A simple and sensitive assay for 9-hydroxyprostaglandin dehydrogenase

The tritium recovery assay of 9-hydroxyprostaglandin dehydrogenase [Pace-Asciak, C. (1975) J. Biol. Chem.250, 2789] has been modified to ensure its applicability to both crude and purified enzyme preparations. The stereospecificity of NAD⁺-dependent 9-hydroxyprostaglandin dehydrogenase with respect to NAD⁺ was determined first and found to be A-side specific. Based on the stereospecificity of the enzyme, a simple and sensitive assay method for 9-hydroxyprostaglandin dehydrogenase has been developed. The assay is able to detect picomole quantities of substrate conversion. When 15-keto-13,14-dihydro-[9β-³H]PGF_2α is employed as substrate, the tritium label of the tritiated prostaglandin is effected to transfer to lactate stereospecifically by coupling 9-hydroxyprostaglandin dehydrogenase with a saturating level of lactate dehydrogenase. The amount of prostaglandin oxidized is quantitated by the radioactivity of the labeled lactate produced, which is separated from labeled prostaglandin by charcoal precipitation. Simultaneous assays with the current tritium-release and thin-layer chromatography methods indicated excellent correlation. Using this method we have found that rat kidney possesses the highest enzyme activity among those tissues examined. Rat kidney enzyme activity is linear for the first 10 min it is studied and is nonlinear with increasing amounts of crude enzyme extract, indicating the possible presence of endogenous inhibitor(s). The apparent K_m for 15-keto-13,14-dihydro-PGF_2α is 0.66 μm. The enzyme is activated by imipramine, inhibited by indomethacin, but not affected by furosemide and ethacrynic acid. These results confirm previous findings reported in the literature.     

An NADP-linked prostacyclin dehydrogenase in rabbit kidney

An NADP-linked 15-hydroxyprostaglandin dehydrogenase specific for prostacyclin was purified 1,300-fold from rabbit kidney. Prostaglandins E₂, F_2α, and 6-Keto PGF_1α and thromboxane B₂ were oxidized by the purified enzyme with rates of reaction less than 4% that of PGI₂. Unlike other rabbit kidney NADP-linked 15-hydroxyprostaglandin dehydrogenases, this enzyme catalyzes oxido reduction more rapidly at the 15- position than at the 9- position and does not utilise NAD as a cofactor. It has a molecular weight of 62,000 and migrates on polyacrylamide disc gel electrophoresis as a single diffuse band. The reaction product was identified by thin-layer chromatography as 6,15-diketo PGF_1α. Prostacyclin dhydrogenase is the first 15-hydroxyprostaglandin dehydrogenase described which is specific for the metabolism of prostacyclin.     

Cloning and sequencing of the cDNA for rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) is an NAD(P)(+)-dependent oxidoreductase which will terminate androgen action by converting 5 alpha-dihydrotestosterone to 3 alpha-androstanediol. It is identical to dihydrodiol dehydrogenase and it can function as a 9-, 11-, and 15-hydroxyprostaglandin dehydrogenase. Its reactions are potently inhibited by the nonsteroidal anti-inflammatory drugs (NSAIDs). A cDNA (2.1 kilobases) for 3 alpha-HSD was cloned from a rat liver cDNA expression library in lambda gt11. Portions of the cDNA insert which contained an internal EcoRI site were subcloned into pGEM3, and dideoxysequencing revealed that the cDNA contains an open reading frame of 966 nucleotides which encode a protein of 322 amino acids with a monomer Mr of 37,029. The identity of this clone was confirmed by locating two tryptic peptides and two endoproteinase Lys-C peptides from purified 3 alpha-HSD within the nucleotide sequence. The amino acid sequence of rat liver 3 alpha-HSD bears no significant homology with 3 beta-, 17 beta- or 11 beta-hydroxysteroid dehydrogenases but has striking homology with bovine lung prostaglandin F synthase (69% homology at the amino acid level and 74% homology at the nucleotide level) which is a member of the aldehyde/aldose reductase family. This sequence homology supports previous correlates which suggest that in rat 3 alpha-HSD may represent an important target for NSAIDs. The nucleotide sequence also contains three peptides that have been identified by affinity labeling with either 3 alpha-bromoacetoxyandrosterone (substrate analog) or 11 alpha-bromoacetoxyprogesterone (glucocorticoid analog) to comprise the active site (see accompanying article (Penning, T. M., Abrams, W. R., and Pawlowski, J. E. (1991) J. Biol. Chem. 266, 8826-8834]. The sequence data presented suggests that 3 alpha-HSD, prostaglandin F synthase, and aldehyde/aldose reductases are members of a common gene family.     

