Two-step purification of cytochrome P-450 from adult pig testis by isoelectric focusing.

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing preparative isoelectrofocusing. Purification was achieved 1132 times with a yield of 4.82%. 17alpha-hydroxylase activity was shown to be 14.5 nmol of product/min/nmol of P-450. The cytochrome P-450 was determined to have an isoelectric point of 6.45 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450.     

Purification of cytochrome P-450 from pig testis by aniline-sepharose 4B and isoelectric focusing

Testicular cytochrome P-450 was purified by a procedure including preparative isoelectrofocusing. The cytochrome P-450 was determined to have an isoelectric point of 6.47 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited absorption spectrum of a typical cytochrome P-450. 284-fold purification was achieved with an yield of 10.6%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing Aniline-Sepharose 4B column chromatography and preparative isoelectric focusing.     

Purification of a Single Major Form of Microsomal Cytochrome P-450 from Tulip Bulbs (Tulipa gesneriana L.)

Microsomal cytochrome P-450 from tulip bulbs (Tulipa gesneriana L., Balalaika) was purified to an almost electrophoretically homogeneous preparation. The specific content of cytochrome P-450 in the final preparation was 6.68 nmol/mg protein, which was 30-fold enriched from that of the solubilized fractions of microsomes. The molecular weight of purified cytochrome P-450 by SDS-gel electrophoresis is 52,500. The Oxidized form of the purified cytochrome P-450 had absorption peaks at 392, 552, and 645 nm and the absolute reduced CO spectrum peaked at 448 nm. Judged spectrally, the purified cytochrome P-450 is in high spin in the oxidized state. Antiserum against this cytochrome P-450 previously has shown to be highly specific for its antigen but showed a single precipitin line with solubilized microsomal proteins from tulip bulbs of several other cultivars. The physiological role of this cytochrome P-450, however, is unknown in these dormant tulip bulbs.     

Cytochrome P-450 from lodderomyces elongisporus: Its purification and some properties of the highly purified protein

An effective method, based on the chromatography on ω-aminooctyl Sepharose 4B, for the purification of the alkane-induced cytochrome P-450 is described. The purified cytochrome P-450 was homogeneous in SDS/polyacrylamide gel electrophoresis. In the oxidized state it showed a low spin type absorption spectrum. The reduced CO-complex is characterized by a Soret peak at 447 nm. The alkane hydroxylating enzyme system could be reconstituted combining purified cytochrome P-450 with partially purified NADPH-cytochrome P-450 reductase from the yeast microsomal fraction.     

Purification and properties of cytochrome P-450 (SCC) from pig testis mitochondria

Cytochrome P-450 was purified from pig testis mitochondria to a specific content of 13.1 n mol/mg of protein. The purified preparation was found to contain a single species of P-450, on sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular weight of about 53000 +/- 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of conversion of cholesterol to pregnenolone was 6.2 n mol/min/n mol of P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum at 448 nm. The purified P-450 was subjected to microsequence analysis and its NH2-terminal amino acid sequence was found to show considerable homology with that of bovine adrenal P-450 (SCC).     

Purification of the major cytochrome P-450 of liver microsomes from rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Cytochrome P-450 was isolated in highly purified form from liver microsomes of adult male rabbits treated with 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin (TCDD). Preparations average 17.8 ± 0.8 nmoles cytochrome P-450 per mg protein and have an estimated molecular weight of 54,500. The visible absorption spectrum of the purified cytochrome displays absorption spectral maxima characteristic of high spin forms of cytochrome P-450. When reconstituted with highly purified NADPH-cytochrome P-450 reductase, this cytochrome catalyzes the hydroxylation of acetanilide and the O-deethylation of 7-ethoxyresorufin, two activities induced by TCDD.     

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus.

A soybean flour-induced, soluble cytochrome P-450 (P-450soy) was purified 130-fold to homogeneity from Streptomyces griseus. Native cytochrome P-450soy is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450soy exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450soy had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin:NADP+ oxidoreductase, purified cytochrome P-450soy catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450soy delta.     

Three immunoidentical cytochromes P-450 from liver microsomes of phenobarbital-treated rats.

