Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7488-7492, November 1987 Biochemistry Inhibition of hydroxymethylglutaryl-coenzyme A synthase by L-659,699 (inhibitor/fi-lactone/natural product/Hep G2 cells) MICHAEL D. GREENSPAN*, JOEL B. YUDKOVITZ, CHIA-YEE L. Lo, JULIE S. CHEN, ALFRED W. ALBERTS, VINCENT M. HUNT, MICHAEL N. CHANG, SHU SHU YANG, KATHRYN L. THOMPSON, YUAN-CHING P. CHIANG, JOHN C. CHABALA, RICHARD L. MONAGHAN, AND ROBERT L. SCHWARTZ Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065 Communicated by P. Roy Vagelos, July 20, 1987

ABSTRACT A ,4lactone isolated from Fusarium sp. has been shown to be a potent specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase by,eCOOH [(S)-3-hydroxy-3-methylglutaryl-CoA acetoacetyl-CoA-lyase HO (CoA-acetylating), EC, 4.1.3.5] from rat liver. The structure 0 of this 8-lactone, termed L-659,699, is (E,E)-11-[3-(hydroxy- 0 methyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienen- oic acid. A partially purified preparation of cytoplasmic FiG. 1. The structure of L-659,699. Stereochemistry is relative. HMG-CoA synthase from rat liver was inhibited by L-659,699 with an IC50 of 0.12 ,uM. The enzymes HMG-CoA reductase, lated independently from Fusarium sp. and Scopulariopsis 13-ketoacyl-CoA thiolase, acetoacetyl-CoA synthetase, and fat- sp., is a specific inhibitor of HMG-CoA synthase (17) (see ty acid synthase were not inhibited to any extent by this Fig. 1 for the structure of L-659,699). This compound was compound. In cultured Hep G2 cells, the compound inhibited originally isolated in 1971 from Cephalosporin sp. as 1233A the incorporation of [14C]acetate into sterols with an IC-% of 6 and was reported as an antibacterial product by Aldridge et ,uM, while incorporation of [3H]mevalonate into sterols in these al. (18). cells was not affected. The activity of HMG-CoA reductase in In the present report,' the activity of L-659,699 and its the cultured Hep G2 cells was induced in a dose-dependent analogs as inhibitors of HMG-CoA synthase is described. manner by incubation with L-659,699. A 37-fold increase in Also discussed are the effects of L-659,699 on cholesterol reductase was observed after a 24-hr incubation with 62 1M synthesis 'in cultured cells and in animals. L-659,699. The effect of a number of analogs of L-659,699 on HMG-CoA synthase is also discussed. MATERIALS AND METHODS. The fungal metabolites lovastatin and compactin are specific L-659,699 was isolated from Fusarium sp. in the Merck Sharp competitive inhibitors ofthe enzyme 3-hydroxy-3-rnethylglu- & Dohme Research Laboratories (Rahway, NJ). In all taryl coenzyme A (HMG-CoA) reductase (1-3). Administra- experiments described, the compound was dissolved in tion of these compounds to cultured animal cells (4), to intact dimethyl sulfoxide. [1-1"C]Acetyl-CoA and [5_3H]mevalon- animals in vivo (1, 5), and to humans (6, 7) resulted in an ate were from ICN and New England Nuclear, respectively; overall decrease in the synthesis of cholesterol. Thus, inhi- CoA esters were from Pha'rmacia P-L Biochemicals; and all bition of a specific enzyme in the cholesterol biosynthetic other biochemical reagents were from Sigma or Boehringer pathway (HMG-CoA reductase) was directly correlated with Mannheim. Other chemicals used were of analytical grade the activity of the overall pathway in a number of different and were obtained from local sources. biological systems. Preparation of Cytoplasmic HMG-CoA Synthase and(- A number of studies also have suggested that HMG-CoA Ketoacyl-CoA Thiolase. The enzymes were prepared from the synthase [(S)-3-hydroxy-3-methylglutaryl-CoA acetoacetyl- livers of Sprague-Dawley rats fed for 1 week with Purina rat CoA-lyase (CoA-acetylating), EC, 4.1.3.5], the first com- chow containing 0.075% lovastatin to induce the enzymes (9). mitted step in isoprenoid