Dehydrogenation of androsterone by purified 3α-hydroxy steroid-dependent nicotinamide–adenine dinucleotide (phosphate)-transhydrogenating enzyme of rat liver

1. An enzyme from rat liver, catalysing 3alpha-hydroxy steroid-dependent NAD(P) transhydrogenation and NAD-linked and NADP-linked dehydrogenation of 3alpha-hydroxy steroids, has been purified 100-fold by chromatography on DEAE-cellulose and calcium phosphate gel. 2. No separation of these activities into different protein fractions has been achieved. 3. The properties of the enzyme in catalysing NAD-linked and NADP-linked dehydrogenation have been compared, with androsterone as substrate. Differences were found in pH optima, affinity for coenzyme and steroid, equilibrium constants and effects of salts. 4. NAD-linked dehydrogenation is inhibited by NADPH(2) but is protected from this inhibition by chloride, which alone is itself an inhibitor. 5. The relevance of these findings to the problem of the number of enzymes involved in catalysis of 3alpha-hydroxy steroid-dependent transhydrogenation is discussed.     

A comparison of selected physical properties of hepatic sorbitol dehydrogenases [L-iditol: NAD oxidoreductases] from four mammalian species.

1. The sorbitol dehydrogenases [L-iditol: NAD oxidoreductase] from livers of cow, man, rat and sheep each possess molecular weights of about 140,000. The beef, rat and sheep liver enzymes are composed of subunits of molecular weight 40,000. 2. The sorbitol dehydrogenases from livers of these four species each possess an isoelectric point of 7.3. 3. The four enzyme preparations show identical mobilities upon disc-gel electrophoresis and yield a single band of enzymic activity. 4. Sorbitol dehydrogenase activity is activated by the presence of ampholines or by increasing ionic strengths, with maximal activation at about 0.5 M salt concentration. These factors may cause the Km for NAD to be lowered.     

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.     

Biosynthesis of hydroxyl-linked glucuronides of short-chain bile acids by rat liver 3-hydroxysteroid UDP-glucuronosyltransferase

Microsomal preparations from livers of Sprague-Dawley rats catalyze the glucuronidation of 3 alpha-hydroxy-5 beta-H (3 alpha, 5 beta) short-chain bile acids (C20-C23), predominantly at the hydroxyl group, while the glucuronidation of 3 beta, 5 beta short-chain bile acids occurs exclusively at the carboxyl group. A similar pattern of conjugation was also observed in Wistar rats having normal levels of 3-hydroxysteroid UDP-glucuronosyltransferase. Significant reductions of formation rates for hydroxyl-linked, but not carboxyl-linked, short-chain bile acid glucuronides were observed in hepatic microsomes from Wistar rats with low 3-hydroxysteroid UDP-glucuronosyltransferase activity. 3-Hydroxysteroid UDP-glucuronosyltransferase, purified to homogeneity from Sprague-Dawley liver microsomes, catalyzed the 3-O-glucuronidation of 3 alpha, 5 beta C20-23 bile acids, as well as of lithocholic and isolithocholic acids (C24). The apparent Michaelis constants (KM) for short-chain bile acids were similar to the value obtained for androsterone. 3 alpha, 5 beta-C20 and 3 beta, 5 beta-C20 competitively inhibited glucuronidation of androsterone by the purified 3-hydroxysteroid UDP-glucuronosyltransferase. Purified 17 beta-hydroxysteroid and p-nitrophenol UDP-glucuronosyltransferases did not catalyze the glucuronidation of bile acids. In addition, none of the purified transferases catalyzed the formation of carboxyl-linked bile acid glucuronides. The results show that 3-hydroxysteroid UDP-glucuronosyltransferase, an enzyme specific for 3-hydroxyl groups of androgenic steroids and some conventional bile acids, also catalyzes the glucuronidation of 3 alpha-hydroxyl (but not carboxyl) groups of 3 alpha, 5 beta short-chain bile acids.     

