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Temperature dependence of the microsomal oxidation of ethanol by cytochrome P450 and hydroxyl radical-dependent reactions
ARCHIVESOF BIOCHEMISTRYAND BIOPHYSICS Vol. 269, No. 2, March, pp. 569-575,1989
Temperature Dependence of the Microsomal Oxidation of Ethanol by Cytochrome P450 and Hydroxyl Radical-Dependent Reactions SUSANA Department
PUNTARULO
of Biochemistry,
AND ARTHUR
I. CEDERBAUM’
Mount Sinai School of Medicine,
New York, New York 10029
Received August 15,1988, and in revised form October 20,1988
The temperature dependence and activation energies for the oxidation of ethanol by microsomes from controls and from rats treated with pyrazole was evaluated to determine whether the overall mechanism for ethanol oxidation by microsomes was altered by the pyrazole treatment. Arrhenius plots of the temperature dependence of ethanol oxidation by pyrazole microsomes were linear and exhibited no transition breaks, whereas a slight break was observed at about 20 * 2.5”C with control microsomes. Energies of activation (about 15-17 kcal/mol) were identical for the two microsomal preparations. Although transition breaks were noted for the oxidation of substrates such as dimethylnitrosamine and benzphetamine, activation energies for these two substrates were similar for control microsomes and microsomes from the pyrazole-treated rats. The addition of ferric-EDTA to the microsomes increased the rate of ethanol oxidation by a hydroxyl radical (‘OH)-dependent pathway. Arrhenius plots of the ‘OH-dependent oxidation of ethanol by both microsomal preparations were linear with energies of activation (about 7 kcal/mol) that were considerably lower than values found for the P450dependent pathway. These results suggest that, at least in terms of activation energy, the increase in microsomal ethanol oxidation by pyrazole treatment is not associated with any apparent change in the overall mechanism or rate-limiting step for ethanol oxidation but likely reflects induction of a P450 isozyme with increased activity toward ethanol. The lower activation energy for the ‘OH-dependent oxidation of ethanol suggests that different steps are rate limiting for oxidation of ethanol by ‘OH and by P450, which may reflect the different enzyme components of the microsomal electron transfer system involved in these reactions. 0 1989 Academic Press, Inc.
The oxidation of ethanol by liver microsomes may contribute toward the overall metabolism of ethanol and assumes increased significance at high concentration of ethanol and after chronic consumption of ethanol (l-6). The microsomal oxidation of ethanol is catalyzed by a cytochrome P450dependent reaction and chronic consumption of ethanol increases microsomal oxidation of ethanol by inducing an iso1To whom correspondence should be addressed at: Box 1020, Department of Biochemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. 569
zyme of cytochrome P450 with increased activity toward ethanol as substrate (7-9). Recent studies have shown that several other agents induce the same isozyme of cytochrome P450 as ethanol does (10-14). Pyrazole, a classic inhibitor of alcohol dehydrogenase, was shown to increase the microsomal oxidation of ethanol and other alcohols (15), as well as several other substrates such as aniline (16), climethylnitrosamine (17,18), and pyrazole itself (19). These increases in activity were associated with induction of a cytochrome P450 isozyme (20) which appears to be identical to the isozyme induced by ethanol and other 0003-986X39 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
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inducers of cytochrome P450IIEl in rat liver (11-14). In the presence of iron chelates, especially ferric-EDTA, microsomes generate ‘OH or ‘OH-like species during NADPHdependent electron transfer and under these conditions ethanol can be oxidized by a ‘OH-dependent reaction, in addition to the cytochrome P450-dependent reaction (21-24). The latter pathway has been shown to not be mediated by free ‘OH (8, 22). The generation of ‘OH by microsomes appears to involve NADPH-cytochrome P450 reductase as the major enzyme component for interacting with ferric-EDTA (21-23), although under certain circumstances, there may be some contribution by cytochrome P450 (25,26). Since the rate of microsomal ethanol oxidation is elevated by pyrazole treatment (15), studies on the temperature dependence and activation energy for ethanol oxidation by microsomes could provide information as to whether the overall mechanism for ethanol oxidation is altered by the pyrazole treatment. Differences in activation energies may be considered as an indication of different rate-limiting steps in a complex reaction mechanism (27). The ‘OH-dependent oxidation of ethanol was also evaluated to determine whether the activation energy for this reaction was similar to that of the P450-catalyzed oxidation of ethanol or whether the two reaction pathways could be distinguished from each other via different temperature dependence or activation energies. MATERIALS
AND
METHODS
Rat liver microsomes were isolated from male Sprague-Dawley rats weighing about 150 g by differential centrifugation, utilizing a buffer containing 0.25 M sucrose-O.01 M Tris, pH 7.4-0.001 M EDTA-50 pM desferrioxamine. The microsomes were washed twice with 125 mM KCI, suspended in 125 mM KCl, and stored at -70°C. Microsomes were also prepared from rats treated with pyrazole (200 mg/kg body wt per day) for 2 days and starved overnight. The production of ‘OH or components with the oxidizing power of ‘OH was assayed by following the generation of ethylene gas
