ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 198, No. 1, November, pp. 110-116, 1979

Studies

on the Biosynthesis of Cytochrome P-450 in Rat LiverA Probe with Phenobarbital’ KOLARI S. BHAT2 AND GOVINDARAJAN

PADMANABAN

Department of Biochemistry, Indian Institute of Science, Bangalore-

012, India

Received April 9, 1979; revised June 25, 1979 Cytochrome P-450 has been purified from phenobarbital-treated rat livers to a specific content of 17 nmol/mg protein. The major species purified has a molecular weight of 48,000. Using the purified antibody for the cytochrome P-450 preparation it has been shown that the major product synthesized in wivo and in the homologous cell-free system in vitro is the 48,000 molecular weight species. Poly(A)-containing RNA isolated from phenobarbitaltreated animals codes for the synthesis of the 48,000 molecular weight species in the wheat germ and reticulocyte lysate cell-free systems. It is concluded that cytochrome P-450 synthesis does not involve processing of a polyprotein precursor, although certain minor modifications including glycosylation of the primary translation product are not ruled out. Phenobarbital treatment of the animal results in a significant increase in the cytochrome P-450 messenger activity as measured in the wheat germ cell-free system.

Cytochrome P-450 of the liver endoplasmic reticulum plays a central role in drug hydroxylation and is induced by a wide variety of chemicals (1, 2). It is now recognized that cytochrome P-450 encompasses a group of proteins. Phenobarbital and 3-methylcholanthrene constitute two prototype inducers, the former inducing the cytochrome P-450 species and the latter cytochrome P-448 species (3, 4). More species of this cytochrome have also been reported (5-7). Although the possibility that the different species of cytochrome P-450 are but interconvertible forms has been considered (8, 91, it has been suggested that their formation involves de novo synthesis of the concerned species (10). Using SDS3-gel electrophoresis LThis work formed a part of the Ph.D. thesis of K.S.B. 2 Present address: Department of Microbiology, College of Medicine, Pennsylvania State University, The Milton S. Hershey Medical Center, Hershey, Pa. 17033. 3 Abbreviations used: SDS, sodium dodecyl sulfate; IgG, immunoglobulin; DTT, dithiothreitol; TCA, trichloroacetic acid; Hepes, 4-(2hydroxyethyl)-lpiperazineethanesulfonic acid; EGTA, ethylene glycol bis(p-aminoethyl ether)N,N’-tetraacetic acid; PPO, polyphenylene oxide; cDNA, complementary DNA. 0003-9861/79/130110-07$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

to separate the microsomal proteins, it has been shown that the drugs such as phenobarbital, 3-methylcholanthrene, and p-naphthaflavone enhance labeled amino acid incorporation into the cytochrome P-450 region (10-12). With the use of cytochrome P-450-specific antibody, it has recently been shown that phenobarbital enhances the rate of synthesis of this protein (13). De novo synthesis of the different species of cytochrome P-450 can still be accounted for on the basis of a drug-specified differential processing of a common precursor protein. If such a common precursor giving rise to the different species of cytochrome P-450 were to exist, it should be possible to detect such a precursor when liver messenger RNA is translated in heterologous cell-free systems. This is on the basis that the heterologous cell-free system would lack the machinery to process the precursor protein and that the cytochrome P-450-specific antibody would recognize the precursor formed. In the present study, the possible existence of a precursor protein giving rise to the different cytochrome P-450 species has been examined by translating liver poly(A)containing RNA, isolated from phenobarbital-

