20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: purification and some properties

20 beta-Hydroxysteroid dehydrogenase was purified from a cytosol fraction of neonatal pig testes to homogeneity as demonstrated by polyacrylamide gel electrophoresis (PAGE) and by isoelectric focusing. The molecular weight was estimated to be 30,500 using PAGE with sodium dodecyl sulfate and the gel filtration method. Molecular estimations showed that the purified enzyme consisted of a single polypeptide chain. It catalyzed the reduction of 17 alpha-hydroxyprogesterone to 17 alpha,20 beta-dihydroxypregn-4-en-3-one with NADPH. Furthermore, the C21-steroids, such as progesterone, pregnenolone, 17 alpha-hydroxypregnenolone, deoxycorticosterone, and deoxycortisol were also reduced by the purified enzyme. Apparent Km values for 17 alpha-hydroxyprogesterone, progesterone, pregnenolone, and deoxycorticosterone were 9.4, 1.5, 4.0, and 8.6 microM, respectively. The enzyme did not show 20 alpha-hydroxysteroid dehydrogenase activity. The maximum rate of enzyme activity was observed at 45 degrees C and optimum pH was at pH 5.5. The enzyme activity was strongly inhibited by heavy metal ions such as Hg2+ and Cu2+.     

Cytochrome P-450(17 alpha,lyase)-mediating pathway of androgen synthesis in bovine adrenocortical cultured cells.

Cytochrome P-450(17 alpha,lyase) mediating pathway of dehydroepiandrosterone (DHA) formation from pregnenolone was investigated in primary cultures of bovine adrenocortical fasciculata-reticularis cells. To determine whether DHA formation proceeds predominantly by successive monooxygenase reactions without 17 alpha-hydroxypregnenolone leaving P-450(17 alpha,lyase) the cells were incubated with [14C]pregnenolone and 17 alpha-[3H]hydroxypregnenolone in the presence of Trilostane. Results of the double-substrate double-label experiments indicate that in the presence of high concentration of pregnenolone most of DHA was formed, directly from pregnenolone by the successive reactions. Since the concentration of pregnenolone usually exceeds that of 17 alpha-hydroxypregnenolone in the adrenal glands, DHA is concluded to be formed predominantly by successive reactions from pregnenolone without 17 alpha-hydroxypregnenolone leaving P-450(17 alpha,lyase) in vivo. By chronic ACTH treatment, the activities of 17 alpha-hydroxylation and DHA formation in adrenocortical cultured cells became higher concomitantly with the increase of P-450(17 alpha,lyase) content. Most of DHA was found to be formed by successive reactions from pregnenolone even under such conditions.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.     

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in canine pancreas

Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with steroid-delta 5-4-isomerase was demonstrated for the first time in the pancreas. The enzyme complex was assayed by measuring the conversion of pregnenolone to progesterone as well as of dehydroepiandrosterone to androstenedione and found to be localized primarily in the mitochondrial fraction of dog pancreas homogenates. The delta 5-3 beta-hydroxysteroid dehydrogenase used either NAD+ or NADP+ as co-substrates, although maximal activity was observed with NAD+. In phosphate buffer, pH 7.0 and 37 degrees C, the apparent Km values of the dehydrogenase were 6.54 +/- 0.7 microM for pregnenolone and 9.61 +/- 0.8 microM for NAD+. The apparent Vmax was determined as 0.82 +/- 0.02 nmol min-1 mg-1. Under the same conditions the Km values for dehydroepiandrosterone and NAD+ were 3.3 +/- 0.2 microM and 9.63 +/- 1.6 microM, respectively, and the apparent Vmax was 0.62 +/- 0.01 nmol min-1 mg-1.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Study of the metabolism of steroids in larvae of the fleshfly Sarcophaga bullata

1. Larvae of the fleshfly Sarcophaga bullata were injected with several 3H C21 and C19 steroids. After different incubation times, the larvae were homogenized and the metabolites were extracted and fractionated by Sephadex LH 20-, paper- and thin-layer chromatography. The chromatographic mobility of the labeled zones was compared with that of standard steroids. 2. Progesterone and 17 alpha-hydroxypregnenolone were metabolized to 17 alpha-hydroxyprogesterone. Androstenedione, 17 alpha-hydroxyprogesterone and dehydroepiandrosterone were converted to testosterone. Transformation of pregnenolone to progesterone or 17 alpha-hydroxypregnenolone was not observed. 3. C21 or C19 steroid formation from cholesterol could not be demonstrated. 4. Sixteen metabolites, different from all our standard substances have been found. Their structure remains to be elucidated.     