CEDERBAUM from 2-keto-4-thiomethylbutyrate (KMB).2 The basic reaction system consisted of 100 mM potassium phosphate buffer, pH 7.4,1 mM sodium azide (to inhibit catalase present as a contaminant in isolated microsomes), 10 mM KMB, 50 @Mferric ammonium sulfate100 pM EDTA (ferric-EDTA, 1:2 chelate) and about 1 mg microsomal protein in a final volume of 1 ml. Reactions were initiated by the addition of 2.0 mM NADPH and were terminated with 1 N HCl after a IO-min period. Ethylene production was assayed by a head-space gas chromatography procedure (24). NADPH was utilized in place of an NADPH-generating system to avoid any complexities which could result from the effect of temperature on the generating system itself (e.g., activity of glucose-6-phosphate dehydrogenase). Ethanol oxidation was determined by assaying for the production of acetaldehyde. Reactions were carried out in the system described above except that 50 mM ethanol was added instead of KMB, and in some experiments, the ferric-EDTA was omitted. Reactions were carried out for 5 min and acetaldehyde was determined by a head-space gas chromatography procedure (24). The demethylation of 1 mM benzphetamine and 2 mM dimethylnitrosamine (DMN) was determined in the same basic reaction system. The formaldehyde produced was determined by a fluorometric modification (28) of the method of Nash (29), using an excitation wavelength of 415 nm and an emission wavelength of 505 nm. All values were corrected for zero-time controls in which HCl or TCA was added prior to the microsomes. Experiments were conducted in Dubnoff shakers at the indicated temperatures, which were maintained &O.l”C of the appropriate temperature. Energies of activation were calculated by linear regression analysis of the slopes of the Arrhenius plots. RESULTS
Ethanol oxidation by microsomes, in the absence of ferric-EDTA, is mainly due to a cytochrome P450-catalyzed reaction (8, 9, 22,30). In the presence of ferric-EDTA the rates of acetaldehyde production are increased, and these rates represent the oxidation of ethanol by cytochrome P450dependent plus ‘OH- (or oxidants with similar oxidizing potential) dependent pathways. Differences in the rates of ethanol oxidation in the absence and presence of ferric-EDTA reflect the ‘OH-dependent ’ Abbreviations used: KMB, 2-keto-4-thiomethylbutyrate; DMN, dimethylnitrosamine; TCA, trichloroacetic acid.
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FIG. 1. Effect of temperature on the microsomal oxidation of ethanol. (A) Reactions were carried out as described under Materials and Methods utilizing microsomes from chow-fed controls (0) or from rats treated with pyrazole (0). In some experiments, 50 pM ferric-EDTA was added to control microsomes (A) or to the microsomes from the pyrazole-treated rats (a). (B) Rates refer to the difference in rates for reactions carried out in the absence and presence of ferricEDTA for control microsomes (0) and for microsomes from pyrazole-treated rats (0).
oxidation of ethanol (22-24). Rates of ethanol oxidation were determined in the absence and presence of ferric-EDTA as a function of increasing temperature. The rate of acetaldehyde production from ethanol was increased as the temperature was elevated over the range of 8 to 30°C; at all temperatures, the rate was higher with microsomes isolated from rats treated with pyrazole as compared to chow-fed controls although this was most notable at the higher temperatures (Fig. 1A). The addition of ferric-EDTA, the iron chelate which was found to be most effective in stimulating microsomal ‘OH generation (23), increased the oxidation of ethanol by both microsomal preparations (Fig. 1A). The ‘OH-dependent rate of ethanol oxidation, as calculated from the difference in rates of ethanol oxidation in the absence and presence of ferric-EDTA, also increased as the temperature was elevated (Fig. 1B). Arrhenius plots were calculated from these results and are shown in Fig. 2. The cytochrome P450-dependent rate of ethanol oxidation by control microsomes displayed a slight, but consistent, break in the
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Arrhenius plot at a temperature of about 20.3 f 2.5”C, whereas the Arrhenius plot for ethanol oxidation by microsomes from pyrazole-treated rats was linear (Fig. 2A). In the presence of ferric-EDTA, the Arrhenius plots were linear for both microsomal preparations (Fig. 2A, top 2 curves). The Arrhenius plots for the ‘OH-dependent oxidation of ethanol by both microsomal preparations were also linear (Fig. 2B). Energies of activation for the cytochrome P450, the total, and the-OH-dependent oxidation of ethanol were calculated from the slopes of the plots shown in Fig. 2. The energy of activation for the cytochrome P450-catalyzed oxidation of ethanol was identical for microsomes isolated from rats treated with pyrazole and controls (Table I). In a similar manner, energies of activation for the total and ‘OH-dependent rates of ethanol oxidation were identical for both microsomal preparations. However, the energy of activation for ethanol oxidation by a ‘OH-dependent pathway was markedly lower than that required for ethanol oxidation by the cytochrome P450-dependent pathway (Table I). Parallel experiments with substrates such as benzphetamine and DNM were conducted, as a comparison for results ob-
-s
7-7
5[ 4t
FIG. 2. Arrhenius plot of the microsomal oxidation of ethanol. (A) Results were calculated from the data of Fig. 1A. Control mierosomes (0); mierosomes from pyrazole-treated rats (0); control microsomes plus 50 pM ferric-EDTA (A.); pyrazole microsomes plus 50 FM ferric-EDTA (a). (B) Results were calculated from the data of Fig. 1B and refer to the ‘OH-dependent oxidation of ethanol by control microsomes (0) and by microsomes from pyrazole-treated rats (0).
