Structure/function of the inhibition of human 3beta-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane.

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3beta-HSD1 is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a major target enzyme for the treatment of breast cancer. 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to evaluate the role of the 2alpha-cyano group on trilostane (2alpha-cyano-4alpha,5alpha-epoxy-17beta-ol-androstane-3-one) and determine which amino acids may be critical for 3beta-HSD1 specificity. Trilostane without the 2alpha-cyano group, 4alpha,5alpha-epoxy-testosterone, was synthesized. Using our structural model of 3beta-HSD1, trilostane or 4alpha,5alpha-epoxy-testosterone was docked in the active site using Autodock 3.0, and the potentially critical residues (Met187 and Ser124) were identified. The M187T and S124T mutants of 3beta-HSD1 were created, expressed and purified. Dixon analyses of the inhibition of wild-type 3beta-HSD1, 3beta-HSD2, M187T and S124T by trilostane and 4alpha,5alpha-epoxy-testosterone suggest that the 2alpha-cyano group of trilostane is anchored by Ser124 in both isoenzymes. Kinetic analyses of cofactor and substrate utilization as well as the inhibition kinetics of M187T and the wild-type enzymes suggest that the 16-fold higher-affinity inhibition of 3beta-HSD1 by trilostane may be related to the presence of Met187 in 3beta-HSD1 and Thr187 in 3beta-HSD2. This structure/function information may lead to the production of more highly specific inhibitors of 3beta-HSD1 to block the hormone-dependent growth of breast tumors.

Cyclical oxidation-reduction of the C3 position on bile acids catalyzed by rat hepatic 3 alpha-hydroxysteroid dehydrogenase. I. Studies with the purified enzyme, isolated rat hepatocytes, and inhibition by indomethacin.

We recently identified that the Y' bile acid binders are 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSD). In the present studies, purified 3 alpha-HSD catalyzed rapid 3H loss from [3 beta-3H, C24-14C]lithocholic and chenodeoxycholic acids without net conversion to 3-oxo bile acids under physiologic pH and redox conditions. [3 beta-3H]Cholic acid was a poor substrate. The Y' fraction of hepatic cytosol was exclusively responsible for this activity and 3H was transferred selectively to NADP+. Time-dependent 3H loss was also seen in isolated hepatocytes. Further hydroxylation products of lithocholic and chenodeoxycholic acids lost 3H at the same rate, whereas 3H loss from lithocholic acid rapidly ceased, which suggests compartmentation of this bile acid in hepatocytes. Indomethacin inhibited 3H loss from bile acids either in incubations with the pure enzyme or in isolated hepatocytes. Indomethacin did not alter the initial uptake rate of bile acids by hepatocytes, but caused a redistribution of unconjugated bile acids into the medium at early time points (2.5 and 5.0 min) and that of conjugated bile acids at later time intervals (30 min). 3H loss from the 3 beta position therefore can be used to probe the interaction between bile acids and cytosolic 3 alpha-HSD in intact cells, and indomethacin is capable of inhibiting this interaction.

3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.

Rat Y' bile acid binders (33 kD) have been previously recognized as cytosolic bile acid binding proteins (Sugiyama, Y., T. Yamada, and N. Kaplowitz, 1983, J. Biol. Chem., 258:3602-3607). We have now determined that these Y' binders are 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSD), bile acid-metabolizing enzymes. 3 alpha-HSD activity copurified with lithocholic acid-binding activity after sequential gel filtration, chromatofocusing, and affinity chromatography. Three peaks of 3 alpha-HSD activity (I, II, III) were observed in chromatofocusing and all were identified on Western blot by a specific Y' binder antiserum. 3 alpha-HSD-I, the predominant form, was purified and functioned best as a reductase at pH 7.0 with a marked preference for NADPH. Michaelis constant values for mono- and dihydroxy bile acids were 1-2 microM, and cholic acid competitively inhibited the reduction of 3-oxo-cholic acid. Under normal redox conditions, partially purified 3 alpha-HSD-I and freshly isolated hepatocytes catalyzed the rapid reduction of 3-oxo-cholic to cholic acid without formation of isocholic acid, whereas the reverse reaction was negligible. The Y' bile acid binders are therefore 3 alpha-HSD, which preferentially and stereospecifically catalyze the reduction of 3-oxo-bile acids to 3 alpha-hydroxy bile acids.