Regulation of human placental progesterone synthesis in vitro by naturally occurring steroids

A regulatory model of human placental progesterone synthesis is based on studies with isolated placental enzymes. Steroids causing a dose-dependent inhibition are listed in the standing order of their inhibitory potency (I50 (microM)/Ki value (microM)/type of inhibition: c = competitive and nc = non competitive). Cholesterol side chain cleavage enzyme (mitochondria): Mainly regulated by hydroxylated cholesterol derivates. No inhibition was observed by cholesterylesters and by other naturally occurring steroids tested. 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (mitochondria): 6 beta-hydroxyprogesterone (nc), dehydroepiandrosterone (0.32/0.82/c), 20 alpha-dihydroprogesterone (0.38/-/nc), progesterone (0.46/-), estrone (0.56/0.1/c), estradiol (0.1/0.8/c), 17 alpha-hydroxyprogesterone (2.1/-/nc), 17 alpha-hydroxypregnenolone (0.4/-/c), dehydroepiandrosterone sulfate (2.5/-/c), cortisone (5.0/-), cortisol (100/-). 20 alpha-hydroxysteroid dehydrogenase (cytoplasmic): estrone (0.26/0.7/c), estradiol (0.28/0.9/c), pregnenolone (4.4/9.2/c), 5 alpha-pregnan-3 beta-ol-20-one (4.6/-/nc), estriol (5.1/11.5/c); dehydroepiandrosterone (7.2/14.0/c), 5 alpha-dihydrotestosterone (26.0/-/nc), progesterone (33.0/48.0/c), dehydroepiandrosterone sulfate (50.0/23.0/nc), and testosterone (59.0/63.0/c). An autoregulatory mechanism of placental progesterone synthesis is postulated which is in good agreement with data published by others proving that placental progesterone synthesis is independent of the endocrine organs of the mother and the fetus.     

Inhibition of rat ovarian 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase and 17,20 lyase by progestins and danazol

The site of action of synthetic progestins or danazol in the treatment of endometriosis is considered to be mainly the hypothalamo-pituitary level, but the direct action to the uterine endometrium and the ovary is also suggested. We investigated the effect of these synthetic steroids to rat ovarian steroidogenic enzymes. The effect of norethisterone, levonorgestrel, danazol, gestrinone, desogestrel and 3-keto-desogestrel was studied in vitro. The sources of the enzymes were prepared from ovaries of immature rats treated either with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) for 3 beta-hydroxy steroid dehydrogenase (3 beta-HSD), or with PMS for 17 alpha-hydroxylase and 17,20 lyase. The substrates used were pregnenolone (P5) for 3 beta-HSD, progesterone (P4) for 17 alpha-hydroxylase, and 17 alpha-hydroxy-progesterone (17 alpha-OH-P4) for 17,20 lyase. The substrates were incubated with the enzyme sources and coenzymes, and the products formed were measured. All the steroids inhibited 3 beta-HSD, and the inhibition by gestrinone (Ki = 3.0 microM) and 3-keto-desogestrel (17.5 microM) was particularly marked. Only desogestrel (Ki = 30.3 microM) and danazol (168 microM) inhibited 17 alpha-hydroxylase. All the steroids inhibited 17,20 lyase, and the inhibition by desogestrel (Ki = 0.70 microM), danazol (0.80 microM), and gestrinone (30 microM) was particularly marked.     