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It was difficult to accurately measure the low rates of DMN mttabolism by control microsomes at temperatures much below the transition temperature, which could contribute toward the very high activation Energy of activation energy calculated under these conditions (keaVmo1) (93.5 kcal/mol, Table I). The finding of a discontinuity in the Arrhenius plot for the Pyrazole Substrate oxidation of DMN or benzphetamine, but Controls treated not for ethanol oxidation, by microsomes Ethanol from pyrazole-treated rats suggests an imMinus ferricportant role of the substrates in the overall EDTA 14.5 + 1.2” 17.4 + o.@ 14.9 f 2.2 pattern. This is reminiscent of results by Plus ferricPeterson et al, (31) who found that ArrheEDTA 10.3 f 0.7 8.7 * 1.4 nius plots for the slow phase of reduction A (‘OH of cytochrome P450 by NADPH exhibited 7.4 + 1.1” dependent) 6.3 + 1.3" a discontinuity with some substrates 12.ga40.8* DMN 11.8"93.6* Benzphetamine 14.8"47.4b (hexobarbital and ethylmorphine) but 11.9”37.4b were linear with more hydrophobic subNote. The energies of activation were calculated by strates. linear regression analysis of the slopes of the ArrhenIn view of the necessity of calculating ius plots. Results, where indicated, refer to means differences of rates of ethanol oxidation in f SE. the absence and presence of ferric-EDTA “Calculated for results above the temperature to obtain the ‘OH-dependent rates, experibreak. *Calculated for results below the temperature ments were conducted to determine the break. effect of temperature on the oxidation of ‘P < 0.05 as compared to the minus ferric-EDTA the potent ‘OH scavenger, KMB, to ethylvalues. ene. Oxidation of KMB by microsomes is very low in the absence of ferric-EDTA tained with ethanol. DMN, but not benz- and proceeds by a cytochrome P450-indephetamine, is a good substrate for oxida- pendent pathway (21). Inhibition of KMB tion by cytochrome P450IIEl (12, 14, 1’7, oxidation by catalase or glutathione plus 18). The pyrazole treatment resulted in glutathione peroxidase or a variety of competitive ‘OH scavengers implicates a role about a twofold increase in the oxidation of DMN, whereas benzphetamine demeth- for ‘OH, or an ‘OH-like species, in the miylase activity was decreased about 10 to crosomal oxidation of KMB to ethylene (23, 20% (data not shown). With both micro- 32, 33). Rates of ethylene generation by somal preparations, there was a break in both microsomal preparations increased the Arrhenius plot for the temperature de- as the temperature was increased (Fig. pendence of DMN or benzphetamine oxida- 3A). Arrhenius plots for both microsomal tion (data not shown). The transition tem- preparations were linear and displayed no perature for the oxidation of both sub- transition breaks (Fig. 3B). Energies of activation for the ‘OH-dependent oxidation strates by either microsomal preparation was the same, about 20°C. Activation ener- of KMB were 8.5 and 10.1 kcal/mol for congies for both substrates, above and below trol microsomes and microsomes from the transition temperature, are shown in pyrazole-treated rats, respectively. Table I. Similar to results found for ethanol oxidation, there were no significant DISCUSSION differences in the activation energies for In view of the higher molar ratio of cytothe oxidation of DMN or benzphetamine by control microsomes as compared to mi- chrome P450 compared to NADPH-cytocrosomes from the pyrazole-treated rats. chrome P450 reductase, there has been TABLE
I
ENERGIES OF ACTIVATION FOR THE OXIDATION OF ETHANOL, DMN, AND BENZPHETAMINE BY MICROSOMES
ENERGY
Co+ TEMPERATURE (Tl
OF ACTIVATION
-‘&,I ’ ’ 1 ’ ’ 4 0330335 340 345 ?JM355 360 I,, I !03CK.‘1
FIG. 3. Effect of temperature on the oxidation of KMB to ethylene by control microsomes (0) and by microsomes from pyrazole-treated rats (0). Reactions were carried out as described under Materials and Methods. B is the Arrhenius plot of the results shown in A.
considerable interest in the lateral diffusion of these two major enzyme components of the hepatic mixed-function oxidase system, and the effect of temperature on mixed-function oxidase reactions (31, 34-37). Schenkman (38) reported that there was no transition temperature for NADPH-cytochrome P450 reductase activity nor for the oxidation of drug substrates such as aminopyrine, ethylmorphine, or aniline, although each substrate possessed a different activation energy (13-21 kcal/mol). Other workers have reported transition breaks (20-25°C) in the Arrhenius plots associated with the demethylation of ethylmorphine, benzphetamine, or aminopyrine (39), or the metabolism of ethoxycoumarin or p-nitroanisole (40). The slow, but not the rapid, phase of reduction of cytochrome P450 by NADPH demonstrated a break in the Arrhenius plot at about 20°C (31). The activation energy for ethanol oxidation by control microsomes and by microsomes from pyrazole-treated rats was found to be identical, although the rate of ethanol oxidation was several-fold higher with the latter. Energies of activation for ethanol oxidation in the absence of added ferric-EDTA were about 15 kcal/mol, which is comparable to values found for the oxidation of DMN and benzphetamine (above the transition temperature) by the same microsomes and for other substrates oxidized by a cytochrome P450-dependent
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process (38-40). The increase in ethanol oxidation after pyrazole treatment reflects the induction of an isozyme of cytochrome P450, P450IIEl. Although ethanol is a preferred substrate for oxidation by cytochrome P450IIE1, it can also be oxidized at lower rates by other isozymes of P450 including the phenobarbital-induced isozyme (8,22,41). The comparable activation energies for ethanol oxidation by control and by induced microsomes suggests that a similar rate-limiting step is common for the overall oxidation of ethanol by the various isozymes present in control microsomal preparations and by the pyrozoleinduced P450. The addition of ferric-EDTA increases the rate of ethanol oxidation by an ‘OH-dependent mechanism (22-24). There was a striking lowering of the activation energy for ethanol oxidation by both microsomal preparations in the presence of ferricEDTA. The activation energy for the ‘OHdependent oxidation of ethanol was similar to the activation energy for the oxidation of KMB, and was about one-half that for the cytochrome P450-dependent oxidation of ethanol. These results suggest different overall mechanisms or rate-limiting steps for the oxidation of ethanol by ‘OH and by cytochrome P450. This would be in agreement with previous results indicating that the reductase and not cytochrome P450 played the major role in the interaction with ferric-EDTA and the subsequent generation of ‘OH (21,22). Linear Arrhenius plots were generated when assaying the activity of the reductase (3%40), and it was suggested that this reflected interaction of the reductase with water-soluble acceptors such as cytochrome c or dichlorophenol indophenol (42). The linear Arrhenius plots generated for the ‘OH-dependent oxidation of ethanol and KMB may, in an analogous manner, reflect the interaction of the reductase with watersoluble ferric-EDTA and the generation of ‘OH in solution. Therefore, changes in the physical state of the membrane as caused by temperature should not alter the reductaseferric-EDTA-catalyzed generation of ‘OH.