Three forms of cytochrome P-450 were purified to homogeneity from liver microsomes of Wistar-strain rats treated with phenobarbital. They had minimum mol.wts. of 52 000, 53 000 and 54 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and are designated as P-450(L), P-450(M) and P-450(H) respectively. They were shown to be immunoidentical by Ouchterlony double-diffusion analysis. Several criteria, such as isoelectric points, substrate specificities and sensitivities to tryptic digestion, however, indicated that these cytochromes are distinct isoenzymes of cytochrome P-450. Whereas P-450(L) was highly active on various substrates, P-450(H) had generally low catalytic activities, except on aminopyrine. The cytochromes purified by immunoaffinity chromatography using anti-P-450(L) showed a marked variation in their distribution depending on the strain and colony of rat. Limited tryptic digestion of P-450(H) gave one tryptic peptide showing the same mobility as P-450(L) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their primary structures were very similar. The result suggests a possibility that such limited proteolysis is involved in the post-translational modification of the cytochrome or its destruction.     

Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli

We have expressed in Escherichia coli a cDNA encoding rabbit liver cytochrome P-450IIE1, the ethanol-inducible P-450. The expressed P-450 is located primarily in the bacterial inner cell membrane and comprises 3% of the E. coli total membrane protein. The partially purified cytochrome exhibits a reduced CO difference spectrum with a maximum at 452 nm, characteristic of P-450IIE1, and solubilized membranes or partially purified P-450 preparations reconstituted with NADPH-cytochrome P-450 reductase and phosphatidylcholine catalyze the deethylation of N-nitrosodiethylamine with a turnover number equal to that of purified liver P-450IIE1 (approximately 4.5 nmol/min/nmol of P-450). A modified IIE1 cDNA that encodes a protein lacking amino acids 3-29, a proposed membrane anchor for cytochrome P-450, was also expressed in E. coli and, unexpectedly, the shortened protein was also found to be predominantly located in the bacterial inner membrane rather than the cytosol. Like the full-length protein, this truncated cytochrome has a reduced CO difference spectrum characteristic of P-450IIE1 and is fully active in the deethylation of N-nitrosodiethylamine. These results demonstrate that the NH2-terminal hydrophobic segment is not solely responsible for attachment to the membrane and evidently is not required for proper protein folding or catalytic activity.     

Existence of multiple forms of cytochrome P-450scc purified from bovine adrenocortical mitochondria

Three fractions of cytochrome P-450scc (denoted as fractions a, b, and c) were purified by a new procedure from bovine adrenocortical mitochondria. The amino-acid content analyses of these three fractions showed no difference. NH2-terminal amino-acid sequences of cytochrome P-450scc fractions, a and b agreed completely with the sequence deduced by nucleotide sequence of cDNA of cytochrome P-450scc mRNA (Morohashi, K., Fujii-Kuriyama, Y., Okada, Y., Sogawa, K., Hirose, T., Inayama, S. and Omura, T. (1984) Proc. Natl. Acad. Sci. USA 81, 4647-4651), whereas the sequence of fraction c showed a missing of isoleucine at the NH2-terminal. COOH-terminal ámino-acid sequences of fractions a, b and c were -Gln-Ala-COOH, identical with the deduced sequence from the cDNA. Measurements of the enzymatic activities of cholesterol side-chain cleavage reaction revealed no distinct difference among these three fractions. Although each of these fractions appeared as a single protein staining band upon SDS-polyacrylamide gel electrophoresis, these fractions showed heterogeneities upon two-dimensional electrophoresis and chromatofocusing. Fraction a contained the major form of cytochrome P-450scc, and its isoelectric point was estimated to be pH 7.8 by isoelectric focusing under both native and denatured conditions, and this value was confirmed by chromatofocusing. Neither of the carbohydrate-specific stainings (such as periodic acid-Schiff staining and lectin-peroxidase stainings using concanavalin A, wheat-germ agglutinin, and soybean agglutinin) of purified cytochrome P-450scc fractions after the electrophoretic resolution on SDS-polyacrylamide gel could show cytochrome P-450scc fractions as glycoproteins, suggesting that the heterogeneities were not due to the glycosylation state.     