Development of affinity labeling agents based on nonsteroidal anti-inflammatory drugs: labeling of the nonsteroidal anti-inflammatory drug binding site of 3 alpha-hydroxysteroid dehydrogenase.

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their effect by inhibiting the target enzyme cyclooxygenase (prostaglandin H2 synthase); however, little is known about the peptides comprising its NSAID binding site. Hydroxyprostaglandin dehydrogenases also bind NSAIDs, but their NSAID binding sites have not been well characterized. Using existing synthetic strategies, we have incorporated the bromoacetoxy affinity labeling moiety around the perimeter of two potent NSAIDs, indomethacin and mefenamate, a N-phenylanthranilate. The compounds synthesized were 1-(4-(bromoacetamido)benzyl)-5-methoxy-2-methylindole-3-acetic acid (1), 3-(2-(2-bromoacetoxy)ethyl)-1-(4-chlorobenzyl)-5-methoxy-2-methylindole (2), 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (3), N-(3-(bromoacetamido)phenyl)-anthranilic acid (4), and N-(4-(bromoacetamido)phenyl)anthranilic acid (5). To access whether these compounds have general utility in labeling NSAID binding sites, the compounds were evaluated as affinity labeling agents for 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from rat liver cytosol. This enzyme displays 9-, 11-, and 15-hydroxyprostaglandin dehydrogenase activity, is inhibited potently by NSAIDs, and is homologous to bovine lung prostaglandin F synthase. Compounds 1-5 were shown to affinity label the NSAID binding site of 3 alpha-HSD. They inactivated 3 alpha-HSD through an E.I complex in a time- and concentration-dependent manner with t1/2 values ranging from seconds to hours. Ligands that compete for the active site of 3 alpha-HSD (NAD+ and indomethacin) afforded protection against inactivation, and the inactivators could demonstrate competitive kinetics against 3 alpha-hydroxysteroid substrates by forming an E.NAD+.I complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of human hepatic 3 alpha-hydroxysteroid dehydrogenase.

3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) was purified greater than 500-fold from human liver cytosol. The purification was monitored using 5 beta-[3H]dihydrocortisol (5 beta-DHF) as substrate. Electrophoretically homogeneous enzyme was obtained using a procedure that involved ammonium sulfate precipitation and three successive column chromatography steps: DEAE-cellulose, hydroxylapatite and Blue-Sepharose. The enzyme is a monomer since the native molecular weight was found to be 37,000, using a calibrated Sephadex G-75 column, and the denatured subunit molecular weight was determined to be 38,500, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The enzyme had a pI of 5.6-5.9. The 3-ketosteroids: cortisol, testosterone, progesterone and androstenedione, were not substrates for 3 alpha-HSD indicating that a saturated 4,5 double bond was required for substrate activity. The conformation at the 5 position, however, did not influence substrate activity since 5 alpha- and 5 beta-DHF and 5 alpha-dihydrotestosterone were all reduced at similar rates. The purified enzyme preferred NADPH to NADH as a cofactor and showed a broad peak of activity in the pH range of 6.8-7.4. The apparent Km for 5 beta-DHF was 18 microM. The enzyme was markedly stabilized by 50 mM phosphate buffer containing 10 to 20% glycerol at 4 degrees C. Freezing and thawing of the enzyme resulted in a large loss of activity during early stages of the purification. This is the first report of the purification to homogeneity of 3 alpha-HSD from human tissue.     