Purification by cibacron blue F3GA dye affinity chromatography and comparison of NAD(P)H:quinone reductase (E.C.1.6.99.2) from rat liver cytosol and microsomes

The dicoumarol-sensitive NAD(P)H:quinone reductase (E.C.1.6.99.2), often referred to as DT-diaphorase, has been purified from both the cytosolic and microsomal fractions from rat liver using a novel, highly efficient, two-step purification procedure utilizing immobilized Cibacron Blue F3GA dye affinity chromatography as the principal step. Under the conditions reported here, this dye affinity resin, generally recognized as preferentially binding nucleotide-dependent proteins, was highly selective in the recovery of up to 95% of the NAD(P)H:quinone reductase directly from the cytosol as a preparation which was often greater than 90% pure. Further purification by gel exclusion chromatography resulted in pure protein preparations with final recoveries approaching 80%. Similar results were obtained during the purification of this quinone reductase activity from microsomal extracts. Evidence is presented which suggests that the enzyme isolated from each cellular fraction are highly homologous, if not identical; data are consistent with genetic evidence.     

Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.     

Oxidative release of nitrite from 2-nitrotoluene by a three-component enzyme system from Pseudomonas sp. strain JS42.

Pseudomonas sp. strain JS42 utilizes 2-nitrotoluene (2NT) as the sole source of carbon and energy for growth. Intact cells catalyze the oxidation of 2NT to 3-methylcatechol and nitrite in a reaction that requires molecular oxygen. Cell extracts oxidized 2NT to 3-methylcatechol and nitrite in the presence of NAD(P)H and ferrous iron. Ion-exchange chromatography yielded three protein fractions (A, B, and C) which were all required for the oxidation of 2NT to 3-methylcatechol and nitrite. Component B (reductase2NT) catalyzed a NAD(P)H-dependent reduction of cytochrome c. Solutions of component A (ISP2NT) were brown and showed absorption maxima at 458 and 324 nm. Two major bands with M(r)s 52,500 and 28,000 were observed when ISP2NT was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component C could be replaced by ferredoxin NAP from the Pseudomonas putida NCIB 9816-4 naphthalene dioxygenase system and was given the designation ferredoxin2NT. Experiments with 18O2 showed that both oxygen atoms were added to the aromatic ring of 2NT to yield 3-methylcatechol. The enzyme is a new multicomponent enzyme system which we have designated 2NT 2,3-dioxygenase.     

Strain differences in purified rat hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.

Qualitative and quantitative differences of purified hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase were investigated in Wistar and Sprague-Dawley rats. Individual differences in the glucuronidation rate of androsterone and chenodeoxycholic acid were observed in hepatic microsomal fractions from Wistar but not Sprague-Dawley rats. No individual variation was observed in the glucuronidation of testosterone, p-nitrophenol or oestrone. The 3 alpha-hydroxysteroid UDP-glucuronosyltransferases from livers of Wistar and Sprague-Dawley rats were isolated and highly purified by using Chromatofocusing and affinity chromatography. The amount of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase in the liver of Wistar rats exhibiting low rates for androsterone glucuronidation is about 10% or less than that found in hepatic microsomal fractions obtained from Wistar rats having high rates for androsterone glucuronidation. The apparent Km for androsterone with purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase from Wistar rats with high glucuronidation activity (6 microM) was not different from that observed for the enzyme purified from Sprague-Dawley animals, whereas that for the enzyme purified from Wistar rats with low glucuronidation activity was substantially higher (120 microM). Despite the differences in apparent Km values for androsterone, the apparent Km for UDP-glucuronic acid (0.3 mM) was not different in the different populations of rats.     

Purification of a form of mouse liver UDP glucuronosyltransferase which glucuronidates androgens

A form of UDP glucuronosyltransferase active in the glucuronidation of the androgens, testosterone, androsterone and dihydrotestosterone has been purified to apparent homogeneity as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis from the livers of phenobarbital-treated C57BL/6N mice. This UDP glucuronosyltransferase is inactive towards estrone as substrate. Data from chromatofocusing and purification experiments suggest that testosterone and androsterone are glucuronidated primarily by this enzyme form and to a lesser extent by an enzyme form which has a slightly higher isoelectric point. However, this major form is only responsible for about half the capacity to glucuronidate dihydrotestosterone.     

A comparative study of bile acid CoA:amino acid:N-acyltransferase (BAT) from four mammalian species.

1. Bile acid CoA:amino acid:N-acyltransferase (BAT) was partially purified from dog, human, pig and rat livers. The interspecies variation in substrate specificity and kinetics were determined for glycine and taurine. 2. BAT activity from dog liver formed bile acid conjugates with taurine exclusively, whereas BAT activity from each of the other species formed conjugates with both taurine and glycine. 3. Biliary composition of glycine and taurine bile acid conjugates could partly be accounted for by substrate affinity (Km) and turnover number (Vmax) of BAT activity. 4. A monospecific anti-human BAT polyclonal antibody reacted on Western blot analysis with a 40 kDa band in a 100,000 g supernatant fraction from rat liver. 5. Immunoabsorption chromatography using an anti-human BAT antibody-Sepharose affinity column showed that both the immunoreactive protein band and BAT activity were removed from the 100,000 g supernatant fraction from human and rat livers.     