The role of the hydrophobic fragment of cytochrome b5 in the interaction with cytochrome P-450

The interaction of highly purified liver microsomal cytochrome P-450 from phenobarbital-induced rabbits and cytochrome b5 has been investigated by the difference and second derivative difference spectroscopy. The addition of cytochrome b5 to cytochrome P-450 results in transition of cytochrome P-450 heme iron from low to high spin state. The interaction is accompanied by the changes in the second derivative spectrum of cytochrome P-450, which point to the participation of tryptophanyl residues in this process. The hydrophilic fragment of cytochrome b5 is unable to form a complex with cytochrome P-450 as judged by the absence of the difference spectrum and any changes in the second derivative UV-spectrum of cytochrome P-450. The evidence obtained indicates that the hydrophobic tail of the cytochrome b5 molecule responsible for its binding to membrane is also indispensable for forming a functional cytochrome P-450-cytochrome b5 complex.     

Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells.

The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-450(25] from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253, 549-552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol.mg of protein-1, and the protein showed a single spot with an apparent isoelectric point of 7.4 and an Mr of 50,500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124 pmol.min-1.nmol of cytochrome P-450-1 and towards 1 alpha-hydroxyvitamin D3 it was 1375 pmol.min-1.nmol-1. The preparation also catalysed the 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol at a rate of 1000 pmol.min-1.nmol of cytochrome P-450-1 and omega-1 hydroxylation of lauric acid at a rate of 200 pmol.min-1.nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(25) from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5 beta-cholestane-3 alpha,7 alpha-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-450(25) was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-450(25) from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.     

Electrophoretic, spectral, catalytic and immunochemical properties of highly purified cytochrome P-450 from sheep lung

1. Cytochrome P-450LgM2 was purified from sheep lung microsomes in the presence of detergents, Emulgen 913 and cholate. 2. The purification procedure involved the chromatography of the detergent solubilized microsomes on DEAE-cellulose and hydroxylapatite. 3. Cytochrome P-450LgM2 was further purified on second DEAE-cellulose and hydroxylapatite columns. 4. The specific content of the highly purified P-450LgM2 was 16-18 nmol P-450/mg protein and purified 164-fold. 5. The yield was 16% of the initial content in microsomes. 6. The SDS-polyacrylamide slab gel electrophoresis (PAGE) of the purified lung cytochrome P-450LgM2 showed one protein band having the monomer molecular weight of 49,500. 7. The absolute CO-difference spectrum of dithionate-reduced P-450LgM2 gave a peak at 451 nm. 8. When sheep lung cytochrome P-450LgM2 and P-450LM2 purified from liver of phenobarbital (PB)-induced rabbit were subjected to Western Blotting and visualized immunochemically with anti-P-450LM2, they showed identical mobilities. 9. P-450LgM2 was found to be very active in N-demethylation of benzphetamine in a reconstituted system containing purified sheep lung reductase and synthetic lipid. 10. Turnover numbers (min-1) for benzphetamine, aniline, ethylmorphine and p-nitrophenol were determined to be 273, 1.2, 15.5 and 1.05, respectively, in a reconstituted microsomal lung monooxygenase system. 11. Spectral, electrophoretic, biocatalytic and immunochemical properties of sheep lung P-450LgM2 were found to be similar to those of P-450 isozyme 2, purified from PB-treated rabbit liver and of rabbit lung microsomes.     

Purification and characterization of two constitutive cytochromes P-450 (F-1 and F-2) from adult female rats: identification of P-450F-1 as the phenobarbital-inducible cytochrome P-450 in male rat liver