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Another possibility for the lowered activation energy for ethanol oxidation by ‘OH compared to cytochrome P450 could reflect the energetics required for the breaking of the carbon-hydrogen bond of carbon one of the ethanol molecule. Oxidation of ethanol by ‘OH proceeds via hydrogen abstraction to yield the hydroxylalkyl radical (CHaCHOH), which upon oxidation or dismutation would give rise to acetaldehyde. Hydroxylation of ethanol by an oxidizing intermediate of P450 presumably also requires breaking a carbon-hydrogen bond, followed by insertion of an oxygen atom to yield hydrated acetaldehyde. If carbon-hydrogen bond breakage is a ratelimiting step, the lower activation energy associated with the oxidation of ethanol by ‘OH compared to cytochrome P450 may reflect the relative ease by which each of the oxidants catalyze cleavage of this bond. In summary, at least in terms of activation energy, the induction of P450IIEl by pyrazole treatment is not associated with any apparent change in the overall mechanism or rate-limiting step for ethanol oxidation. The increase in ethanol oxidation produced by pyrazole treatment appears therefore to be associated with an increased amount of an ethanol-preferring isozyme of P450. The ‘OH-dependent oxidation of ethanol is associated with a lower energy of activation than the cytochrome P450-dependent oxidation, suggesting that the latter pathway proceeds via a more complex mechanism than the ‘OH-dependent pathway, and that different steps are rate limiting for the oxidation of ethanol by either of the two reaction pathways. ACKNOWLEDGMENTS These studies were supported by United States Public Health Service Grants AA-06610 and AA03312 from The National Institute on Alcohol Abuse and Alcoholism. We thank Ms. Roslyn C. King for typing the manuscript.
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CEDERBAUM 2. LIEBER, C. S., AND DECARLI, L. M. (1972) J. PharmacoL Exp. Ther. 181,279-287. 3. VIND, C., AND GRUNNET, N. (1985) Biochem. PharmacoL 34,655-661. 4. TESCHKE, R., AND GELLERT, J. (1986) Alcoholism Clin Exp. Res. 10,2OS-32s. 5. LIEBER, C. S. (1984) Hepatology 4,1243-1260. 6. SHIGETA, Y., NOMURA, F., IIDA, S., LEO, M. A., FELDER, M. A., AND LIEBER, C. S. (1984) Biothem. Phamnacol33,807-814. 7. OHNISHI, K., AND LIEBER, C. S. (1977) J. Biol. Chem. 252,7124-7131. 8. MORGAN, E. T., KOOP, D. R., AND COON, M. J. (1982) J BioL Chem. 257,13951-13957. 9. KOOP, D. R., MORGAN, E. T., TARR, G. E., AND COON, M. J. (1982) J. Biol. Chem. 257,8472-8480. 10. KOOP, D. R., CRUMP, B. L., NORDBLOM, G. D., AND COON, M. J. (1985) Proc. NatL Acad Sci. USA. 82,4066-4069. 11. RYAN, D. E., RAMANATHAN, L., IIDA, S., THOMAS, P. E., HANIU, M., SHIVELY, J. E., LIEBER, C. S., AND LEVIN, W. (1985) J. BioL Chem 260,63856393. 12. RYAN, D. E., KOOP, D. R., THOMAS, P. E., COON, M. J., AND LEVIN, W. (1986) Arch Biochem. Birr phys. 246,633-644. 13. KOOP, D. R., AND CASAZZA, J. P. (1985) J. BioL Chem. 260,13607-13612. 14. PATTEN, C. J., NING, S. M., Lu, A. Y. H., AND YANG, C. S. (1986) Arch. B&hem. Biophys. 251,629638. 15. KRIKUN, G., AND CEDERBAUM, A. I. (1984) Biochim. Biophys. Ada 801,131-137. 16. POWIS, G., AND GRANT, L. (1976) Biochem. PharmacoL 25,2197-2201. 17. Tu, Y. Y., SONNENBERG, J., LEWIS, K. F., AND YANG, C. S. (1981) B&hem. Biophys. Res. Commun. 103,905-912. 18. EVARTS, R. P., HALIDAY, E., NEGESHI, M., AND HJELMELAND, L. M. (1982) B&hem. PharmaCOL31,1245-1249. 19. FEIERMAN, D. E., AND CEDERBAUM, A. I. (1987) LIrug Metab. Dispos. 15,634-639. 20. PALAKODETY, R. B., CLEJAN, L., KRIKUN, G., FEIERMAN, D. E., AND CEDERBAUM, A. I. (1988) J Biol. Chem. 263,878-884. 21. WINSTON, G. W., AND CEDERBAUM, A. I. (1983) J. Biol. Chem. 258,1508-1513. 22. WINSTON, G. W., AND CEDERBAUM, A. I. (1983) J. Biol. Chem. 258,1514-1519. 23. WINSTON, G. W., FEIERMAN, D. E., AND CEDERBAUM, A. I. (1984) Arch. Biochem. Biwhys. 232, 378-390. 24. CEDERBAUM, A. I., AND COHEN, G. (1984) in Methods in Enzymology (Packer, L., Ed.), Vol. 105, pp. 516-522, Academic Press, San Diego.
