important in the 70 thiol o = KIDNEY reactions catalyzed by both enzymes, but that it is markedly o = E. coli hindered (or "buried") in the active site of the E. coli 0- 60 enzyme, appears unlikelyJ It is probable that the thiol plays rCn a role in catalysis by the kidney enzyme and that this 0- 2 4 6 8 10 function is performed by another structure in the E. coli GSH (mM) enzyme. An active center thiol seems not to be involved in inactivation by buthionine sulfoximine, a phenomenon asso- FIG. 2. Inhibition by glutathione of y-glutamylcysteine synthe- ciated with phosphorylation of buthionine sulfoximine by tase activities of E. coli and rat kidney. Enzyme activity was ATP that is presumably analogous to y-glutamyl phosphate determined as described in the text in the presence of various formation. concentrations of glutathione (GSH) or of 10 mM L-y-glutamyl-L-a- The possibility that the active center thiol of the kidney aminobutyrylglycine (y-GLU-aABA-GLY). enzyme has a function apart from catalysis needs to be Previous studies have shown that glutathione inhibits the considered. The data indicate that such a function is unre- activity of y-glutamylcysteine synthetase of rat kidney in a lated to feedback inhibition by glutathione, but perhaps manner that is competitive with glutamate (2). This is thought another metabolic phenomenon is connected with the action to provide a physiologically significant feedback inhibition of kidney 'y-glutamylcysteine synthetase; the nature of such mechanism, which explains, for example, the finding of a metabolic link is not now clear. 5-oxoprolinuria in severe congenital One must consider the idea that the rat kidney enzyme and deficiency (31, 32). Glutathione inhibits the activity of y- the E. coli enzyme, although they catalyze the same overall glutamylcysteine synthetase from E. coli strain KM to about reaction, perform their catalytic functions by use of different the same extent as found with the rat kidney enzyme. Neither active center structures. Although bacterial and mammalian enzyme is inhibited appreciably by y-glutamyl-a-amino- enzymes have commonly been found to have somewhat butyrylglycine. different properties, it is generally thought that the struc- tures of the respective active sites are similar. This may not be the case for the E. coli and kidney enzymes considered DISCUSSION here; thus, two different active sites might catalyze the same The catalytic properties of the y-glutamylcysteine synthe- . tases from E. coli and rat kidney are very similar. This is No information is currently available that relates to the evident from a comparison of the turnover numbers, sub- evolution of y-glutamylcysteine synthetase. If both enzymes strate specificities, and apparent Km values. Furthermore, arose from an ancestral y-glutamylcysteine synthetase, pos- both enzymes are inactivated to about the same extent when sibly the kidney enzyme evolved to a form possessing an incubated with buthionine sulfoximine and ATP (Table 2). active site thiol. The E. coli enzyme seems to have evolved Another point of similarity concerns the inhibition of the toward a more streamlined molecule that might be expected enzyme activities by glutathione. As indicated in Fig. 2, the to have greater survival properties. Thus, the E. coli enzyme quantitative aspects ofglutathione inhibition are virtually the does not contain subunits, has a smaller molecular mass, same for the E. coli and rat kidney 'y-glutamylcysteine and, importantly, cannot be inactivated by reactions involv- synthetases. It is interesting to observe that such inhibition ing an active center thiol. (See Note Added in Proof ii.) requires the thiol group of glutathione; thus, the analog y-glutamyl-a-aminobutyrylglycine is ineffective in producing inhibition. Data previously published (2) led to the conclu- tThe kidney enzyme is substantially inhibited by incubation for 5 min with 5 mM iodoacetamide [partially protected by L-glutamate sion that inhibition by glutathione is "non-allosteric" be- (10, 33)] and p-hydroxymercuribenzoate and p-chloromercuriben- cause inhibition is competitive toward L-glutamate. How- zenesulfonate at concentrations of 0.002 mM and 0.02 mM, respec- ever, glutathione, but not y-glutamyl-a-aminobutyrylgly- tively (33). The E. coli strain KM enzyme is not affected by cine, inhibits. This suggests that glutathione binds not only incubation for 10 min with 5 mM iodoacetamide or 10 mM N- at the glutamate binding site of the active center but also at ethylmaleimide; after incubation for 10 min with 1 mM p-hydroxy- another site that interacts with the thiol moiety of gluta- mercuribenzoate, it was about 65% inhibited in the presence or thione. The latter site does not bind a in which the absence of 10 mM L-glutamate. thiol of glutathione