Inhibitory effects of diesel exhaust components and flavonoids on 20alpha-hydroxysteroid dehydrogenase activity in mouse tissues

The inhibitory effects of diesel exhaust components and flavonoids on 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity were examined in cytosolic fractions from the liver, kidney and lung of male mice. 9,10-Phenanthrenequinone (9,10-PQ) and 1,2-naphthoquinone (1,2-NQ), which are contained in diesel exhaust particles (DEPs), potently inhibited 20alpha-HSD activity in liver cytosol. 9,10-PQ also inhibited the enzyme activity in lung cytosol. However, 20alpha-HSD activity in kidney cytosol was little inhibited by 9,10-PQ or 1,2-NQ. Flavonoids such as quercetin, fisetin and kaempferol exhibited high inhibitory potencies for 20alpha-HSD activity in liver cytosol, whereas these flavonoids were poor inhibitors for the enzyme activity in kidney cytosol. It is likely that several diesel exhaust components and flavonoids augment the signaling of progesterone in the liver cells, by potently inhibiting 20alpha-HSD activity in mouse liver cytosol. The possibility that there are distinct enzymes catalyzing 20alpha-HSD activity in the non-reproductive tissues of male mice is also discussed.     

Functional expression, purification, and characterization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) catalyzes the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralization of the steroid nucleus to CO(2) and H(2)O in Comamonas testosteroni. By this activity, 3alpha-HSD provides the basis for C. testosteroni to grow on steroids as sole carbon and energy source. 3alpha-HSD was cloned and overexpressed in E. coli and purified to homogeneity by an affinity chromatography system as His-tagged protein. The recombinant enzyme was found to be functional as oxidoreductase toward a variety of steroid substrates, including androstanedione, 5alpha-dihydrotestosterone, androsterone, cholic acid, and the steroid antibiotic fusidic acid. The enzyme also catalyzes the carbonyl reduction of nonsteroidal aldehydes and ketones such as metyrapone, p-nitrobenzaldehyde and a novel insecticide (NKI 42255), and, based on this pluripotent substrate specificity, was named 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR). It is suggested that 3alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. Antibodies were generated in rabbits against the entire 3alpha-HSD/CR protein, and may now be used for evaluating the pattern of steroid induction in C. testosteroni on the protein level. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein revealing for the first time the dimeric nature of 3alpha-HSD/CR of C. testosteroni.     

Characteristics and inhibition by flavonoids of 20alpha-hydroxysteroid dehydrogenase activity in mouse tissues

Progesterone was stereoselectively reduced to a metabolite 20alpha-hydroxy-4-pregnen-3-one in the cytosolic fraction from the liver of male mice, indicating that the reduction of progesterone is catalyzed by 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). The cytosolic 20alpha-HSD activity was observed not only in the liver, but also in the kidney and lung. In liver cytosol, both NADPH and NADH were effective as cofactors for 20alpha-HSD activity, although NADPH was better than NADH for the enzyme activity. On the other hand, 20alpha-HSD activity in kidney cytosol required only NADPH as a cofactor. No significant sex-related difference of 20alpha-HSD activity was observed in liver and kidney cytosols. Flavonoids have been reported to inhibit the biosynthesis and metabolism of steroids. However, little is known about inhibitory effects of flavonoids on 20alpha-HSD activity. Thus, the effects of 16 flavonoids on 20alpha-HSD activity were examined, using liver cytosol of male mice. Among flavonoids tested, fisetin, apigenin, naringenin, luteolin, quercetin and kaempferol exhibited high inhibitory potencies for the 20alpha-HSD activity. We propose the possibility that these flavonoids augment progesterone signaling by inhibiting potently 20alpha-HSD activity in non-reproductive tissues.     

NAD+-15-hydroxyprostaglandin dehydrogenase distribution in rat kidney.