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25. INGELMAN-SIJNDBERG, M., AND JOHANSSON, I. (1981) J. Bio/. Chem. 256,6321-6326. 26. EKSTROM, G., CRONHOLM, T., AND INGELMANSUNDBERG, M. (1986) Biochem. J 233,755-‘761. 27. GUTFREUND, H., Ed. (1972) Enzymes: Physical Principles, pp. 157-167, Wiley-Interscience, London. 28. STEFFEN, C., AND NETTER, K. J. (1979) Z'o~icol Appl. Pharmacol47,593-602. 29. NASH,T. C. (1953) Biochem. J. 55,416-421. 30. KOOP, D. R., NORDBLOM, G. D., AND COON, M. J. (1984) Arch. Biochem. Biophys. 235,228-238. 31. PETERSON, J. A., EBEL, R. E., O’KEEFFE, D. H., MATSUBARA, T., AND ESTABROOK, R. W. (1976) J. Biol. Chem 251,4010-4016. 32. COHEN, G., AND CEDERBAUM, A. I. (1980) Arch. Biochem. Biophys. 199,438-447. 33. BELOQUI, O., AND CEDERBAUM, A. I. (1986) Bie them. Pharmacol. 35,2663-2669. 34. ESTABROOK, R. W., WERRINGLOER, J., MASTERS, B. S. S., JONEN, H., MATSUBARA, T., EBEL, R.,
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O’KEEFE, D., AND PETERSON, J. A. (1976) in The Structural Basis of Membrane Function (Hatefi, Y., and Diavadi-Ohaniance, L., Eds.), pp. 429-445, Academic Press, New York. Wu, E. S., AND YANG, C. S. (1984) Biochemistry 23, 28-33. DEPIERRE, J. W., AND DALLNER, G. (1975) Biochim. Biophys. Acta 415,411-472. YANG, C. S. (1975) FEBSLett. 54,61-64. SCHENKMAN, J. B. (1972) MoL Pharmacol 8,178188. YANG, C. S., STRICKHART, F. S., AND KICHA, L. P. (1977) Biochim. Biophys. Acta 465,362-370. DUPPEL, W., AND ULLRICH, V. (1976) Biochim. Biophys. Acta 426,399-407. MIWA, G. T., LEVIN, W., Lu, A. Y. H., AND THOMAS, P. E. (1978) Arch. Biochem. Biophys. 187,464473. TANIGUCHI, H., IMAI, Y., AND SATO, R. (1987) Bie chemistry 26,7084-‘7090.
Temperature Dependence of the Microsomal Oxidation of Ethanol by Cytochrome P450 and Hydroxyl Radical-Dependent Reactions SUSANA Department
PUNTARULO
of Biochemistry,
AND ARTHUR
I. CEDERBAUM’
Mount Sinai School of Medicine,
New York, New York 10029
Received August 15,1988, and in revised form October 20,1988
The temperature dependence and activation energies for the oxidation of ethanol by microsomes from controls and from rats treated with pyrazole was evaluated to determine whether the overall mechanism for ethanol oxidation by microsomes was altered by the pyrazole treatment. Arrhenius plots of the temperature dependence of ethanol oxidation by pyrazole microsomes were linear and exhibited no transition breaks, whereas a slight break was observed at about 20 * 2.5”C with control microsomes. Energies of activation (about 15-17 kcal/mol) were identical for the two microsomal preparations. Although transition breaks were noted for the oxidation of substrates such as dimethylnitrosamine and benzphetamine, activation energies for these two substrates were similar for control microsomes and microsomes from the pyrazole-treated rats. The addition of ferric-EDTA to the microsomes increased the rate of ethanol oxidation by a hydroxyl radical (‘OH)-dependent pathway. Arrhenius plots of the ‘OH-dependent oxidation of ethanol by both microsomal preparations were linear with energies of activation (about 7 kcal/mol) that were considerably lower than values found for the P450dependent pathway. These results suggest that, at least in terms of activation energy, the increase in microsomal ethanol oxidation by pyrazole treatment is not associated with any apparent change in the overall mechanism or rate-limiting step for ethanol oxidation but likely reflects induction of a P450 isozyme with increased activity toward ethanol. The lower activation energy for the ‘OH-dependent oxidation of ethanol suggests that different steps are rate limiting for oxidation of ethanol by ‘OH and by P450, which may reflect the different enzyme components of the microsomal electron transfer system involved in these reactions. 0 1989 Academic Press, Inc.