is replaced by a methyl group. In striking contrast to the several points of similarity Notes Added in Proof. (i) We have recently learned from K. Murata between the two enzymes summarized above, properties of (personal communication) that he has made observations that agree the kidney enzyme that may be attributed to the presence of with these conclusions-i.e., that the apparent inhibition of the E. an active site thiol are lacking in the E. coli enzyme. Thus, coli enzyme by cystamine does not reflect the presence of an the E. coli enzyme is not inactivated by cystamine, 4- enzyme thiol but is related to the method of assay. (ii) We have methylene glutamate, S-sulfohomocysteine, or S-sulfocys- recently established that the antigenic determinants of the E. coli teine. and kidney enzymes are different; polyclonal antibodies obtained The finding of a thiol at or close to the active site of the from rabbits immunized against the purified enzymes do not cross- kidney enzyme led to the previous suggestion that a thiol react on Ouchterlony plates. group is involved in the mechanism of the catalytic reaction, We thank Dr. Kousaku Murata for the strains ofE. coli used in this and thus a y-glutamyl-S-enzyme intermediate was consid- work. We are indebted to Dr. Andrew P. Seddon for the amino acid ered (14). The present findings on the E. coli enzyme analyses. We thank Dr. Mary E. Anderson for valuable discussions of indicate that a thiol is not involved in the reaction catalyzed this research. We thank Drs. Seddon and Anderson for their con- by this enzyme; this may be taken as an indication that the structive criticisms of this manuscript. This research was supported Downloaded by guest on October 4, 2021 2468 Biochemistry: Huang et al. Proc. Natl. Acad. Sci. USA 85 (1988) in part by a grant (2R37DK12034) from the U.S. Public Health 17. Simondsen, R. P. & Meister, A. (1986) J. Biol. Chem. 261, Service, National Institutes of Health. 17,134-17,137. 18. Moore, W., Wiener, H. L. & Meister, A. (1987) J. Biol. Chem. 1. Meister, A. (1974) in The Enzymes, ed. Boyer, P. (Academic, 262, 16771-16777. New York), 3rd Ed., Vol. 10. 19. Huang, C.-S., Moore, W. R. & Meister, A. (1987) Fed. Proc. 2. Richman, P. & Meister, A. (1975) J. Biol. Chem. 250, 1422- Fed. Am. Soc. Exp. Biol. 46, 1757 (abstr.). 1426. 20. Murata, K. & Kimura, A. (1982) Appl. Environ. Microbiol. 43, 3. Ronzio, R. & Meister, A. (1968) Proc. Nati. Acad. Sci. USA 289-297. 59, 164-170. 21. Gushima, H., Miya, K., Murata, K. & Kimura, A. (1983) J. 4. Manning, J. M., Moore, S., Rowe, W. B. & Meister, A. (1969) Appl. Biochem. 5, 43-52. Biochemistry 8, 2681-2685. 22. 5. Richman, P. G., Orlowski, M. & Meister, A. (1973) J. Biol. Murata, K., Abbott, W. A., Bridges, R. J. & Meister, A. Chem. 248, 6684-6690. (1985) Anal. Biochem. 150, 235-237. 6. Griffith, 0. W., Anderson, M. E. & Meister, A. (1979) J. Biol. 23. Moore, W. R. & Meister, A. (1987) Anal. Biochem. 161, Chem. 254, 1205-1210. 487-493. 7. Griffith, 0. W. & Meister, A. (1979) J. Biol. Chem. 254, 24. Seelig, G. F. & Meister, A. (1985) Methods Enzymol. 113, 7558-7560. 379-390. 8. Griffith, O. W. (1982) J. Biol. Chem. 257, 13,704-13,712. 25. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 9. Meister, A. (1978) in Enzyme-Activated Irreversible Inhibitors, 26. Sekura, R. & Meister, A. (1977) J. Biol. Chem. 252, 2599-2605. eds. Seiler, N., Jung, M. J. & Koch-Weser, J. (Elsevier-North 27. Watanabe, K., Yamano, Y., Murata, K. & Kimura, A. (1986) Holland Biomedical, Amsterdam), pp. 187-211. Nucleic Acids Res. 14, 4393-4400. 10. Sekura, R. & Meister, A. (1977) J. Biol. Chem. 252, 2606-2610. 28. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406- 11. Khedouri, E., Anderson, P. M. & Meister, A. (1966) Biochem- 4412. istry 5, 3552-3557. 29. Bidlingmeyer, B. A., Cohen, S. A. & Tarvin, T. L. (1984) J. 12. Griffith, 0. W., Larsson, A. & Meister, A. (1977) Biochem. Chromatogr. 336, 93-104. Biophys. Res. Commun. 79, 919-925. 30. Watanabe, K., Murata, K. & Kimura, A. (1986) Agric. Biol. 13. Lebo, R. V. & Kredich, N. M. (1978) J. Biol. Chem. 253, Chem. 50, 1925-1930. 2615-2623. 31. Meister, A. (1983) in Metabolic Basis of Inherited Diseases, 14. Beamer, R. L., Griffith, 0. W., Gass, J. D., Anderson, M. E. eds. Stanbury, J. B., Wyngaarden, J. B., Frederickson, D. S., & Meister, A. (1980) J. Biol. Chem. 255, 11,721-11,726. Goldstein, J. L., & Brown, M. S. (McGraw-Hill, New York), 15. Seelig, G. F. & Meister, A. (1982) J. Biol. Chem. 257, 5092- 5th Ed., pp. 348-359. 5096. 32. Wellner, V. P., Sekura, R., Meister, A. & Larsson, A. (1974) 16. Seelig, G. F. & Meister, A. (1984) J. Biol. Chem. 259, 3534- Proc. Natl. Acad. Sci. USA 71, 2505-2509. 3538. 33. Orlowski, M. & Meister, A. (1971) Biochemistry 10, 372-380. Downloaded by guest on October 4, 2021