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14C-labeled chlorella protein hydrolysate to male rats 1 h before sacrifice. The animals received phenobarbitone (80 mgikg) 6 hours before the label. The livers were homogenized in 5 vol of 1.15% (w/v) KC1 and postmitochondrial supernatant was spun at 100,OOOgfor 90 min to isolate the microsomal fraction. Microsomes were labeled in vitro by the procedure of Weinstein (16). For this purpose livers were homogenized in 3 vol of 0.25 M sucrose containing 0.01 M Tris-HCl buffer, pH 7.6,0.06 M KCl, 5 mM MgQ, and 6 mM P-mercaptoethanol (TKM buffer). The homogeMATERIALS AND METHODS nate was spun at 12,OOOgfor 15 min and the superfor 2 h to collect the microMaterials. c[G3H]Leucine (7 Ci/mol) and 14C-labeled natant was spun at 105,OOOg somal fraction. A portion of the postmicrosomal fraction chlorella protein hydrolysate (27 mCi/matom C) were was passed through a Sephadex G-25 column to get purchased from BARC, Bombay. L-[4,5-3H]Leucine the S,,, fraction. (60 Ciimmol) and L-[U-‘4C]leucine (358 mCi/mmol) were from Amersham, Bucks, U. K. Emulgen was The reaction mixture in a total volume of 0.25 ml a gift from KAO Soap Company, Ltd., Japan. All other contained: 0.5 mM ATP; 0.25 mM GTP; 2.5 mM phosbiochemicals were purchased from Sigma Chemical phoenol pyruvate; amino acid mixture minus leucine, Company, St. Louis, Missouri. 40 Fgiml; 10 mM Tris-HCl, pH 7.6 buffer; 5 mM MgCl,; Putijcation of cytochrome P-450 and preparation 60 mM KCl; 6 mM P-mercaptoethanol; 1.2 mg of So0 of antibody. Cytochrome P-450 from phenobarbitalprotein/ml; 13H]leucine (7 Ciimmol); 100 &i/ml, and injected rat liver was purified by the method of Ryan the microsomal suspension. The mixture was incubated et al. (14). The animals were given four injections of at 37°C for 30 min. phenobarbital (80 mgikg), one injection per day. The Isolation of polysomes and poly(A)-containing RNA. protein was purified to a specific content of 17 nmolimg Rat liver polysomes were isolated by the magnesium precipitation method as described by Palmiter (17). protein. Antibody was raised in rabbits by giving two priPoly(A)-containing RNA was directly isolated from the mary injections of 250 pg of the protein in complete polysomes using oligo-dT cellulose chromatography as Freund’s adjuvant intradermally at multiple sites at described by Krystosek et al. (18). In some experiments &day intervals. Again two booster injections at poly(A)-containing RNA was isolated from total RNA X-day intervals were given. The rabbits were bled isolated from the polysomes. Total RNA was isolated 1 week after the third injection. Subsequently the am- from polysomes using phenol-chloroform as described mals were given 100 rg of the protein every 4 weeks by Penman (19). Translation of poly(A)-containing RNA in the wheat and bled 1 week after every injection. The antisera were pooled and the IgG fraction was prepared using germ system. Wheat germ SsOextract was prepared ammonium sulfate fractionation and DEAE-cellulose according to Roberts and Paterson (20) except that chromatography. Further purification of the cyto- the preincubation step was omitted. The incubation chrome P-450 antibody was carried out by the general mixture in 0.2 ml contained; 0.06 ml (1 A,,,) of the S,, procedure described by McCans et al. (15). To 10 ml fraction; 0.02 M Hepes, pH 7.6; 2 mM DTT; 1 mM ATP; of the cytochrome P-450 antiserum containing 3 mg of 20 pM GTP; 8 mM creatine phosphate; 8 pg creatine phenylmethylsulfonylfluoride, a protease inhibitor, phosphokinase; 40 &M each of 19 unlabeled amino acids 4.4 mg of cytochrome P-450 (in 2 ml of 0.05 M potassium except leucine; 5 FCi of [SH]leucine (60 Ci/mmol); 80 mM phosphate buffer, pH 7.5, containing 20% glycerol, KCl; 2.25 mM magnesium acetate; 300 pM phenyl10m3M EDTA, and 10d4M DTT) was added. After methylsulfonylfluoride, and rat liver poly(A)-containing incubation at 27°C for 1 h and then at 4°C overnight, RNA. The mixture was incubated at 30°C for 60 min. the immunoprecipitate was collected by centrifugation. At the end of the incubation 5~1 aliquots were plancheted The immunoprecipitate was washed thrice with 10 ml on to filter paper discs to measure incorporation into of 0.02 M Tris-HCl buffer, pH 7.5, containing 0.15 M TCA-precipitable proteins and the rest used for immunoprecipitation. NaCi. The antigen-antibody complex was dissociated with 5 ml of 10 mM glycine buffer, pH 2.8, containing Translation of poly(A)-containing RNA in the 0.14 M NaCI, and incubated at 4°C for 1 h. It was then reticulocyte lysate system. Reticulocyte lysate was centrifuged at 10,OOOg for 15 min to pellet the antigen. prepared according to the method of Hunt et al. (21). The supernatant containing the anti-cytochrome P-450 The lysate incubation conditions including micrococcal antibody was dialyzed extensively against 0.02 M n&ease treatment were as described by Stewart et al. Tris-HCI buffer, pH 7.4, containing 0.15 M NaCl and (22). Nuclease treatment of lysate (40 pgiml) was 0.02% sodium azide. carried out in presence of 1 mM CaCI, and inactivated Labeling of microsomes in vivo and in vitro. Microwith 2 mM EGTA. The incubation mixture in 0.2 ml somes were labeled in viva by injecting 50 WCi of contained: 0.02 ml of poly(A)-containing RNA and