Lyophilized Clostridium perfringens 3 alpha- and Clostridium bifermentans 7 alpha-hydroxysteroid dehydrogenases: two new stable enzyme preparations for routine bile acid analysis.

Preparations of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) from Clostridium perfringens were successfully lyophilized into a stable powder form. Purification of the enzyme was achieved using triazine dye affinity chromatography. C. perfringens 3 alpha-hydroxysteroid dehydrogenase was purified 24-fold using Reactive Red 120 (Procion Red) -cross-linked agarose (70% yield). Quantitative measurement of bile acids with the purified enzymes, 3 alpha-hydroxysteroid dehydrogenase and 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) from Clostridium bifermentans (strain F-6), was achieved spectrophotometrically. Standard curves with chenodeoxycholic acid (CDC) and cholic acid were linear within a concentration range of 20-100 microM. Analysis of mixtures of ursodeoxycholic acid and CDC showed the additive nature of the 3 alpha-hydroxysteroid dehydrogenase and showed also that 7 alpha-hydroxyl groups were independently quantified by the 7 alpha-hydroxysteroid dehydrogenase. Bile acids in Folch extracts of human bile samples were measured using purified preparations of Pseudomonas testosteroni 3 alpha-hydroxysteroid dehydrogenase, C. perfringens 3 alpha-hydroxysteroid dehydrogenase, Escherichia coli 7 alpha-hydroxysteroid dehydrogenase and C. bifermentans (strain F-6) 7 alpha-hydroxysteroid dehydrogenase. Statistical comparison validated the use of C. perfringens 3 alpha- and C. bifermentans 7 alpha-hydroxysteroid dehydrogenases for the quantification of bile acids in bile.

Hydroxysteroid dehydrogenases of Pseudomonas testosteroni. Separation of a 17 beta-hydroxysteroid dehydrogenase from the 3(17) beta-hydroxysteroid dehydrogenase and comparison of the two enzymes.

When a crude extract of Pseudomonas testosteroni induced with testosterone was subjected to polyacrylamide gel electrophoresis, six bands that stained for 17 beta-hydroxysteroid dehydrogenase activity was observed. A protein fraction containing the enzyme corresponding to the fastest migrating band and devoid of the other hydroxysteroid dehydrogenase activities has been obtained. This preparation appears to be distinct from the previously isolated 3(17) beta-hydroxysteroid dehydrogenase (EC 1.1.1.51) in its chromatography properties on DEAE-cellulose, substrate and cofactor specificity, immunological properties and heat stability. The preparation appears devoid of 3alpha-, 3beta-, 11beta-, 17alpha-, 20alpha-, and 20beta-hydroxysteroid dehydrogenase activities. The enzyme transfers th 4-pro-S-hydrogen of NADH from estradiol-17beta (1,3,5(10)estratriene-3,17beta-diol) to estrone (3-hydroxy-1,3,5(10)-estratriene-17-one).

Characterization of delta 4-3-ketosteroid-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase in cell extracts of Clostridium innocuum.

Cell extracts prepared anaerobically from Clostridium innocuum and Clostridium paraputrificum reduced delta 4-3-ketosteroids to 3 beta 5 beta and 3 alpha 5 beta derivatives, respectively. delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) from both organisms required NADH for activity. 5 beta-Reductase from C. innocuum had a pH optimum of 5.0. The substrate concentration at half-maximal reaction velocity was 4.2 microM, and a specific activity of 17 nmol product formed/h per mg protein was determined using 4-pregnen-3,20-dione (progesterone) as a substrate. delta 4-3-Ketosteroid-5 beta-reductase from C. innocuum reduced progesterone and testosterone, but not 4-cholesten-3-one, to corresponding 3-keto-5 beta derivatives. A relative molecular (Mr) weight of 80 000 was estimated for 5 beta-reductase using HPLC-gel filtration chromatography. 3 beta-Hydroxysteroid dehydrogenase in cell extracts of C. innocuum was oxygen sensitive and required NADH for activity. An Mr of 80 000 was estimated for 3 beta-hydroxysteroid dehydrogenase. However, 5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase activities were separated using an HPLC-DEAE chromatography technique.