biosynthesis, may be an important Cytoplasmic HMG-CoA synthase was purified through the site ofcontrol for the cholesterol pathway. The activity ofthis DEAE-cellulose step essentially as described by Mehrabian enzyme is affected by agents that alter cholesterol synthesis et al. (19). This procedure was modified slightly by carrying in the livers ofchickens (8) and rats (8-10). Studies in Chinese out the 30-50% ammoniumi sulfate precipitation of the hamster ovary cells (11, 12), HeLa (13), C-6 glia cells (14, 15), enzyme and by dialysis prior to the DEAE-cellulose step. As and L-M cells (16) have also demonstrated that cholesterol described by Mehrabian ea al. (19), the 0.06 M and 0.1 M biosynthesis was sensitive to changes in the HMG-CoA potassium phosphate eluates contained the f3-ketothiolase synthase activity. and the synthase, respectively. These samples were concen- trated by ammonium sulfate, precipitation, dissolved in the With this in mind, and with the experience of finding buffer used for elution, and stored at -70'C. inhibitors of HMG-CoA reductase, an effort was undertaken HMG-CoA synthase and 8-ketoacyl-CoA thiolase were to discover agents that have a specific inhibitory effect on assayed as described by Clinkenbeard et al. (20). Unless HMG-CoA synthase. This search led to the discovery that stated otherwise, inhibition of HMG-CoA synthase was L-659,699 {(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]- measured as follows: of the enzyme was incubated in 3,5,7-trimethyl-2,4-undecadienenoic acid}, a P-lactone iso- ==24,4g Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme The publication costs of this article were defrayed in part by page charge A; L-659,699, (E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7- payment. This article must therefore be hereby marked "advertisement" trimethyl-2,4-undecadienenoic acid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

7488 Downloaded by guest on October 2, 2021 Biochemistry: Greenspan et al. Proc. Natl. Acad. Sci. USA 84 (1987) 7489 a vol of 85 1.l containing 118 mM Tris HCl (pH 8.0), 1.18 mM EDTA, 11.8 mM MgCl2, and 0.59 mM dithiothreitol. L-659,699 was added. to the incubation mixture in no more than 2 tkl dimethyl sulfoxide. This solution was incubated for 5 min at 306C, after which time 15 Al of a solution containing 80 acetoacetyl-CoA and [1-14C]acetyl-CoA (2.5 ,.Ci/umol; 1 Ci = 37 GBq) was added and the reaction continued for another 10 min at 30'C. The final concentrations in the assay were 100 z 60 F mM Tris-HCI (pH 8.0), 1.0 mM EDTA, 10 mM MgCl2, 0.5 0 mM dithiothreitol, 0.1 mM acetoacetyl-CoA, and 0.4 mM [1-14C]acetyl-CoA. The indicated concentrations of I L-659,699 were calculated on the basis ofthe 100-pAI vol in the z final 10-min assay. The reaction was stopped by the removal 40 F of 50 Al of the reaction mixture and by adding this to 200 ,ul of 6 M HCI in a glass scintillation vial. This solution was heated at 110°C for 1 hr. At the end of the hour, an additional 200 ul of 6 M HCI was added and the heating continued for 20 F an additional hour. One milliliter of water and 10 ml of Aquasol 2 were added to the vial and the radioactivity was determined in a liquid scintillation counter. Other Enzymes. Mitochondrial HMG-CoA synthase was 0.6 0.8 1 2 4 prepared through the DEAE-cellulose procedure and as- sayed by the method ofReed et al. (21). HMG-CoA reductase L-659,699 [M X was prepared and assayed by the method of Alberts et al. (1), 10-7] fatty acid synthase was prepared by the method of Alberts et FIG. 2. Inhibition of HMG-CoA synthase by L-659,699. Incuba- al. (22), and acetoacetyl-CoA synthetase was prepared as tions were carried out as described. described by Bergstrom et al. (9). Tissue Culture. Human Hep G2 cells obtained from Barbara Knowles (Wistar Institute, Philadelphia) were grown this enzyme (80%o inactivated) was diluted into the assay in 25-cm2 flasks in the presence of minimal essential medium mixtures with either no compound or enough L-659,699 to and 10% fetal calf serum. Forty-eight hours before the make final concentrations of0.04, 0.08, 0.12, and 0.4OAAM, the experiment, the cells were switched to serum-free Higuchi inhibition was reduced proportionally. Indeed, the inhibition medium (23). Incubations were carried out in the Higuchi of the enzyme by preincubation with L-659,699 coincided medium containing L-659,699, sodium [1-14C]acetate (1 very closely to a control in which the enzyme was preincu- mCi/35.7 mg), 2 /Ci/ml, and [5-3H]mevalonate lactone (5 bated without the inhibitor. These studies indicated that ,uCi/,mol), 5 ,u/ml. After 3 hr, the reaction was stopped by although 0.4 ,M L-659,699 inhibited the synthase almost aspiration of the medium and rinsing the cells three times with 80%, this inhibition could be reversed simply by dilution of 3 ml of saline. The cells were treated for 30 min with 2 ml of the enzyme-inhibitor complex. 1 M NaOH and then this mixture was transferred to screw cap Studies on the specificity of the inhibition of HMG-CoA tubes and heated at 95°C for 1 hr. A portion (0.2 ml) was synthase by L-659,699 are shown in Table 1. Enzymes cata- removed for protein determination by a microbiuret method lyzing different reactions of both fatty acid and cholesterol (24), the remainder of the solution was acidified with 0.2 ml biosynthesis as well as the mitochondrial HMG-CoA synthase of concentrated HCI and extracted three times with 4 ml each were assayed with L-659,699. The inhibition of both the of petroleum ether. The petroleum ether extracts were mitochondrial and the cytosolic HMG-CoA synthase were combined, evaporated to dryness under nitrogen, and redis- similar, with IC50 values of0.1 ALM, while HMG-CoA reductase, solved in 0.2 ml of ethyl acetate. A 0.1-ml aliquot was chromatographed on silica gel G thin-layer plates, which were developed in petroleum ether/diethyl ether/acetic acid (75:25:1). The plates were stained with iodine and those areas corresponding to standard cholesterol, oleic acid, and WI squalene were scraped into scintillation vials; 10 ml of 0. Aquasol 2 was added and the radioactivity was determined. CL Inhibition of Cholesterol Synthesis in Rats. Studies on the .-I incorporation of [1-14C]acetate into plasma cholesterol were carried out as described (1). O- RESULTS The 43-lactone L-659,699 inhibited HMG-CoA synthase with an IC50 of 0.12 ,uM, and a semilogarithmic plot of the activity of the enzyme versus inhibitor concentration is shown in Fig. L-659,699 (Mx1O-7) 2. The inhibition of HMG-CoA synthase by L-659,699 was FIG. 3. Effect of dilution on L-659,699-inhibited HMG-CoA demonstrated to be reversible in the experiment described in synthase. HMG-CoA synthase (120 Ag) was incubated in a vol of 50 Fig. 3. In this study, the enzyme was preincubated in the pl containing either no (e) or 0.4AM (o) L-659,699 at 300C for 5 miri. At the end of this time, a 10-Al aliquot of each of the enzymes was absence and the presence of enough L-659,699 (0.4 ,uM) to added to 75 Al of the standard reaction mixture, which contained produce almost complete inhibition of the synthase. When sufficient L-659,699 to bring the concentration of L-659,699 to 0.04, the same concentration of 0.4 AM was maintained in the 0.08, 0.12, and 0.4/AM. After an additional 5 min incubation at 30TC, assay system following the 5Cmin preincubation with the 15 ,Al of acetoacetyl-CoA and [1-_4C]acetyl-CoA were added and the inhibitor, the enzyme was inhibited -80o. However, when assay was carried out as described. Downloaded by guest on October 2, 2021 7490 Biochemistry: Greenspan et al. Proc. Natl. Acad. Sci. USA 84 (1987) Table 1. Inhibition of rat liver lipogenic enzymes