Rat kidney NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was measured in zones and substructure of the rat kidney nephron. This was accomplished utilizing an assay procedure based upon determining the amount of prostaglandin E1 present before and after the reaction with the 15-hydroxyprostaglandin dehydrogenase contained in the tissue sample. The enzyme activity was assayed in freeze dried, quick frozen rat kidney sections and its distribution within the rat kidney was determined. In kidney zones, it was localized to medullary rays and inner cortex. In kidney substructure, activity was highest in collecting tubule, pars recti tubule, distal convoluted tubule and the ascending limb of Henle (14.2, 11.5, 6.4 and 9.2 mM kg-1hr-1, respectively). Activity in glomeruli, proximal convoluted tubule and small arteries was lower (2.1, 2.8 and 2.1 mM kg-1hr-1, respectively). The assay procedure was verified by established assays (spectrophotometric, fluorometric and radiometric TLC) which are often used in homogenate and purified PGDH preparations.     

Inhibition of 15-hydroxyprostaglandin dehydrogenase by papaverine.

Papaverine was found to inhibit NAD⁺-linked 15-hydroxyprostaglandin dehydrogenase partially purified from guinea pig lung. The inhibition was noncompetitive with prostaglandin E₂, uncompetitive with NAD⁺, and reversible. The Ki was calculated to be 26 μM. Papaverine also inhibited the enzyme from swine lung, chicken and dog heart, and rat and dog kidney. The inhibitory effects of papaverine on the 15-hydroxyprostaglandin dehydrogenase were compared with those on cyclic AMP phosphodiesterases in these tissues.     

Purification and characterization of rat ovarian 20 alpha-hydroxysteroid dehydrogenase

To investigate the regulatory mechanism of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) (EC 1.1.1.149) activity in ovarian tissue, the enzyme was purified from ovaries of normal mature female rats. Column chromatography of the cytosolic fraction from ovaries on DEAE-Toyopearl 650M revealed two peaks of the 20 alpha-HSD activity at different ionic strengths. These peaks were designated HSD1 and HSD2, respectively. Each of the active fractions was further purified to homogeneity by dye-affinity chromatography using Matrex Green A and AF Red-Toyopearl. Both the fractions appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (at Mr = 33,000 under reducing conditions). Under non-reducing conditions, similar values were obtained on gel-exclusion HPLC, indicating that the enzyme fractions were single-stranded, monomeric polypeptides. Homogeneous HSD1 and HSD2 were purified 361-fold and 509-fold, respectively, and differed in their substrate preference. The two enzyme fractions had Km values of 4.75 microM and 5.16 microM for 20 alpha-dihydroprogesterone, respectively, and showed almost the same RF values on reverse-phase HPLC and free-zone capillary electrophoresis. However, amino acid composition was slightly different, i.e. lysin content was higher in HSD1 than HSD2. Thus, it was clarified that two types of 20 alpha-HSD with very similar molecular structures are present in the rat ovary.     

9-Hydroxyprostaglandin dehydrogenase in rat kidney cortex converts prostaglandin I2 into 15-keto-13,14-dihydro 6-ketoprostaglandin E1

15-Keto-13,14-dihydro 6-ketoprostaglandin E1 was positively identified by gas chromatography-mass spectrometry with negative-ion chemical ionisation detection from samples of rat kidney high-speed supernatant incubated with prostaglandin I2 in the presence of NAD+. A decreased formation of this product was observed when NAD+ was substituted with NADP+ and none was observed in the absence of nucleotide or substrate prostaglandin I2. Experiments with [9 beta-3H]prostaglandin I2 showed a time- and concentration-dependent loss of tritium which appeared as tritiated water, typical of reaction of [9 beta-3H]prostaglandin substrates with the enzyme, 9-hydroxyprostaglandin dehydrogenase. Time-course measurements of the appearance of tritiated water showed similar rates with 6-keto[9 beta-3H]prostaglandin F1 alpha and 15-keto-13,14-dihydro 6-keto[9 beta-3H]prostaglandin F1 alpha as substrates. These experiments suggest that the transformation of prostaglandin I2 and 6-ketoprostaglandin F1 alpha into the 15-keto-13,14-dihydro 6-ketoprostaglandin E1 catabolite occurs in this in vitro preparation via the corresponding 15-keto-13,14-dihydro catabolite of 6-ketoprostaglandin F1 alpha.     