treated animals, in reticulocyte and wheat germ cell-free systems and immunoprecipitating the product with the antibody specific for cytochrome P-450, purified from phenobarbital-treated livers. The wheat germ system has also been used to quantitate the effect of the drug on the cytochrome P-450 functional messenger RNA content.

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0.18 ml of nuclease-treated lysate providing 25 mM Tris-HCl, pH 7.6; 100mM KCI; 2 mM MgCl,; 1 mM ATP; 0.25 mM GTP; 8 mM creatine phosphate; 20 pg creatine phosphokinase; 1 mM DTT; 30 pM each of 19 unlabeled amino acids except leucine; 1 j&i of [‘YJleucine; 30 pM hemin. The mixture was incubated at 30°C for 60 min. At the end of the incubation, aliquots were used for measurement of total protein and immunoprecipitable protein synthesis. Immunoprecipitation procedures. Microsomal samples were solubilized in 0.1 M sodium phosphate buffer, pH 7.4, containing 20% glycerol, 1 mM EDTA, and 1% sodium cholate. Samples from in vitro protein synthesis were made 1% with respect to cholate and nonradioactive leucine was also added to 10 mM concentration. The samples were clarified by centrifugation at 2500 rpm for 20 min. Immunoprecipitation was carried out in a final volume of 0.5 ml containing the clarified preparation, a twofold excess of anti-cytochrome P-450 y-globulin, 0.5% cholate, and 1 mM phenylmethylsulfonylfluoride. To samples from in vitro protein synthesis, 20 pg of carrier cytochrome P-450 was added before the addition of antibody. The mixtures were incubated at 27°C for 1 hand at 4°C overnight. The immunoprecipitates were collected by centrifugation, washed twice with 0.5 ml of 0.02 M Tris-HCl buffer, pH 7.4 containing 0.15 M NaCl. The precipitate was then suspended in 0.4 ml of the same buffer and layered over 1 ml of Tris-HCl buffer, pH 7.4 containing 0.5 M sucrose and 1% deoxycholate. The precipitate was again collected by centrifugation and finally washed with Tris-saline buffer. The immunoprecipitate was solubilized using 0.01 M Tris-HCl buffer, pH 7.4, containing2% SDS (w/v) and 5% P-mercaptoethanol (w/v). The solution was kept in a boiling water bath for 2 min and then analyzed using SDS-gel electrophoresis. SDS-gel electrophoresis. SDS-gel electrophoresis was carried out according to the procedure of Laemmli (23). The protein bands were stained with Coomassie blue. In the case of the labeled immunoprecipitates, after the run the gels were sliced (l-mm slices) and the slices were digested with 0.5 ml of 30% (v/v) Hz02 and the radioactivity measured using 0.5% PPO in Triton X-IOO-toluene (1:2, v/v) cocktail. Filter disks were counted using 0.5% (w/v) PPO in toluene. The radioactivity measurements were made in a Beckman LS-100 liquid scintillation counter and under the experimental conditions 14Cand 3H were counted with efficiencies of 35 and 5%, respectively. RESULTS

Phenobarbital-induced cytochrome P-450 has been purified by the method of Ryan et aZ. (14) to a specific content of 17 nmol/mg protein. The final preparation on SDS-gel electrophoresis shows a major band cor-