The oxidation of ethanol by liver microsomes may contribute toward the overall metabolism of ethanol and assumes increased significance at high concentration of ethanol and after chronic consumption of ethanol (l-6). The microsomal oxidation of ethanol is catalyzed by a cytochrome P450dependent reaction and chronic consumption of ethanol increases microsomal oxidation of ethanol by inducing an iso1To whom correspondence should be addressed at: Box 1020, Department of Biochemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. 569
zyme of cytochrome P450 with increased activity toward ethanol as substrate (7-9). Recent studies have shown that several other agents induce the same isozyme of cytochrome P450 as ethanol does (10-14). Pyrazole, a classic inhibitor of alcohol dehydrogenase, was shown to increase the microsomal oxidation of ethanol and other alcohols (15), as well as several other substrates such as aniline (16), climethylnitrosamine (17,18), and pyrazole itself (19). These increases in activity were associated with induction of a cytochrome P450 isozyme (20) which appears to be identical to the isozyme induced by ethanol and other 0003-986X39 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
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inducers of cytochrome P450IIEl in rat liver (11-14). In the presence of iron chelates, especially ferric-EDTA, microsomes generate ‘OH or ‘OH-like species during NADPHdependent electron transfer and under these conditions ethanol can be oxidized by a ‘OH-dependent reaction, in addition to the cytochrome P450-dependent reaction (21-24). The latter pathway has been shown to not be mediated by free ‘OH (8, 22). The generation of ‘OH by microsomes appears to involve NADPH-cytochrome P450 reductase as the major enzyme component for interacting with ferric-EDTA (21-23), although under certain circumstances, there may be some contribution by cytochrome P450 (25,26). Since the rate of microsomal ethanol oxidation is elevated by pyrazole treatment (15), studies on the temperature dependence and activation energy for ethanol oxidation by microsomes could provide information as to whether the overall mechanism for ethanol oxidation is altered by the pyrazole treatment. Differences in activation energies may be considered as an indication of different rate-limiting steps in a complex reaction mechanism (27). The ‘OH-dependent oxidation of ethanol was also evaluated to determine whether the activation energy for this reaction was similar to that of the P450-catalyzed oxidation of ethanol or whether the two reaction pathways could be distinguished from each other via different temperature dependence or activation energies. MATERIALS
AND
METHODS
Rat liver microsomes were isolated from male Sprague-Dawley rats weighing about 150 g by differential centrifugation, utilizing a buffer containing 0.25 M sucrose-O.01 M Tris, pH 7.4-0.001 M EDTA-50 pM desferrioxamine. The microsomes were washed twice with 125 mM KCI, suspended in 125 mM KCl, and stored at -70°C. Microsomes were also prepared from rats treated with pyrazole (200 mg/kg body wt per day) for 2 days and starved overnight. The production of ‘OH or components with the oxidizing power of ‘OH was assayed by following the generation of ethylene gas
CEDERBAUM from 2-keto-4-thiomethylbutyrate (KMB).2 The basic reaction system consisted of 100 mM potassium phosphate buffer, pH 7.4,1 mM sodium azide (to inhibit catalase present as a contaminant in isolated microsomes), 10 mM KMB, 50 @Mferric ammonium sulfate100 pM EDTA (ferric-EDTA, 1:2 chelate) and about 1 mg microsomal protein in a final volume of 1 ml. Reactions were initiated by the addition of 2.0 mM NADPH and were terminated with 1 N HCl after a IO-min period. Ethylene production was assayed by a head-space gas chromatography procedure (24). NADPH was utilized in place of an NADPH-generating system to avoid any complexities which could result from the effect of temperature on the generating system itself (e.g., activity of glucose-6-phosphate dehydrogenase). Ethanol oxidation was determined by assaying for the production of acetaldehyde. Reactions were carried out in the system described above except that 50 mM ethanol was added instead of KMB, and in some experiments, the ferric-EDTA was omitted. Reactions were carried out for 5 min and acetaldehyde was determined by a head-space gas chromatography procedure (24). The demethylation of 1 mM benzphetamine and 2 mM dimethylnitrosamine (DMN) was determined in the same basic reaction system. The formaldehyde produced was determined by a fluorometric modification (28) of the method of Nash (29), using an excitation wavelength of 415 nm and an emission wavelength of 505 nm. All values were corrected for zero-time controls in which HCl or TCA was added prior to the microsomes. Experiments were conducted in Dubnoff shakers at the indicated temperatures, which were maintained &O.l”C of the appropriate temperature. Energies of activation were calculated by linear regression analysis of the slopes of the Arrhenius plots. RESULTS
Ethanol oxidation by microsomes, in the absence of ferric-EDTA, is mainly due to a cytochrome P450-catalyzed reaction (8, 9, 22,30). In the presence of ferric-EDTA the rates of acetaldehyde production are increased, and these rates represent the oxidation of ethanol by cytochrome P450dependent plus ‘OH- (or oxidants with similar oxidizing potential) dependent pathways. Differences in the rates of ethanol oxidation in the absence and presence of ferric-EDTA reflect the ‘OH-dependent ’ Abbreviations used: KMB, 2-keto-4-thiomethylbutyrate; DMN, dimethylnitrosamine; TCA, trichloroacetic acid.
ENERGY
OF ACTIVATION
FIG. 1. Effect of temperature on the microsomal oxidation of ethanol. (A) Reactions were carried out as described under Materials and Methods utilizing microsomes from chow-fed controls (0) or from rats treated with pyrazole (0). In some experiments, 50 pM ferric-EDTA was added to control microsomes (A) or to the microsomes from the pyrazole-treated rats (a). (B) Rates refer to the difference in rates for reactions carried out in the absence and presence of ferricEDTA for control microsomes (0) and for microsomes from pyrazole-treated rats (0).