3 alpha-hydroxysteroid dehydrogenase activity catalyzed by purified pig adrenal 20 alpha-hydroxysteroid dehydrogenase.

In earlier studies, two distinct molecules, 20 alpha-HSD-I and 20 alpha-HSD-II, responsible for 20 alpha-HSD activity of pig adrenal cytosol were purified to homogeneity and characterized [S. Nakajin et al., J. Steroid Biochem. 33 (1989) 1181-1189]. We report here that the purified 20 alpha-HSD-I, which mainly catalyzes the reduction of 17 alpha-hydroxyprogesterone to 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, catalyzes 3 alpha-hydroxysteroid oxidoreductase activity for 5 alpha (or 5 beta)-androstanes (C19), 5 alpha (or 5 beta)-pregnanes (C21) in the presence of NADPH as the preferred cofactor. The purified enzyme has a preference for the 5 alpha (or 5 beta)-androstane substrates rather than 5 alpha (or 5 beta)-pregnane substrates, and the 5 beta-isomers rather than 5 alpha-isomers, respectively. Kinetic constants in the reduction for 5 alpha-androstanedione (Km; 3.3 microM, Vmax; 69.7 nmol/min/mg) and 5 beta-androstanedione (Km; 7.7 microM, Vmax; 135.7 nmol/min/mg) were demonstrated for comparison with those for 17 alpha-hydroxyprogesterone (Km; 26.2 microM, Vmax; 1.3 nmol/min/mg) which is a substrate for 20 alpha-HSD activity. Regarding oxidation, the apparent Km and Vmax values for 3 alpha-hydroxy-5 alpha-androstan-17-one were 1.7 microM and 43.2 nmol/min/mg, and 1.2 microM and 32.1 nmol/min/mg for 3 alpha-hydroxy-5 beta-androstan-17-one, respectively. 20 alpha-HSD activity in the reduction of 17 alpha-hydroxyprogesterone catalyzed by the purified enzyme was inhibited competitively by addition of 5 alpha-DHT with a Ki value of 2.0 microM. Furthermore, 17 alpha-hydroxyprogesterone inhibited competitively 3 alpha-HSD activity with a Ki value of 150 microM.     

Rat liver 3 alpha-hydroxysteroid dehydrogenase

3 alpha-HSD appears to be a multifunctional enzyme. In addition to its traditional role of catalyzing early steps in androgen metabolism, it will also oxidoreduce prostaglandins and detoxify trans-dihydrodiols (proximate carcinogens). Since these novel reactions have been quantified using homogeneous enzyme it is necessary to interpret the role of the enzyme in these processes in vivo with some caution. However, it is rare that such observations on a purified hydroxysteroid dehydrogenase have led to such important questions. Is the 3 alpha-HSD the only steroid dehydrogenase that transforms prostaglandins and trans-dihydrodiols? Are hydroxysteroid dehydrogenases and prostaglandin dehydrogenases the same enzymes in certain tissues? Does 3 alpha-HSD protect against chemical carcinogenesis in vivo? The inhibition of the purified dehydrogenase by therapeutically relevant concentrations of anti-inflammatory drugs also deserves comment. Is this hydroxysteroid dehydrogenase really an in vivo target for anti-inflammatory drug action? Could these drugs exert some of their pharmacological effect either by preventing glucocorticoid metabolism in some tissues or by preventing the transformation of PGF2 alpha (non-inflammatory prostanoid) to PGE2 (a pro-inflammatory prostanoid)? Could these drugs, by inhibiting trans-dihydrodiol oxidation, potentiate the initiation of chemical carcinogenesis? These and other important questions can be answered only by developing specific inhibitors for the dehydrogenase to decipher its function in vivo.     