Identification of 7 alpha,12 alpha-dihydroxy-5 beta-cholestan-3-one 3 alpha-hydroxysteroid dehydrogenase.

A reductase catalyzing the reduction of the 3-ketone group of 7 alpha,12 alpha-dihydroxy-5 beta-cholestan-3-one and 7 alpha-hydroxy-5 beta-cholestan-3-one, which are the intermediates in the conversion of cholesterol to cholic acid and chenodeoxycholic acid, respectively, into the 3 alpha-hydroxyl group, was purified about 250-fold as judged by the activity from the 100,000 X g supernatant of rat liver homogenate. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretography was 32,000. The absorption spectrum of the purified enzyme showed only a peak at 280 nm due to aromatic amino acids, precluding the presence of a chromophoric prosthetic group in the molecule. The enzyme showed activity toward a variety of substrates, including 3-oxo-5 beta-cholanoic acid, androsterone, 9,10-phenanthrenquinone, p-nitrobenzaldehyde, but not toward glucuronic acid, DL-glyceraldehyde, and glycolaldehyde. The optimal pH for the reduction of 7 alpha-hydroxy-5 beta-cholestan-3-one was 7.4, and the cofactor required was either NADPH or NADH, though the former gave the higher activity. Judging from the chromatography behavior as well as substrate specificity, the enzyme was identified as 3 alpha-hydroxysteroid dehydrogenase (3 alpha-hydroxysteroid:NAD(P)+ oxidoreductase, EC 1.1.1.50).

Metabolism of glycyrrhetic acid by rat liver microsomes: glycyrrhetinate dehydrogenase.

Glycyrrhetic acid, derived from a main component of liquorice, was converted to 3-ketoglycyrrhetic acid reversibly by rat liver homogenates in the presence of NADPH or NADP+. Glycyrrhetic acid-oxidizing and 3-ketoglycyrrhetic acid-reducing activities were localized in microsomes among the subcellular fractions of rat liver. Glycyrrhetic acid-oxidizing activity and 3-ketoglycyrrhetic acid-reducing activities showed pH optima at 6.3 and 8.5, respectively, and required NADP+ or NAD+ and NADPH or NADH, respectively, indicating that these activities were due to glycyrrhetinate dehydrogenase. The dehydrogenase was not solubilized from the membranes by the treatment with 1 M NaCl or sonication, indicating that the enzyme is a membrane component. The dehydrogenase was solubilized with detergents such as Emalgen 913, Triton X-100 and sodium cholate, and then separated from 3 beta-hydroxysteroid dehydrogenase (5 beta-androstan-3 beta-ol-17-one-oxidizing activity) by butyl-Toyopearl 650 M column chromatography. Partially purified enzyme catalyzed the reversible reaction between glycyrrhetic acid and 3-ketoglycyrrhetic acid, but was inactive toward 3-epiglycyrrhetic acid and other steroids having the 3 beta-hydroxyl group. The enzyme required NADP+ and NADPH for the highest activities of oxidation and reduction, respectively, and NAD+ and NADH for considerable activities, similar to the results with microsomes. From these results the enzyme is defined as glycyrrhetinate dehydrogenase, being quite different from 3 beta-hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestine, which is active for both glycyrrhetic acid and steroids having the 3 beta-hydroxyl group.

Purification and properties of 3 alpha-hydroxysteroid dehydrogenase as a 3-keto bile acid reductase from human liver cytosol.