by L-659,699 Table 3. Effect of L-659,699 on lipid synthesis in Hep G2 cells Enzyme IC50, M % inhibition of incorporation HMG-CoA synthase ['4C]Acetate [3HI]Mevalonate Cytosolic 1.0 x 1O-7 L-659,699, Fatty Fatty Mitochondrial 0.8 x 10-7 x 10-5 M Cholesterol acids Cholesterol acids HMG-CoA reductase >3.5 x 10-4 P-Ketoacyl-CoA thiolase >3.5 x 10-4 0.3 48.0 3.0 0 0 Acetoacetyl-CoA synthetase >3.5 x 10-4 3.0 85.4* 4.0 18 0 Fatty acid synthase 4.1 x 10-5 *Average of six determinations from 81.4-92.4% inhibition. ,3-ketoacyl-CoA thiolase, and acetoacetyl-CoA synthetase 85%, whereas mevalonate incorporation was essentially were not inhibited at 0.35 mM, the highest concentration tested. unaffected. Inhibition of acetate incorporation into choles- Slight inhibition was observed with fatty acid synthase, which terol was concentration dependent with an IC50 value of 6.0 was inhibited by L-659,699 with an IC50 of 41 ,M, a value AM. In addition, there was very little effect of L-659,699 on 1/400th that observed with HMG-CoA synthase. the incorporation of [14C]acetate into fatty acids. The pattern A series of analogs of L-659,699 were prepared and their of inhibition by L-659,699 of acetate and mevalonate into inhibition ofHMG-CoA synthase was compared to L-659,699 squalene was also examined and the results were similar to (Table 2). The saturated derivative (L-660,281; IC50, 0.4 ,tM) those observed for acetate and mevalonate incorporation into was .25% as active as the native polyunsaturated com- cholesterol in the Hep G2 cells. This study provides direct pound. The methyl ester (L-660,347) and the corresponding evidence that a block of HMG-CoA synthase activity will methyl ether (L-662,750) exhibited inhibitory activities com- lead to a block in overall cholesterol biosynthesis. That a parable to L-659,699, whereas acylation of the hydroxyl block in HMG-CoA synthase resulted in inhibition of sterol (L-660,343) or oxidative cleavage of the E-4 double bond to synthesis was first demonstrated by Schnitzer-Polokoff et al. form a ketone (L-662,786) reduced the inhibition consider- (12) with a mutant Chinese hamster ovary cell defective in ably. A loss of 1/3rd of the activity was observed with the HMG-CoA synthase. ethyl ether (L-663,225) as compared to the methyl ether. Incubation of L-659,699 with the Hep G2 cells not only Opening of the ,-lactone ring (L-660,282) resulted in com- inhibited cholesterol biosynthesis from acetate (synthesis plete loss of inhibitory activity. was inhibited >90% at a concentration of 15.5 ,uM) but also The effect of L-659,699 on sterol biosynthesis from acetate appeared to induce the activity of HMG-CoA reductase in a and mevalonate was examined in Hep G2 cells, and the dose-dependent manner (Table 4). The effects of L-659,699 results are described in Table 3. At a concentration 30 uM, on the activity of HMG-CoA synthase and ketothiolase were L-659,699 inhibited acetate incorporation into cholesterol by not measured in this experiment. Table 2. Inhibition of rat liver HMG-CoA synthase by L-659,699 and analogs L number ICo,, M

HO COOH 659,699 1 X 10-7 0

HO COOH 660,281 4 x 10-7 0

HO COOH 660,282 No activity O OH OH AcO COOH 660,343 2.0 x 10-6 0

HOO0 COOCH3 660,347 1 X 10-7 0

662,750 2.3 x 10-7

H0 O 662,786 1.6 x 10-6