FIG. 1. SDS-gel electrophoresis pattern of the purified cytochrome P-450 preparation. Fifteen micrograms of the protein was loaded on the gel.

responding to a molecular weight of 48,000 (Fig. 1). In some preparations two minor bands corresponding to 53,000 and 47,000 molecular weights are also seen. All the bands stain for heme with benzidine. It is now recognized that phenobarbital induces multiple cytochrome P-450 species. Recently, three species of cytochrome P-450 have been detected in the purified preparation obtained from phenobarbital-treated rats (24). The antibody prepared for the purified cytochrome P-450 preparation has been characterized by Ouchterlony immunodiffusion technique (Fig. 2). The IgG fraction gives a single precipitin line with the purified cytochrome P-450 preparation as well as with solubilized microsomes. It also shows a weaker cross-reactivity with a partially purified preparation obtained from 3-methylcholanthrene-treated rat livers. The purified antibody also shows a similar pattern. Immunotitration data revealed that at equivalence point 1 mg of the IgG fraction

BIOSYNTHESIS

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FIG. 2. Ouchterlony double diffusion analysis of anticytochromeP-450 antibody. The central wall contained 75 ~1 of anti-P-450 antiserum. The outer walls contained the different antigen preparations: (1) and (9, purified phenobarbitone-induced cytochrome P-450, 10 and 30 Kg proteins, respectively. (3) Partially purified uninduced cytochrome P-450 (140 pg of protein). (4) Partially purified phenobarbitone-induced cytochrome P-450 (140 Fg of protein). (5) Partially purified 3-methylcholanthrene-induced cytochrome P-450 (140 kg/ of protein). (6) Solubilized normal microsome (350 fig of protein).

and the purified antibody can precipitate 45 and 180 eg of cytochrome P-450, respectively.

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The specificity of the cytochrome P-450 antibody has been further examined by subjecting the labeled immunoprecipitate obtained from the microsomes labeled in viva as well asin vitro, to SDS-gel electrophoresis. It is clear from Fig. 3a that the immunoprecipitable products obtained under in vivo and in vitro conditions are quite similar. There is a major radioactivity peak corresponding to the molecular weight species of 48,000 and a minor peak corresponding to the 53,000 molecular weight species. Perhaps, antibodies to more than one species are present in the IgG preparations. The primary translation product has been characterized in the reticulocyte lysate cellfree system. The micrococcal nucleasetreated reticulocyte lysate shows a low endogenous rate of protein synthesis and shows about a 50-fold stimulation on addition of poly(A)-containing RNA. Figure 3b reveals the radioactivity profile in SDS-gels of the labeled immunoprecipitate obtained from the reticulocyte lysate translation system. It can be seen that the profile shows a peak closely corresponding to the cytochrome P-450 species with the molecular weight of 48,000.

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No

FIG. 3. Radioactivity profile after SDS-gel electrophoresis of the labeled immunoprecipitates obtained from cytochrome P-450 synthesized in vim and in vitro. (a) Cytochrome P-450 labeled in vivo with ‘%-labeled chlorella protein hydrolysate (---); cytochromeP-450 labeled in vitro with 13H]leucine in isolated microsome (- - -); (b) cytochrome P-450 synthesized in the reticulocyte lysate system labeled with [L4C]leucine.

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Next, poly(A)-containing RNA from normal and phenobarbital-treated rat livers has been translated in the wheat germ cellfree system. The Mg2+ and K+ requirements for the synthesis of the total trichloroacetic acid-precipitable protein as well as for the synthesis of cytochrome P-450 as measured by immunoprecipitation, in the wheat germ system have been found to be the same. The optimum Mg2+ and K+ concentrations are 2.25 and 80 mM, respectively. The poly(A)containing RNA preparations from normal and phenobarbital-treated animals stimulate total protein synthesis to a similar extent at different RNA concentrations tested (Fig. 4). Figure 5a reveals the radioactivity profile after SDS-gel electrophoresis of the labeled immunoprecipitate obtained after translation of poly(A)-containing RNA in the wheat germ system. The labeling has been carried out with [3H]leucine and the immunoprecipitate has been coelectrophoresed with the 14C-chlorella protein hydrolysate-labeled immunoprecipitate obtained from rat liver microsomes labeled in viva. It is clear that this product obtained in the wheat germ system shows a radioactivity peak closely corresponding to the major cytochrome P-450 species synthesized in rat liver in response to phenobarbital under in vivo conditions. Finally, the poly(A)-containing RNA preparations isolated from normal and phenobarbital-treated rats have been compared for the messenger activity to code for cytochrome P-450 in the wheat germ system. The results presented in Fig. 5b indicate that at a nonsaturating RNA concentration of 20 pg/ml, the RNA preparation from drug-treated livers permits threefold more synthesis of cytochrome P-450 than that of the preparation from normal animals. Similar results have been obtained at an RNA concentration of 10 pg/ml.