The NADPH-dependent 3 alpha-hydroxysteroid dehydrogenases (peaks 1, 2 and 3) acting on 3-keto-5 beta-cholanoic acid separated by carboxymethyl-cellulose chromatography from human liver cytosol were purified to homogeneous protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using Affi-Gel blue, phenyl-Sepharose CL-4B, TSKgel G3000 SW chromatography and chromatofocusing. The overall purifications of the enzymes from cytosol were 316-fold (peak 1), 232-fold (peak 2) and 345-fold (peak 3) and the recoveries of the enzymes were 0.4% (peak 1), 7.1% (peak 2) and 3.7% (peak 3). The isoelectric points of the enzymes were found to be 7.34, 7.46 and 7.88 by chromatofocusing. The molecular weights of the enzymes were similar and estimated to be about 32,000 by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymological properties were nearly identical among the three forms. The reaction was reversible and the optimum pH of the enzymes for oxidation was about 8.4 and that for reduction was about 7.4. The enzymes could not reduce 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid, 7 alpha-hydroxy-5 beta-cholestan-3-one and 7 alpha, 12 alpha-dihydroxy-5 beta-cholestan-3-one to the corresponding 3 alpha-hydroxysteroids, whereas the enzymes could reduce 3,7-disubstituted 3-keto bile acids. Thus, the enzymes purified in this study were found to have a stereospecific character for some 3-ketosteroids. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of dithiothreitol to the reaction mixture, indicating that the enzyme required a sulfhydryl group for activity.

Enzymes involved in the formation of 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid from dehydrocholic acid by Ruminococcus sp. obtained from human intestine.

Ruminococcus sp. PO1-3 from human intestinal flora reduced dehydrocholic acid to 3 beta-hydroxy-7,12-dioxo-5 beta-cholanic acid by means of the enzyme 3 beta-hydroxysteroid dehydrogenase (Akao, T., Akao, T., Hattori, M., Namba, T. and Kobashi, K. (1986) J. Biochem. (Tokyo) 99, 1425-1431). This bacterium and its crude extract gave rise to another product, showing a lower RF value on TLC, from dehydrocholic acid. The product was identified as 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid. The crude extract reduced 7-ketolithocholic acid and its methyl ester, but not 6-ketolithocholic acid and 12-ketochenodeoxycholic acid, in the presence of NADPH, and oxidized ursodeoxycholic acid and beta-muricholic acid, but not cholic acid, chenodeoxycholic acid, deoxycholic acid and hydrocholic acid, in the presence of NADP+. Therefore, besides 3 beta-hydroxysteroid dehydrogenase, 7 beta-hydroxysteroid dehydrogenase was shown to be present in this bacterium. The two dehydrogenases were clearly separated from each other by butyl-Toyopearl 650 M column chromatography. From dehydrocholic acid, 7 beta-hydroxy-3,12-dioxo-5 beta-cholanic acid was produced by 7 beta-hydroxysteroid dehydrogenase and 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid was produced by combination of two enzymes, 7 beta- and 3 beta-hydroxysteroid dehydrogenase.

Full length cDNA structure and deduced amino acid sequence of human 3 beta-hydroxy-5-ene steroid dehydrogenase.

Polyclonal antibodies raised against 3 beta-hydroxysteroid dehydrogenase isolated from human placenta were used to screen a lambda gt11 expression cDNA library from the same tissue. The protein deduced from cDNA sequences contains 372 amino acids with a calculated mol wt of 42,216. Since 3 beta-hydroxysteroid dehydrogenase is the enzyme catalyzing the formation of all classes of hormonal steroids, the availability of the cDNA encoding this enzyme opens new possibilities for a detailed investigation of the factors regulating the expression and activity of this crucial enzyme in adrenal, gonadal as well as peripheral tissues.

Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase.

Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family.

[Enzymes involved in modification of the steroid nucleus of industrial mycobacterial strains: isolation, functions, and properties].

The key enzymes involved in modification of the steroid nucleus of sterol-transforming mycobacteria--3beta-hydroxysteroid oxidase (3-OH-SO, EC 1.13.1.2) and 17beta-hydroxysteroid dehydrogenase (17-OH-SDH, EC 1.1.1)--were isolated and characterized. It is shown that 3-OH-SO is a multifunctional enzyme catalyzing oxidation of the 3beta-OH group, delta5 --> delta4 isomerization, and 6-hydroxylation. Two forms of intracellular 17-OH-SDH that catalyze redox reactions at C17 were found, and their properties were determined. The presence of an extracellular 17-OH-SDH in Mycobacterium spp. (VKM Ac-1815 D and Et1) was demonstrated for the first time.