Molecular cloning of a novel type of rat cytoplasmic 17beta-hydroxysteroid dehydrogenase distinct from the type 5 isozyme

Rat liver contains two cytosolic enzymes (TBER1 and TBER2) that reduce 6-tert-butyl-2,3-epoxy-5-cyclohexene-1,4-dione into its 4R- and 4S-hydroxy metabolites. In this study, we cloned the cDNA for TBER1 and examined endogenous substrates using the homogenous recombinant enzyme. The cDNA encoded a protein composed of 323 amino acids belonging to the aldo-keto reductase family. The recombinant TBER1 efficiently oxidized 17beta-hydroxysteroids and xenobiotic alicyclic alcohols using NAD+ as the preferred coenzyme at pH 7.4, and showed low activity towards 20alpha- and 3alpha-hydroxysteroids, and 9-hydroxyprostaglandins. The enzyme was potently inhibited by diethylstilbestrol, hexestrol and zearalenone. The coenzyme specificity, broad substrate specificity and inhibitor sensitivity of the enzyme differed from those of rat NADPH-dependent 17beta-hydroxysteroid dehydrogenase type 5, which was cloned from the liver and characterized using the recombinant enzyme. The mRNA for TBER1 was highly expressed in rat liver, gastrointestinal tract and ovary, in contrast to specific expression of 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the liver and kidney. Thus, TBER1 represents a novel type of 17beta-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. In addition, TBER2 was identified as 3alpha-hydroxysteroid dehydrogenase on chromatographic analysis of the enzyme activities in rat liver cytosol and characterization of the recombinant 3alpha-hydroxysteroid dehydrogenase.     

12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) is an excellent substrate for NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was found to be an excellent substrate for NAD+ dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney. Kcat/Km value of HHT was comparable to that of prostaglandin E although HHT is not a prostanoic acid derivative. Product of enzyme catalyzed oxidation of HHT was identified as 12-keto-5,8,10-heptadecatrienoic acid by gas chromatography-mass spectrometry. The fact that HHT is an excellent substrate for 15-hydroxyprostaglandin dehydrogenase suggest that HHT may have profound unrecognized biological actions and its inactivation may be via oxidation of the hydroxyl group.     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

Prostaglandin metabolism. II. Identification of two 15-hydroxyprostaglandin dehydrogenase types.

Homogenates of several mammalian tissues were measured by radioimmunoassay for 15-hydroxyprostaglandin dehydrogenase activity. Two types of enzyme activity were detected. One, which used NAD-plus as cofactor much more effectively than NADP-lus, was found in monkey lung, heart, liver, kidney, and spleen and in chicken heart and dog lung. A second type, which uses NADP-plus as a cofactor more effectively than NAD-plus, was found in monkey and human brain and red blood cells and in swine kidney. These two types of 15-hydroxyprostaglandin dehydrogenase were partially purified from monkey brain and chicken heart. In addition to different cofactor requirements, the two partially purified enzymes could be distinguished by chromatographic properties, their relative affinities for prostaglandin I2 and F2alpha, and their sensitivities to inhibition by reduced pyridine nucleotides, thyroid hormones, and prostaglandin B2.     

Nuclear 3alpha-hydroxysteroid dehydrogenase (3alphaOHD) activity for 5alpha-dihydrotestosterone in the rat prostate.

The in vitro examination of adult male rat prostatic 3alpha-hydroxysteroid dehydrogenase (3alphaOHD) activity using 5alpha-dihydrotestosterone4 as substrate indicates that significant levels of enzyme activity are associated with purified nuclei as well as with the cytosol fractions. Both the purified nuclear and the cytosol fractions exhibited higher levels of 3alphaOHD activity with NADH than with NADPH. The pH activity curves for the NADH and NADPH catalyzed reactions were different for both the nuclear and cytosol fractions. The results suggest the presence of a number of 3alphaOHD enzymes in rat prostate.