Calcium-activated, phospholipid-dependent protein kinases from rat liver: subcellular distribution, purification, and characterization of multiple forms

Three forms of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) were extensively purified from rat liver homogenate. Subcellular fractionation analysis indicated that the majority (approximately 85%) of the activity was associated with particulate fractions of the liver. Among these, the microsomal and nuclear fractions accounted for approximately 63% and approximately 10% of total activity. The remaining 15% of protein kinase C was recovered in the soluble fraction following differential centrifugation. It was also found that most of the membrane-associated protein kinase C was latent, with 4-6-fold stimulation with detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, octyl beta-glucoside, or Triton X-100. The activity of both the bound form and the soluble enzyme was enhanced by the addition of Ca2+ and phosphatidylserine, when histone H1 was used as substrate. The bound protein kinase C activity was dissociated by homogenization of liver in buffer containing ethylene glycol bis(beta-aminoethyl ether)-N,-N,N',N'-tetraacetic acid, ethylenediaminetetraacetic acid, and various proteolytic inhibitors, and the solubilized extract was used to purify multiple forms of the enzyme. The purification procedure sequentially utilized (NH4)2SO4 fractionation, ion-exchange chromatography on DEAE-cellulose, gel permeation chromatography on Fractogel TSK HW-55 (F), ion-exchange chromatography on hydroxylapatite, gel permeation chromatography on Ultrogel AcA34, and affinity chromatography on polyacrylamide-immobilized phosphatidylserine. On hydroxylapatite columns, protein kinase C activity was resolved into three isoenzymic forms designated C-I, C-II, and C-III. The molecular weights of the three isoenzymic forms were in the range of 208,000-225,000 as shown by chromatography on calibrated Ultrogel AcA34 columns and sucrose density gradient centrifugation. Furthermore, all three isoenzymes demonstrated a single peak with a sedimentation coefficient (s20.w) in the range of 9.0-9.2. However, with polyacrylamide gel electrophoresis, all the forms showed a single protein component with average molecular weight of 64K, suggesting that the native isoenzymes may be composed by subunits. Finally, all three isoenzymes exhibited nearly identical enzymatic properties.     

Two aldehyde dehydrogenases from human liver. Isolation via affinity chromatography and characterization of the isozymes.

Human liver extracts show two major bands with aldehyde dehydrogenase (Aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) activity via starch gel electrophoresis at pH 7.0. Both bands have been purified to apparent homogeneity via classical chromatography combined with affinity chromatography on 5'-AMP-Sepharose 4B. The slower migrating band, enzyme 1, when assayed at pH 9.5 has a low Km for NAD (8 micrometer) and a high Km for acetaldehyde (approx. 0.1 mM). It is very strongly inhibited by disulfiram at pH 7.0 with a Ki of 0.2 micrometer. The faster migrating band, enzyme 2, has a low Km for acetaldehyde, (2--3 micrometer at pH 9.5), a higher Km for NAD (70 micrometer at pH 9.5), and is not inhibited by disulfiram at pH 7.0. The two enzymes are very similar to the F1 and F2 isozymes of horse liver purified by Eckfeldt et al. (Eckfeldt, J., Mope, L., Takio, K. and Yonetani, T. (1976) J. Biol, Chem. 251, 236-240) in molecular weight, subunit composition, amino acid composition and extinction coefficient. Preliminary kinetic characterizations of the enzyme are presented.     