Characterisation of an associate 17-beta-hydroxysteroid dehydrogenase activity and affinity labelling of the 3-alpha-hydroxysteroid dehydrogenase of Pseudomonas testosteroni.

The 3-alpha-hydroxysteroid dehydrogenase and the 3-beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni were purified to homogeneity by polyaerylamide gel electrophoresis using the following stages: DEAE cellulose chromatography, affinity chromatography on oestrone-aminocaproate sepharose and Sephadex gel filtration. The pure 3-alpha-hydroxysteroid dehydrogenase was completely devoid of 3-beta-hydroxysteroid dehydrogenase activity but could oxidize estradiol 17-beta at an appreciable rate. This activity accounts for about 40 per cent of the total 17-beta-estradiol dehydrogenase of the crude bacterial extract. Affinity labelling of pure 3-alpha-hydroxysteroid dehydrogenase was carried out using 5-beta-pregnane 3,20-dione-12-alpha-iodoacetate and 5-alpha-androstane 3-one-17-beta-bromoacetate. With both reagents, inactivation was obtained only in the presence of coenzyme, the substrate protected against inactivation and the enzyme was fully inhibited with covalent binding of 1 mole of reagent per mole of subunit suggesting an active site directed inhibition. Histidine and methionine were identified as the labelled aminoacid residues.

Purification and characterization of NADP+-dependent 3 alpha-hydroxysteroid dehydrogenase from mouse liver cytosol.

A monomeric 3 alpha-hydroxysteroid dehydrogenase with a molecular weight of 34,000 was purified to apparent homogeneity from mouse liver cytosol. The enzyme catalyzed the reversible oxidation of the 3 alpha-hydroxy group of C19-, C21-, and C24-steroids, reduced a variety of carbonyl compounds, and was inhibited by SH-reagents, synthetic estrogens, anti-inflammatory drugs, prostaglandins, and delta 4-3-ketosteroids. Although these properties are similar to those of the enzyme from rat liver cytosol, the mouse enzyme exhibited low dehydrogenase activity toward benzene dihydrodiol and some alicyclic alcohols, it showed a strict cofactor specificity for NADP(H), and high substrate inhibition was observed in the reverse reaction. In addition, dexamethasone, deoxycorticosterone, and medroxyprogesterone acetate inhibited the mouse enzyme competitively at low concentrations and noncompetitively at high concentrations, whereas hexestrol, indomethacin, and prostaglandin A1 were competitive inhibitors. Steady-state kinetic measurements in both directions indicated that the reaction proceeds through an ordered bi bi mechanism with the cofactors binding to the free enzyme. The 3-ketosteroid substrates inhibited the enzyme uncompetitively at elevated concentrations, suggesting that the substrates bind to the enzyme.NADPH complex and to the enzyme NADP+ complex.

Purification and properties of 3 alpha-hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum from human intestine.

3 alpha-Hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum, isolated from human intestinal bacteria, was capable of converting 3-ketoglycyrrhetic acid to 3 alpha-hydroxyglycyrrhetic acid. The enzyme was purified to homogeneity by means of butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A, Toyopearl HW-55S, and isoelectric focusing column chromatographies. The purified enzyme showed a specific activity of 156 mumol/min.mg toward 3 alpha-hydroxyglycyrrhetic acid, and showed a single band on SDS-polyacrylamide gel electrophoresis. The apparent molecular weight was 53,000, as estimated by gel filtration, and 30,000, as judged by SDS-polyacrylamide gel electrophoresis. Its isoelectric point was 5.2. The enzyme showed absolute specificity for the 3 alpha-hydroxyl and 3-ketonic groups of 18 alpha- or 18 beta-glycyrrhetic acid and required NADP+ and NADPH as cosubstrates. The enzyme did not act on any 3 alpha-hydroxyl or 3-ketonic group of steroids or bile acids. The enzyme is a novel type of enzyme, defined as 3 alpha-hydroxy-glycyrrhetinate dehydrogenase, being quite different from 3 alpha-hydroxysteroid dehydrogenase [EC 1.1.1.50].