oxidation of ethanol (22-24). Rates of ethanol oxidation were determined in the absence and presence of ferric-EDTA as a function of increasing temperature. The rate of acetaldehyde production from ethanol was increased as the temperature was elevated over the range of 8 to 30°C; at all temperatures, the rate was higher with microsomes isolated from rats treated with pyrazole as compared to chow-fed controls although this was most notable at the higher temperatures (Fig. 1A). The addition of ferric-EDTA, the iron chelate which was found to be most effective in stimulating microsomal ‘OH generation (23), increased the oxidation of ethanol by both microsomal preparations (Fig. 1A). The ‘OH-dependent rate of ethanol oxidation, as calculated from the difference in rates of ethanol oxidation in the absence and presence of ferric-EDTA, also increased as the temperature was elevated (Fig. 1B). Arrhenius plots were calculated from these results and are shown in Fig. 2. The cytochrome P450-dependent rate of ethanol oxidation by control microsomes displayed a slight, but consistent, break in the
FOR ETHANOL
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OXIDATION
Arrhenius plot at a temperature of about 20.3 f 2.5”C, whereas the Arrhenius plot for ethanol oxidation by microsomes from pyrazole-treated rats was linear (Fig. 2A). In the presence of ferric-EDTA, the Arrhenius plots were linear for both microsomal preparations (Fig. 2A, top 2 curves). The Arrhenius plots for the ‘OH-dependent oxidation of ethanol by both microsomal preparations were also linear (Fig. 2B). Energies of activation for the cytochrome P450, the total, and the-OH-dependent oxidation of ethanol were calculated from the slopes of the plots shown in Fig. 2. The energy of activation for the cytochrome P450-catalyzed oxidation of ethanol was identical for microsomes isolated from rats treated with pyrazole and controls (Table I). In a similar manner, energies of activation for the total and ‘OH-dependent rates of ethanol oxidation were identical for both microsomal preparations. However, the energy of activation for ethanol oxidation by a ‘OH-dependent pathway was markedly lower than that required for ethanol oxidation by the cytochrome P450-dependent pathway (Table I). Parallel experiments with substrates such as benzphetamine and DNM were conducted, as a comparison for results ob-
-s
7-7
5[ 4t
FIG. 2. Arrhenius plot of the microsomal oxidation of ethanol. (A) Results were calculated from the data of Fig. 1A. Control mierosomes (0); mierosomes from pyrazole-treated rats (0); control microsomes plus 50 pM ferric-EDTA (A.); pyrazole microsomes plus 50 FM ferric-EDTA (a). (B) Results were calculated from the data of Fig. 1B and refer to the ‘OH-dependent oxidation of ethanol by control microsomes (0) and by microsomes from pyrazole-treated rats (0).
572
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AND CEDERBAUM
It was difficult to accurately measure the low rates of DMN mttabolism by control microsomes at temperatures much below the transition temperature, which could contribute toward the very high activation Energy of activation energy calculated under these conditions (keaVmo1) (93.5 kcal/mol, Table I). The finding of a discontinuity in the Arrhenius plot for the Pyrazole Substrate oxidation of DMN or benzphetamine, but Controls treated not for ethanol oxidation, by microsomes Ethanol from pyrazole-treated rats suggests an imMinus ferricportant role of the substrates in the overall EDTA 14.5 + 1.2” 17.4 + o.@ 14.9 f 2.2 pattern. This is reminiscent of results by Plus ferricPeterson et al, (31) who found that ArrheEDTA 10.3 f 0.7 8.7 * 1.4 nius plots for the slow phase of reduction A (‘OH of cytochrome P450 by NADPH exhibited 7.4 + 1.1” dependent) 6.3 + 1.3" a discontinuity with some substrates 12.ga40.8* DMN 11.8"93.6* Benzphetamine 14.8"47.4b (hexobarbital and ethylmorphine) but 11.9”37.4b were linear with more hydrophobic subNote. The energies of activation were calculated by strates. linear regression analysis of the slopes of the ArrhenIn view of the necessity of calculating ius plots. Results, where indicated, refer to means differences of rates of ethanol oxidation in f SE. the absence and presence of ferric-EDTA “Calculated for results above the temperature to obtain the ‘OH-dependent rates, experibreak. *Calculated for results below the temperature ments were conducted to determine the break. effect of temperature on the oxidation of ‘P < 0.05 as compared to the minus ferric-EDTA the potent ‘OH scavenger, KMB, to ethylvalues. ene. Oxidation of KMB by microsomes is very low in the absence of ferric-EDTA tained with ethanol. DMN, but not benz- and proceeds by a cytochrome P450-indephetamine, is a good substrate for oxida- pendent pathway (21). Inhibition of KMB tion by cytochrome P450IIEl (12, 14, 1’7, oxidation by catalase or glutathione plus 18). The pyrazole treatment resulted in glutathione peroxidase or a variety of competitive ‘OH scavengers implicates a role about a twofold increase in the oxidation of DMN, whereas benzphetamine demeth- for ‘OH, or an ‘OH-like species, in the miylase activity was decreased about 10 to crosomal oxidation of KMB to ethylene (23, 20% (data not shown). With both micro- 32, 33). Rates of ethylene generation by somal preparations, there was a break in both microsomal preparations increased the Arrhenius plot for the temperature de- as the temperature was increased (Fig. pendence of DMN or benzphetamine oxida- 3A). Arrhenius plots for both microsomal tion (data not shown). The transition tem- preparations were linear and displayed no perature for the oxidation of both sub- transition breaks (Fig. 3B). Energies of activation for the ‘OH-dependent oxidation strates by either microsomal preparation was the same, about 20°C. Activation ener- of KMB were 8.5 and 10.1 kcal/mol for congies for both substrates, above and below trol microsomes and microsomes from the transition temperature, are shown in pyrazole-treated rats, respectively. Table I. Similar to results found for ethanol oxidation, there were no significant DISCUSSION differences in the activation energies for In view of the higher molar ratio of cytothe oxidation of DMN or benzphetamine by control microsomes as compared to mi- chrome P450 compared to NADPH-cytocrosomes from the pyrazole-treated rats. chrome P450 reductase, there has been TABLE
I
ENERGIES OF ACTIVATION FOR THE OXIDATION OF ETHANOL, DMN, AND BENZPHETAMINE BY MICROSOMES
ENERGY
Co+ TEMPERATURE (Tl
OF ACTIVATION
-‘&,I ’ ’ 1 ’ ’ 4 0330335 340 345 ?JM355 360 I,, I !03CK.‘1
FIG. 3. Effect of temperature on the oxidation of KMB to ethylene by control microsomes (0) and by microsomes from pyrazole-treated rats (0). Reactions were carried out as described under Materials and Methods. B is the Arrhenius plot of the results shown in A.