Isolation and characterization of hyodeoxycholic-acid: UDP-glucuronosyltransferase from human liver

The enzyme hyodeoxycholic-acid: UDP-glucuronosyltransferase was purified about 230-fold from a solubilized human liver microsomal preparation utilizing anion-exchange chromatography, ampholyte-displacement chromatography and UDP-hexanolamine--Sepharose affinity chromatography. The homogeneity of the final enzyme preparation was judged by two criteria: the appearance of a single band of Mr 52000 in SDS/PAGE; the elution of a single peak in reversed-phase FPLC. The isolated enzyme catalyzed the glucuronidation of the 6 alpha-hydroxy bile acids hyodeoxycholic and hyocholic acids, and of the steroid hormone estriol, with a ratio of relative reaction rates of 13:1:2.7. UDP-glucuronosyltransferase activities toward the 3 alpha-hydroxy bile acid lithocholic acid, androsterone, testosterone, bilirubin and p-nitrophenol were not detectable in the pure enzyme preparation and were shown to be separated from enzyme activity toward hyodeoxycholic acid during ampholyte-displacement chromatography and/or UDP-hexanolamine--Sepharose affinity chromatography. Two-substrate kinetic analysis of hyodeoxycholic-acid-conjugating activity gave a sequential mechanism with apparent Km values of 12 microM and 4 microM for hyodeoxycholic acid and UDP-glucuronic acid, respectively. Phospholipids were required for reconstitution of maximal activity toward hyodeoxycholic acid. Phosphatidylcholine was the most effective activator of enzyme activity.     

Pyridine nucleotide interaction with rat liver dihydropteridine reductase.

The interactions of a homogeneous preparation of rat liver dihydropteridine reductase with NADH, NADPH, NAD+, NADP+, and the 1-N6-ethenoadenine derivative of NAD+ have been investigated by fluorescence titration, circular dichroism, equilibrium dialysis, Sephadex G-25 chromatography, and polyacrylamide gel electrophoresis. The procedures indicate that the dimeric enzyme has a definite preference for NADH, but binds only 1 mol of this nucleotide per mol of enzyme. The binary complex of enzyme with NADH is only partially stable to exhaustive dialysis and gel electrophoresis, where it shows greater mobility (0.26) than the free enzyme (0.21); however, the complex can be isolated by Sephadex G-25 chromatography, and characterized with respect to its absorbance spectrum. No ternary complexes are observed when samples of reductase, preincubated with excess NADH, and either the reaction product, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, or the inhibitor, methotrexate, are subjected to polyacrylamide gel electrophoresis.     

Rat casein: isolation and characterization of two major fractions.

1. In rat milk, casein exists as particles of 77 nm mean diameter, similar in appearance to the casein micelles in the milk of other species. 2. The heterogeneity of rat casein was investigated by polyacrylamide gel electrophoresis and by chromatography on DEAE-cellulose, both in the presence of 8 M-urea. 3. The chromatography yielded two fractions, D2 and D4. 4. In chemical composition, D2 resembles the kappa-caseins of other species, while D4 resembles the alpha 5-caseins of other species. 5. D2 is soluble in 80 mM-CaCl2 at 37 degrees C, but D4 is insoluble under these conditions. 6. In 80 mM-CaCl2 at 37 degrees C, D2 prevents the precipitation of D4 by the formation of particles similar to those found in rat milk.     

Production of monospecific antibodies to rat liver ornithine decarboxylase and their use in turnover studies.

Two forms of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) were purified from the livers of rats which had been treated with thioacetamide for 16 h (for details, see miniprint to Obenrader, M.F., and Prouty, W. F. (1977) J. Biol. Chem. 252, 2860-2865). The enzyme was purified over 7,000-fold from liver cytosol with an overall yield of 8%. Enzyme activity was eluted finally in two distinct fractions by chromatography on activated thiol-Sepharose 4B. Both forms appear to be dimeric proteins having molecular weights of approximately 100,000 by equilibrium sedimentation and analysis on a calibrated Sephadex G-200 column. The apparent subunits are approximately 50,000 daltons as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Since electrophoresis in the presence of detergent is the only method used here to indicate subunits, the possibility that conditions of sample preparation resulted in splitting of a labile protein cannot be excluded from consideration. Ornithine decarboxylase has a very broad pH-activity curve with an optimum that shifts from pH 7.0 to pH 7.8 as the enzyme is purified. The apparent Km values for a highly purified mixture of the two forms of enzyme for L-ornithine and pyridoxal 5'-phosphate were determined to be 0.13 mM and 0.25 micronM, respectively. Both sodium and potassium chloride were shown to inhibit enzymatic activity; 50% inhibition occurred at 270 mM for each when Km amounts or ornithine were used. Rat liver ornithine decarboxylase antiserum was prepared in rabbits using Form I of the enzyme as the antigen. The antibody was shown to precipitate quantitatively the ornithine decarboxylase activity isolated from induced rat liver and rat ventral prostate. The specificity of the antiserum was demonstrated by rocket immunoelectrophoresis and by gel electrophoresis in the presence of sodium dodecyl sulfate using immunoprecipitates obtained from enzyme preparations labeled either in vivo, with [3H]leucine, or in vitro, by reductive methylation using formaldehyde and sodium [3H]borohydride. The antibody preparation has been used in a titration method to assess the half-life of antigen in livers of rats induced for ornithine decarboxylase by injection of thioacetamide. In two experiments, the t1/2 of activity at the height of induction, following injection of cycloheximide, was 19 and 24 min, while the t1/2 of disappearance of antigen was 28 and 33 min, respectively. In each experiment the t1/2 for antigen was significantly longer than the t1/2 for loss of enzyme activity. Enzyme levels appear to be modulated primarily by synthesis and degradation of antigen. Furthermore, the observation that enzyme activity is lost with a shorter t1/2 than antigen is consistent with the idea that denaturation is an initial step in the degradation of this enzyme...     