3 beta-Hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestinal bacteria.

Ruminococcus sp. PO1-3 obtained from human intestinal flora is able to reduce dehydrocholate as well as 3-ketoglycyrrhetinate. From this bacterium dehydrocholate- and 3-ketoglycyrrhetinate-reducing activities were purified one thousand-fold together with 3-ketocholanate-reducing and 3-beta-hydroxyglycyrrhetinate (glycyrrhetic acid) oxidizing activities by means of Matrex Red A, Sephadex G-200 and Octyl-Sepharose column chromatography. The purified enzyme catalyzed the reduction of dehydrocholic acid to 3 beta-hydroxy-7,12-diketocholanic acid and of 3-ketocholanic acid to 3 beta-hydroxycholanic acid. Studies on substrate specificity revealed that the enzyme had absolute specificity for the beta-configuration of a hydroxyl group at the 3 position of bile acid and steroids having no double bond in the A/B ring. This enzyme was neither beta-hydroxysteroid dehydrogenase [EC 1.1.1.51] nor 3 beta-hydroxy-delta 5-steroid dehydrogenase [EC 1.1.1.145], but a novel type of enzyme, defined as 3 beta-hydroxysteroid dehydrogenase.

Identification of two dihydrodiol dehydrogenases associated with 3(17)alpha-hydroxysteroid dehydrogenase activity in mouse kidney.

Dihydrodiol dehydrogenase activity was detected in the cytosol of various mouse tissues, among which kidney exhibited high specific activity comparable to the value for liver. The enzyme activity in the kidney cytosol was resolved into one major and three minor peaks by Q-Sepharose chromatography: one minor form cross-reacted immunologically with hepatic 3 alpha-hydroxysteroid dehydrogenase and another with aldehyde reductase. The other minor form was partially purified and the major form was purified to homogeneity. These two forms, although different in their charges, were monomeric proteins with the same molecular weight of 39,000 and had similar catalytic properties. They oxidized cis-benzene dihydrodiol and alicyclic alcohols as well as trans-dihydrodiols of benzene and naphthalene in the presence of NADP+ or NAD+, and reduced several xenobiotic aldehydes and ketones with NAD(P)H as a cofactor. The enzymes also catalyzed the oxidation of 3 alpha-hydroxysteroids and epitestosterone, and the reduction of 3- and 17-ketosteroids, showing much lower Km values (10(-7)-10(-6) M) for the steroids than for the xenobiotic alcohols. The results of mixed substrate experiments, heat stability, and activity staining on polyacrylamide gel electrophoresis suggested that, in the two enzymes, both dihydrodiol dehydrogenase and 3(17)alpha-hydroxysteroid dehydrogenase activities reside on a single enzyme protein. Thus, dihydrodiol dehydrogenase existed in four forms in mouse kidney cytosol, and the two forms distinct from the hepatic enzymes may be identical to 3(17)alpha-hydroxysteroid dehydrogenases.

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.

Purification and kinetic properties of 3 beta-hydroxysteroid dehydrogenase from bovine adrenocortical microsomes.

3 beta-Hydroxysteroid dehydrogenase was purified from bovine adrenocortical microsomes and its properties were studied. The purified dehydrogenase gave a single homogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed no steroid delta 5-delta-4 isomerase activity. The molecular weight of the dehydrogenase was estimated to be 41,000 for the monomer and the isoelectric point was determined to be at pH 6.3. The Km values of the dehydrogenase were 6.2 microM for NAD+, 4.9 mM for NADP+, 2.0 microM for pregnenolone, and 5.3 microM for 17 alpha-hydroxypregnenolone. The mechanism of inhibition by trilostane of the dehydrogenase was also examined kinetically. The inhibition was found to be competitive, with Ki values of 0.14 microM for 17 alpha-hydroxypregnenolone and 0.38 microM for pregnenolone.