0

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FIG. 4. Effect of poly(A)-containing RNA concentration on protein synthesis in the wheat germ. RNA from phenobarbital-treated animals (-). RNA from control animals (- - -). [3H]Leucine (60 Ci/mmol) was used for labeling. At the end of the incubation, 5 Fg of the incubation mixture was plancheted on the filter paper disks and processed for measurement of total protein radioactivity. The volume of the reaction mixture was 0.1 ml.

Although interconversion of some of the species of cytochrome P-450 is not ruled out, the origin of the basic prototypes involves de novo synthesis of the concerned species (10-12). Peptide mapping of three rat liver cytochrome P-450 species reveals striking differences and it has been suggested that the various cytochrome P-450 for the most part, are different proteins and that differences in physical properties and immunological and substrate specificity are not due to minor alterations of a basic polypeptide structure (25). However, de novo synthesis of the different cytochrome P-450 species can still be explained on the basis of drugspecific processing of a common precursor protein. For example, if one were to assume a common precursor protein of molecular DISCUSSION weight 75,000 (say 750 amino acid residues), The results of the present investigation it can be argued that processing may lead establish two important points. The first one to a fragment consisting of amino acid resiis regarding the origin of the different species dues 1 to 480 giving rise to the phenobarbitalof cytochrome P-450. The second one is on inducible cytochrome P-450 species with the effect of the drug on the cytochrome a molecular weight of 48,000. In the case P-450-specific messenger RNA content. of 3-methylcholanthrene administration,

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FIG. 5. Cytochrome P-450 messenger RNA translation and quantitation in the wheat germ cell-free system. (a) Cytochrome P-450 labeled in vivo with %-labeled chlorella protein hydrolysate (-); cytochrome P-450 synthesized in the wheat germ system labeled in vitro with [“HJleucine (- - -). (b) Cytochrome P-450 synthesized in the wheat germ system labeled with [3H]leucine in response to poly(A)-containing RNA from phenobarbital-treated animals (---) and in response to poly(A)containing RNA from control animals (- - -). The RNA concentration used was 20 @g/ml. The immunoprecipitation procedure is described in the text.

processing may lead to a fragment consisting of amino acid residues 220-750 giving rise to the cytochrome P-448 species with a molecular weight of 53,000. Various other alternative modes of processing are also possible. In such a processing mechanism, it is clear that the bigger the size of the precursor protein, the smaller can be the region of commonality between the different species. The present studies reveal that the product detected in the heterologous cellfree systems is at least very close in size to the in vivo product, if not identical. The present results do not rule out the existence of separate precursors for each cytochrome P-450 species, the precursor differing from the product in the status of glycosylation and the presence of a short peptide fragment, although recent studies reveal that the amino terminal sequences of cytochrome P-450s are similar to that of the “signal peptide” and perhaps cytochrome P-450 synthesis does not involve processing in that sense, the “signal peptide” being retained in the final product (26, 27). The present results, however, rule out the existence of

a big-enough common precursor protein that can give rise to the different cytochrome P-450 species by differential processing. The present results indicate that a drug such as phenobarbital enhances the functional messenger RNA content for cytochrome P-450, which however is a composite effect of the drug on transcription, processing, and stability of messenger RNA. Studies are in progress to purify the cytochrome P-450-messenger RNA with the aim of making the cDNA to answer the question of the origin of the messenger RNA species for the cytochrome P-450 group of proteins. ACKNOWLEDGMENT The financial assistance of the University Grants Commission, New Delhi is gratefully acknowledged. REFERENCES 1. CONNEY, A. H., (1967)Phamzacol.