CH3CH20 COOCH3 663,225 0 7.4 x 10-7 Downloaded by guest on October 2, 2021 Biochemistry: Greenspan et A Proc. Natl. Acad. Sci. USA 84 (1987) 7491 Table 4. Induction of HMG-CoA reductase in Hep G2 cells HMG-CoA reductase, an enzyme that is not the primary site by L-659,699 of inhibition of the compound. HMG-CoA synthase activity, Enzyme activity, Induction in and that of 8-ketothiolase were not assayed in this experi- L-659,699, pmol per min enzyme activity, ment. However, this study suggests that a common feature in x 10-5 M per mg of protein -fold the regulation of these early enzymes of cholesterol biosyn- thesis may exist and that inhibition of any of the early 0 65.6 0 enzymes will lead to changes in the levels of adjacent 0.31 198.9 3.0 enzymes in the pathway. 1.55 466.2 7.1 Although the mechanism of inhibition of HMG-CoA 3.1 1551.5 23.6 synthase by L-659,699 cannot be deduced by a comparison of 6.2 2428.1 37.0 the activities ofthe analogs, it is obvious that the lactone ring Hep G2 cells were grown as described. After 48 hr in serum-free must be intact for activity. The carboxyl end can be tampered Higuchi medium (23), L-659,699 was added to the culture flasks at the with without much loss of activity; however, the inhibition is indicated concentrations for 24 hr. At the end of the incubations, the sensitive to changes in the 3-hydroxyl position. cells were rinsed with 50 mM Tris-HCI/0.15 M NaCl, pH 7.4, Only two other naturally occurring f8-lactones with any scraped, suspended in a small volume of the same buffer, and stored important biological activity have been isolated. These are at -80'C. After thawing, HMG-CoA reductase was solubilized and ebelactone, an inhibitor of pig liver esterase, pig pancreas assayed by the method of Brown et al. (25). lipase, and N-formyl-methionine aminopeptidase (31); and esterastin, which inhibited lysosomal acid lipase (32). The effect ofL-659,699 on cholesterol biosynthesis in animals was also examined. The compound was administered orally to We would like to thank Mrs. Joan Kiliyanski for typing this rats followed 1 hr later by an i.p. administration of [1- manuscript. 14C]acetate. After 50 min more, the animals were bled and the incorporation of radioactivity into plasma cholesterol was 1. Alberts, A. W., Chen, J., Kuron, G., Hunt, V., Huff, J., determined. At a dose of 25 mg/kg, L-659,699 inhibited the Hoffman, C., Rothrock, J., Lopez, M., Joshua, H., Harris, E., incorporation of acetate into plasma sterols by 45% (data not Patchett, A., Monaghan, R., Currie, S., Stapley, E., Albers- shown). Schonberg, G., Hensens, O., Hirshfield, J., Hoogsteen, K., Liesch, J. & Springer, J. (1980) Proc. Natl. Acad. Sci. USA 77, 3957-3%1. DISCUSSION 2. Endo, A., Kuroda, M., Kuroda, M. & Tsujita, A. (1976) J. Antibiot. 29, 1346-1348. 3. Tansawa, K. & Endo, A. (1979) Eur. J. Biochem. 98, 195-201. The fl-lactone L-659,699 has been shown to be a potent 4. Brown, M. S., Faust, J. R., Goldstein, J. L., Kaneko, I. & inhibitor of the enzyme HMG-CoA synthase with an IC50 of Endo, A. (1978) J. Biol. Chem. 253, 1121-1128. 0.12 ,uM. Inhibition of the synthase by this compound was 5. Kovanen, P. T., Bilheimer, D. W., Goldstein, J. L., Jaramillo, specific inasmuch as the enzymes HMG-CoA reductase, J. J. & Brown, M. S. (1981) Proc. Natl. Acad. Sci. USA 78, ,8-ketoacyl-CoA thiolase, acetoacetyl-CoA synthetase, and 1194-1198. fatty acid synthase were not affected by the compound at 6. Bilheimer, D. W., Grundy, S. M., Brown, M. S. & Goldstein, concentrations up to 0.35 mM. The specificity was particu- J. L. (1983) Proc. Natl. Acad. Sci. USA 80, 4124-4128. larly interesting from a mechanistic point of view, since 7. Tobert, J. A., Bell, G. D., Birtwell, J., James, I., Kukovetz, that the the W. R., Pryor, J. S., Buntinz, A., Holmes, J. B., Chao, Y. S. several reports have indicated mechanisms of & Bolognese, J. A. (1982) J. Clin. Invest. 