considerable interest in the lateral diffusion of these two major enzyme components of the hepatic mixed-function oxidase system, and the effect of temperature on mixed-function oxidase reactions (31, 34-37). Schenkman (38) reported that there was no transition temperature for NADPH-cytochrome P450 reductase activity nor for the oxidation of drug substrates such as aminopyrine, ethylmorphine, or aniline, although each substrate possessed a different activation energy (13-21 kcal/mol). Other workers have reported transition breaks (20-25°C) in the Arrhenius plots associated with the demethylation of ethylmorphine, benzphetamine, or aminopyrine (39), or the metabolism of ethoxycoumarin or p-nitroanisole (40). The slow, but not the rapid, phase of reduction of cytochrome P450 by NADPH demonstrated a break in the Arrhenius plot at about 20°C (31). The activation energy for ethanol oxidation by control microsomes and by microsomes from pyrazole-treated rats was found to be identical, although the rate of ethanol oxidation was several-fold higher with the latter. Energies of activation for ethanol oxidation in the absence of added ferric-EDTA were about 15 kcal/mol, which is comparable to values found for the oxidation of DMN and benzphetamine (above the transition temperature) by the same microsomes and for other substrates oxidized by a cytochrome P450-dependent
FOR ETHANOL
OXIDATION
573
process (38-40). The increase in ethanol oxidation after pyrazole treatment reflects the induction of an isozyme of cytochrome P450, P450IIEl. Although ethanol is a preferred substrate for oxidation by cytochrome P450IIE1, it can also be oxidized at lower rates by other isozymes of P450 including the phenobarbital-induced isozyme (8,22,41). The comparable activation energies for ethanol oxidation by control and by induced microsomes suggests that a similar rate-limiting step is common for the overall oxidation of ethanol by the various isozymes present in control microsomal preparations and by the pyrozoleinduced P450. The addition of ferric-EDTA increases the rate of ethanol oxidation by an ‘OH-dependent mechanism (22-24). There was a striking lowering of the activation energy for ethanol oxidation by both microsomal preparations in the presence of ferricEDTA. The activation energy for the ‘OHdependent oxidation of ethanol was similar to the activation energy for the oxidation of KMB, and was about one-half that for the cytochrome P450-dependent oxidation of ethanol. These results suggest different overall mechanisms or rate-limiting steps for the oxidation of ethanol by ‘OH and by cytochrome P450. This would be in agreement with previous results indicating that the reductase and not cytochrome P450 played the major role in the interaction with ferric-EDTA and the subsequent generation of ‘OH (21,22). Linear Arrhenius plots were generated when assaying the activity of the reductase (3%40), and it was suggested that this reflected interaction of the reductase with water-soluble acceptors such as cytochrome c or dichlorophenol indophenol (42). The linear Arrhenius plots generated for the ‘OH-dependent oxidation of ethanol and KMB may, in an analogous manner, reflect the interaction of the reductase with watersoluble ferric-EDTA and the generation of ‘OH in solution. Therefore, changes in the physical state of the membrane as caused by temperature should not alter the reductaseferric-EDTA-catalyzed generation of ‘OH.
574
PUNTARULO
AND
Another possibility for the lowered activation energy for ethanol oxidation by ‘OH compared to cytochrome P450 could reflect the energetics required for the breaking of the carbon-hydrogen bond of carbon one of the ethanol molecule. Oxidation of ethanol by ‘OH proceeds via hydrogen abstraction to yield the hydroxylalkyl radical (CHaCHOH), which upon oxidation or dismutation would give rise to acetaldehyde. Hydroxylation of ethanol by an oxidizing intermediate of P450 presumably also requires breaking a carbon-hydrogen bond, followed by insertion of an oxygen atom to yield hydrated acetaldehyde. If carbon-hydrogen bond breakage is a ratelimiting step, the lower activation energy associated with the oxidation of ethanol by ‘OH compared to cytochrome P450 may reflect the relative ease by which each of the oxidants catalyze cleavage of this bond. In summary, at least in terms of activation energy, the induction of P450IIEl by pyrazole treatment is not associated with any apparent change in the overall mechanism or rate-limiting step for ethanol oxidation. The increase in ethanol oxidation produced by pyrazole treatment appears therefore to be associated with an increased amount of an ethanol-preferring isozyme of P450. The ‘OH-dependent oxidation of ethanol is associated with a lower energy of activation than the cytochrome P450-dependent oxidation, suggesting that the latter pathway proceeds via a more complex mechanism than the ‘OH-dependent pathway, and that different steps are rate limiting for the oxidation of ethanol by either of the two reaction pathways. ACKNOWLEDGMENTS These studies were supported by United States Public Health Service Grants AA-06610 and AA03312 from The National Institute on Alcohol Abuse and Alcoholism. We thank Ms. Roslyn C. King for typing the manuscript.
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