Rev. 19, 317366. 2. ALVARES, A., PARLI, C. J., AND MANNERING, G. J. (1973) Bioch.em. Pharmacol. 22, 1037-1045. 3. SLADEK, N. E., AND MANNERING, G. J. (1966) Biochem. Biophys. Res. Commun. 24,668-674.

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4. MANNERING, G. J., SLADEK, N. E., PARLI, C. J., AND SHOEMAN, D. W. (1969) in Microsomes and Drug Oxidations (Gillette, J. R., Conney, A. H., Cosmides, G. J., E&brook, R. W., Fouts, J. R., and Mannering, G. J., eds.), pp. 303-330, Academic Press, New York. 5. WELTON, A. F., AND AUST, S. D. (1974) Biochem. Biophys. Res. Common. 56, 898-906. 6. WELTON, A. F., ONEAL, F. D., CHANEY, L. C., AND AUST, S. D. (1975) J. Biol. Chem. 250, 5631-5639. 7. THOMAS, P. E., Lu, A. Y. H., WEST, S. B., RYAN, D., MIWA, G. T., AND LEWIN, W. (1977) Mol. Pharrnacol. 13, 819-831. 8. HILDEBRANDT, A. G., AND ESTABROOK, R. W. (1969) in Microsomes and Drug Oxidations (Gillette, J. R., Conney, A. H., Cosmides, G. J., Estabrook, R. W., Fouts, J. R., and Mannering, G. J., eds.), pp. 331-347, Academic Press, New York. 9. IMAI, Y., AND SIEKEVITZ, P. (1971) Arch. Biothem. Biophys. 144, 143-159. 10. HAUGEN, D. A., COON, M. J., AND NEBERT, D. W. (1976) J. Biol. Chem. 251, 1817-1827. 11. DEHLINGER, P. J., AND SCHIMKE, R. T. (1972) J. Biol. Chem. 247, 1257-1264. 12. RAJAMANICKAM, C. R., RAO, M. R. S., AND PADMANABAN, G. (1975) J. Biol. Chem. 250, 2306-2310. 13. BHAT, K. S., AND PADMANABAN, G. (1978) FEBS Lett. 89, 337-340. 14. RYAN, D., Lu, A. Y. H., KAWALEK, J., WEST,

15.

16.

1’7. 18. 19. 20. 21. 22. 23. 24. 25. 26.

27.

S. B., AND LEVIN, W. (1975) Biochem. Biophys. Res. Common. 64, 1134-1141. MCCANS, J. L., LANE, L. K., LINDENMEYER, G. E., BUTLER, V. P. JR., AND SCHWARTZ, A. (1974) Proc. Nat. Acad. Sci. USA 71, 24492452. WEINSTEIN, I. B. (1968) in Methods in Enzymology (Grossmann, L., and Modave, K., eds.), Vol. 12B, pp. 782-787, Academic Press, New York. PALMITER, R. D. (1974) BiochemisWy 13, 36063615. KRYSTOSEK, A., CAWTHON, M. L., AND KABAT, D. (1975) J. Biol. Chem. 250, 6077-6084. PENMAN, S. (1966) J. Mol. Biol. 17, 117-130. ROBERTS, B. E., AND PATERSON, B. M. (1973) Proc. Nat. Acad. Sci. USA 70, 2330-2334. HUNT, T., VANDERHOFF, G. A., AND LONDON, I. M. (1972) J. Mol. Biol. 66, 471-481. STEWART, A. G., LLOYD, M., AND ARNSTEIN, H. R. V. (1977) Eur. J. Biochem. 80,453-459. LAEMMLI, U. K. (1970) Nature (London) 227, 680-685. CRAFT, J. A., COOPER, M. B., AND RABIN, B. R. (1978) FEBS Lett. 88, 62-66. GUENGERICH, F. P. (1978) Biochem. Biophys. Res. Commun. 82, 820-827. HAUGEN, D. A., ARMES, L. G., YASUNOBU, K. T., AND COON, M. J. (1977) Biochem. Biophys. Res. Commun. 77, 967-973. OZOLS, J., GERARD, C., IMAI, Y., AND SATO, R. (1978) Fed. Proc. 37, 1759.