69, 913-919. fatty acid synthase (26) and the 8-ketoacyl-CoA thiolase (27) 8. Clinkenbeard, K. D., Sugiyama, T., Reed, W. D. & Lane, involve acyl-S-enzyme intermediates, as does HMG-CoA M. D. (1975) J. Biol. Chem. 250, 3124-3135. synthase (28, 29). Furthermore, while it has not been firmly 9. Bergstrom, J. F., Wong, G. A., Edwards, P. A. & Edmond, J. established, it is likely that the mechanisms of HMG-CoA (1984) J. Biol. Chem. 259, 14548-14553. reductase and acetoacetyl-CoA synthetase also involve thiol 10. Tanaka, R. D., Edwards, P., Lam, S. F., Knoppel, E. M. & intermediates. Yet, in spite of the presence of a reactive Fogelman, A. M. (1982) J. Lipid Res. 23, 1026-1031. ,3-lactone moiety, L-659,699 had virtually no effect on any of 11. Chang, T. Y. & Limanek, J. S. (1980) J. Biol. Chem. 255, these enzymes except HMG-CoA synthase. The reversibility 5452-5456. since it that 12. Schnitzer-Polokoff, R., Torget, R., Logel, J. & Sinensky, M. of the inhibition is also informative, suggests the (1983) Arch. Biochem. Biophys. 227, 71-80. mechanism of inhibition by L-659,699 is not via alkylation of 13. Ramachandran, C. K., Gray, S. L. & Melnykovych, G. (1978) the enzyme to form a stable alkyl-S-enzyme adduct, which Arch. Biochem. Biophys. 289, 205-211. might have been a consideration given the known pattern of 14. Volpe, J. J. & Obert, K. A. (1981) Arch. Biochem. Biophys. reactivity of simple lactones toward nucleophiles to form 212, 88-97. stable alkylation products. Preliminary studies also suggest 15. Maltese, W. A., Reitz, B. A. & Volpe, J. J. (1980) Biochem. J. that the inhibition may be competitive with acetyl-CoA and 192, 709-717. possibly with acetoacetyl-CoA as well. However, these 16. Maltese, W. A., Reitz, B. A. & Volpe, J. J. (1981) J. Biol. results are complicated by the fact that one of the substrates Chem. 256, 2185-2193. of HMG-CoA synthase, acetoacetyl-CoA, can act as an 17. Omura, S., Toma, H., Kumagai, H., Greenspan, M. D., inhibitor (data not reported). Our finding of substrate inhi- Yudkovitz, J. B., Chen, J., Alberts, A. W., Martin, I., Tubbs Mochales, S., Monaghan, R. L., Chabala, J. C., Schwartz, bition was in agreement with the studies of Lowe and R. E. & Patchett, A. A. (1987) J. Antibiot. 40, 1356-1357. (30), who showed that the mitochondrial HMG-CoA synthase 18. Aldridge, D. C., Gile, D. & Turner, W. B. (1971) J. Chem. was inhibited by acetoacetyl-CoA. Soc., 3888-3891. An especially interesting aspect of these studies with 19. Mehrabian, M., Callaway, K. A., Clarke, C. F., Tanaka, L-659,699 concerns what they have already revealed about R. D., Greenspan, M. D., Lusis, A. J., Sparkes, R. S., the regulation of the early steps of cholesterol biosynthesis. Mohandas, T., Edmond, J., Fogelman, A. M. & Edwards, Inhibition of HMG-CoA reductase by lovastatin concomi- P. A. (1986) J. Biol. Chem. 261, 16249-16255. tantly induced HMG-CoA reductase, HMG-CoA synthase, 20. Clinkenbeard, K. D., Reed, W. D., Mooney, R. A. & Lane, and acetoacetyl-CoA synthetase in rat livers (9), and HMG- M. D. (1975) J. Biol. Chem. 250, 3108-3116. CoA synthase and reductase in cultured cells (12). Similarly, 21. Reed, W. D., Clinkenbeard, K. D. & Lane, M. D. (1975) J. we have observed that L-659,699 induced the activity of Biol. Chem. 250, 3117-3123. Downloaded by guest on October 2, 2021 7492 Biochemistry: Greenspan et al. Proc. Nati. Acad. Sci. USA 84 (1987) 22. Alberts, A. W., Ferguson, K., Hennessey, S. & Vagelos, 28. Stewart, P. R. & Rudney, H. (1966) J. Biol. Chem. 241, P. R. (1974) J. Biol. Chem. 249, 5124-5249. 1222-1225. 23. Higuchi, K. (1970) J. Cell Physiol. 75, 65-72. 29. Middleton, B. & Tubbs, P. K. (1974) Biochem. J. 137, 15-23. 24. Zamenhof, S. (1957) Methods Enzymol. 3, 702. 30. Lowe, D. M. & Tubbs, P. K. (1985) Biochem. J. 227, 591- 25. Brown, M. S., Dana, S. E. & Goldstein, J. E. (1974) J. Biod. 599. Chem. 249, 789-796. 31. Umezawa, H., Aoyagi, T., Uotani, K., Hamada, K., Takeu- 26. Greenspan, M. D., Alberts, A. W. & Vagelos, P. R. (1969) J. chi, T. & Takahashi, S. (1980) J. Antibiot. 33, 1594-1595. Biol. Chem. 244, 6477-6485. 32. Inanaka, T., Moriyana, Y., Ecsedi, G. C., Aogagi, T., 27. Bloxham, D. P., Chalkley, R. A., Coghfin, S. J. & Salam, W. Amanuma-Muta, K., Ohkuma, S. & Takano, T. (1983) J. (1978) Biochem. J. 175, 999-1011. Biochem. 94, 1017-1020. Downloaded by guest on October 2, 2021