Two hepatic microsomal cytochromes P-450, P-450F-1 and P-450F-2 were purified to electrophoretic homogeneity from untreated adult female rats by high-performance liquid chromatography (HPLC) with anion-exchange, cation-exchange, and hydroxyapatite columns. Cytochromes P-450F-1 and P-450F-2 were not adsorbed with the anion-exchange column, but were retained on a cation-exchange column and were separated poorly. These forms separated on hydroxyapatite HPLC. The molecular weights of cytochromes P-450F-1 and P-450F-2 were 50,000 and 49,000, respectively. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of cytochromes P-450F-1 and P-450F-2 were at 450 and 448 nm, respectively. Both forms catalyzed the N-demethylation of benzphetamine and had low catalytic activity for 7-ethoxycoumarin. Cytochrome P-450F-1 had low 2 alpha-hydroxylation activity toward testosterone. Cytochrome P-450F-2 had low 15 alpha-hydroxylation activity. On the basis of these results and those of NH2-terminal sequence analysis, cytochrome P-450F-2 seemed to be the typical female-specific cytochrome P-450. The NH2-terminal sequence of cytochrome P-450F-1 was identical to that of cytochrome P-450PB-2 purified from hepatic microsomes of male rats treated with phenobarbital. Cytochromes P-450F-1 and P-450PB-2 had identical chromatographic properties, minimum molecular weight, spectral properties, and peptide maps. Furthermore, the antibody to phenobarbital-inducible cytochrome P-450PB-2 gave a single immunoprecipitin band with cytochrome P-450F-1 by Ouchterlony double-diffusion analysis.     

Soluble cytochrome P-450 from housefly microsomes. Partial purification and characterization of two hemoprotein forms.

Housefly microsomes contain two spectrally different forms of cytochrome P-450 which we have termed P-450 and P-450I. Methods have been developed for the fractionation and chromatographic purification of these two hemoprotein forms. Microsomes are solubilized first with Triton X-100 in the presence of glycerol, dithiothreitol, ethylenediaminetetra-acetic acid, and phenobarbital. Cytochrome P-450 is recovered in a floating pellet after the addition of 25% ammonium sulfate followed by centrifugation, whereas cytochrome P-450I remains in the 25% ammonium sulfate supernatant fluid. Cytochrome P-450 is purified further by Sephadez G-200 and DEAE-Sephadex A-50 column chromatography, which also allows the isolation of cytochrome b5 and NADPH-dependent cytochrome P-450 reductase in good yields and with little cross-contamination. Cytochrome P-450 apparently is free of cytochromes b5 and P-420 as well as of reductase and is obtained in a final yield of approximately 16% with a 6.9-fold purification. Its maximum absorbance is at 45 mn in the CO-difference spectrum and its average extinction coefficient is 103 cm-1 nm-1. Cytochrome P-450I is purified by Sephadex G-25 column chromatography but still contains some cytochromes b5 and P-420 as well as reductase. Its maximum absorbance is at 448.5 nm in the CO-difference spectrum and its extinction coefficient is 83 to 86 cm-1 mM-1. Both cytochromes hydroxylate type I substrates such as aminopyrine. Sufficient amounts of reductase are present in the cytochrome P-450I preparation to sustain activity, but the reductase has to be added to cytochrome P-450 in a reconstituted system for activity. Cytochrome P-450 is fairly stable, whereas cytochrome P-450I can be isolated only when protected by a substrate (phenobarbital). Detergent-solubilized housefly cytochromes P-450 and P-450I seem to correspond to either aggregates or oligomeric proteins. Cytochrome P-450 appears to correspond to a tetramer, each subunit having a molecular weight of 45,000, whereas cytochrome P-450I may correspond to an aggregate of at least 10 subunits. The cytochrome P-450 aggregate is dissociated by 6 M urea, but cytochrome P-450I remains as such.     

Association of cytochrome b5 and cytochrome P-450 reductase with cytochrome P-450 in the membrane of reconstituted vesicles

A protein-protein association of cytochrome P-450 LM2 with NADPH-cytochrome P-450 reductase, with cytochrome b5, and with both proteins was demonstrated in reconstituted phospholipid vesicles by magnetic circular dichroism difference spectra. A 23% decrease in the absolute intensity of the Soret band of the magnetic CD spectrum of cytochrome P-450 was observed when it was reconstituted with reductase. A difference spectrum corresponding to a 7% decrease in absolute intensity was obtained when cytochrome b5 was incorporated into vesicles that already contained cytochrome P-450 and cytochrome P-450 reductase compared to a decrease of 13% in absolute intensity when cytochrome b5 was incorporated into vesicles that contained only cytochrome P-450. The use of the magnetic circular dichroism confirmed that protein-protein associations that have been detected by absorption spectroscopy between purified and detergent-solubilized proteins also exist in membranes. High ionic strength was shown to interrupt direct electron flow from cytochrome P-450 reductase to cytochrome P-450 but not the electron flow from reductase through cytochrome b5 to cytochrome P-450. Upon incorporation of cytochrome b5 into cytochrome P-450- and cytochrome P-450 reductase-containing vesicles, an increase of benzphetamine N-demethylation activity was observed. The magnitude of this increase was numerically identical to the residual activity of the reconstituted vesicles measured in the presence of 0.3 M KCl. It is concluded that there is a requirement for at least one charge pairing for electron transfer from reductase to cytochrome P-450. These observations are combined in a proposed mechanism of coupled reversible association reactions in the membrane.     

Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1. A cytochrome P-450 species having a nitrogenous ligand trans to thiolate

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.     

25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

A cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 9 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 52,000 upon SDS/polyacrylamide-gel electrophoresis. The preparation showed a single protein spot with an apparent isoelectric point of 7.8 and an Mr of approx. 52,000 upon two-dimensional isoelectric-focusing-polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 5000 times more efficiently than did the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 catalysed, in addition to 25-hydroxylation of vitamin D3, the 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The enzyme did not catalyse side-chain cleavage of cholesterol, 11 beta-hydroxylation of deoxycorticosterone, 1 alpha-hydroxylation of 25-hydroxyvitamin D3, hydroxylations of lauric acid and testosterone or demethylation of benzphetamine. The results raise the possibility that the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of C27 steroids are catalysed by the same species of cytochrome P-450 in liver mitochondria. The possible role of the liver mitochondrial cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

NADPH-cytochrome P-450 reductase from rat liver: purification by affinity chromatography and characterization.

(NADPH)-cytochrome P-450 reductase was purified to apparent homogeneity by a procedure utilizing nicotinamide adenine dinucleotide phosphate (NADP)-Sepharose affinity column chromatography. The purified flavoprotein has a molecular weight of 79 700 and catalyzes cytochrome P-450 dependent drug metabolism, as well as reduction of exogenous electron acceptors. Aerobic titration of cytochrome P-450 reductase with NADPH indicates that an air-stable reduced form of the enzyme is generated by the addition of 0.5 mol of NADPH per mole of flavin, as judged by spectral characteristics. Further addition of NADPH causes no other changes in the absorbance spectrum. A Km value for NADPH of 5 micron was observed when either cytochrome P-450 or cytochrome c was employed as electron acceptor. A Km value of 8 +/- 2 micron was determined for cytochrome c and a Km of 0.09 +/- 0.01 micron was estimated for cytochrome P-450.     

Studies on cytochrome P-450 (P-45017α,lyase)from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome b5

We have reported (Kominami S., Shinzawa K. and Takemori S. (1982) Biochem. Biophys. Res. Commun. 109, 916–921) that a cytochrome P-450 purified from guinea pig adrenal microsomes shows 17α-hydroxylase and C-17,20-lyase activities in a reconstituted system with NADPH-cytochrome P-450 reductase. The homogeneity of the purified cytochrome P-450 was examined with the following methods: isoelectric focusing, immunoelectrophoresis and affinity chromatography on cytochrome b₅-immobilized Sepharose. It was found that progesterone competitively inhibited C-17,20-lyase reaction and that progesterone was converted into androstenedione by 17α-hydroxylation followed by the lyase reaction. These results indicate that the dual activities are carried out by a single enzyme (P-450_17α,lyase). P-450_17α,lyase had the maximum activity at pH 6.1 both for 17α-hydroxylation (6.0 nmol/min per nmol of P-450) and the lyase reaction (11.0 nmol/min per nmol of P-450). Upon addition of cytochrome b₅ to the reconstituted system, the optimal pH for 17α-hydroxylation was shifted to 7.0 and that of the lyase reaction to 6.6. The maximum activities at these optimal pH values were almost the same in the presence or absence of cytochrome b₅. With the addition of cytochrome b₅, both the activities were stimulated above pH 6.3–6.5 and were suppressed below pH 6.3–6.5. These results indicate that cytochrome b₅ plays some important role in controlling the dual activities of P-450_17α,lyase.