Affinity chromatography of 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. Use of N,N-dimethylformamide to prevent hydrophobic interactions between the enzyme and the ligand.

1. The 3alpha-hydroxysteroid: NAD+-oxidoreductase (EC 1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) has been purified by affinity chromatography on Sepharose 4B using glycocholic acid as ligand covalently bound through its carboxyl group to the ethylenediamine spacer. 2. The attachment of the enzyme to the substrate-containing matrix is greatly enhanced by the presence of NAD+ suggesting that this enzyme has a compulsory ordered mechanism where NAD+ binds to the enzyme before the steroid. 3. A NAD+-independent interaction between the enzyme and the ligand was also found. This interaction was mainly hydrophobic and interfered with the NAD+-dependent binding. The NAD+-independent interaction was reduced by N,N-dimethylformamide. 4. By using the affinity column in the presence of 10% N,N-dimethylformamide, highly purified enzyme, as judged from polyacrylamide gel electrophoresis, could be obtained in one step from crude bacterial extracts.     

The distribution of phosphofructokinase isoenzymes in the liver of camel, rat and rabbit

1. The distribution of phosphofructokinase isoenzymes have been compared among camel, rat and rabbit livers. 2. Only a single phosphofructokinase isoenzyme is present in the camel liver which has shown different physical and regulatory properties from the isoenzymes of rat and rabbit liver. 3. The ammonium sulphate precipitation curves of the camel and rabbit enzymes were monophasic, whereas the rat enzyme was biphasic. 4. Rabbit liver phosphofructokinase was slightly more anodic than the rat enzyme, whereas the camel enzyme was the least anodic as shown by the techniques of DEAE-cellulose chromatography and cellulose acetate electrophoresis. 5. Partially purified camel liver phosphofructokinase showed different regulatory properties from the rabbit and rat isoenzymes as the apparent Km values were 0.58, 0.45 and 0.82 mM respectively.     

Purification and characterization of rat adrenal 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene-isomerase

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.     

Molecular cloning of rat liver 3α-hydroxysteroid dehydrogenase and identification of structurally related proteins from rat lung and kidney

3α-Hydroxysteroid dehydrogenase and related enzymes play important roles in the metabolism of endogenous compounds including androgens, corticosteroid, prostaglandins and bile acids, as well as drugs and xenobiotics such as benzo(a)pyrene. Complementary DNA clones encoding 3α-hydroxysteroid dehydrogenase were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. A full-length cDNA clone of 1286 base pairs contained an open reading frame encoding a protein of 322 amino acids with an estimated M(w) of 37 kD. When expressed in E. coli, the encoded protein migrated to the same position on SDS-polycrylamide gels as the enzyme in rat liver cytosols. The protein expressed in bacteria was highly active in androsterone oxidation in the presence of NAD as cofactor and this activity was inhibited by indomethacin, a potent inhibitor of 3α-hydroxysteroid dehydrogenase. The predicted amino acid sequence of 3α-hydroxysteroid d dehydrogenase was related to sequences of several other aldo-keto reductases such as bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase and frog lens epsilon-crystallin, suggesting that these proteins belong to the same gene family. Recently, we have found that monoclonal antibodies against 3α-hydroxysteroid dehydrogenase also recognized multiple antigenically related proteins in rat lung, kidney and testis. Further screening of liver, lung and kidney cDNA libraries using these monoclonal antibodies as probes resulted in the isolation of additional five different cDNAs encoding proteins with high degree of structural homology to rat liver 3α-hydroxysteroid dehydrogenase.