Inhibition by alloxan of mitochondrial aconitase and other enzymes associated with the citric acid cycle

Considerable variations were found in the in vitro effect of alloxan on mouse liver enzymes associated with the citric acid cycle. The following approximative alloxan concentrations induced 50% inhibition of enzyme activity: 10(-6)M for aconitase, 10(-4)M for NAD-linked isocitrate dehydrogenase, glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase and fumarase, and 10(-3)M for citrate synthase and NADP-linked isocitrate dehydrogenase. Pyruvate dehydrogenase, succinate dehydrogenase and malate dehydrogenase were not inhibited by 10(-3)M alloxan. The inhibition of aconitase was competitive both when using mouse liver and purified porcine heart enzyme. The Ki values for the purified enzyme in the presence of 5 microM alloxan were 0.22 microM with citrate, 4.0 microM with cis-aconitate and 0.62 microM with isocitrate as substrate. The high sensitivity of aconitase for inhibition by alloxan probably plays a prominent role for the toxic effects of alloxan.     

Partial purification and some kinetic properties of glucose-6-phosphate dehydrogenase from Phycomyces blakesleeanus

Glucose-6-phosphate dehydrogenase from sporangiophores of Phycomyces blakesleeanus NRRL 1555 (-) was partially purified. The enzyme showed a molecular weight of 85 700 as determined by gel-filtration. NADP+ protected the enzyme from inactivation. Magnesium ions did not affect the enzyme activity. Glucose-6-phosphate dehydrogenase was specific for NADP+ as coenzyme. The reaction rates were hyperbolic functions of substrate and coenzyme concentrations. The Km values for NADP+ and glucose 6-phosphate were 39.8 and 154.4 microM, respectively. The kinetic patterns, with respect to coenzyme and substrate, indicated a sequential mechanism. NADPH was a competitive inhibitor with respect to NADP+ (Ki = 45.5 microM) and a non-competitive inhibitor with respect to glucose 6-phosphate. ATP inhibited the activity of glucose-6-phosphate dehydrogenase. The inhibition was of the linear-mixed type with respect to NADP+, the dissociation constant of the enzyme-ATP complex being 2.6 mM, and the enzyme-NADP+-ATP dissociation constant 12.8 mM.     

Soybean nodule xanthine dehydrogenase: a kinetic study

Xanthine dehydrogenase was purified from soybean nodules and the kinetic properties were studied at pH 7.5. Km values of 5.0 +/- 0.6 and 12.5 +/- 2.5 microM were obtained for xanthine and NAD+, respectively. The pattern of substrate dependence suggested a Ping-Pong mechanism. Reaction with hypoxanthine gave Km's of 52 +/- 3 and 20 +/- 2.5 microM for hypoxanthine and NAD+, respectively. The Vmax for this reaction was twice that for the xanthine-dependent reaction. The pH dependence of Vmax gave a pKa of 7.6 +/- 0.1 for either xanthine or hypoxanthine oxidation. In addition the Km for xanthine had a pKa of 7.5 consistent with the protonated form of xanthine being the true substrate. Km for hypoxanthine varied only 2.5-fold between pH 6 and 10.7. Product inhibition studies were carried out with urate and NADH. Both products gave mixed inhibition with respect to both substrates. Xanthine dehydrogenase was able to use APAD+ as an electron acceptor for xanthine oxidation, with a Km at pH 7.5 of 21.2 +/- 2.5 microM and Vmax the same as that obtained with NAD+. Reduction of APAD+ by NADH was also catalyzed by xanthine dehydrogenase with a Km of 102 +/- 15 microM; Vmax was approximately 2.5 times that for the xanthine-dependent reaction, and was independent of pH between 6 and 9. Reaction with group-specific reagents indicated the possibility of an essential histidyl group. A thiol-modifying reagent did not cause inactivation of the enzyme. A role for the histidyl side chain in catalysis is proposed.     

Substrate and nucleotide specificity of placental microsomal 3 beta-hydroxysteroid dehydrogenase

Recent kinetic studies on the placental microsomal 3 beta-hydroxysteroid dehydrogenase have shown that apparent Km values for 3 beta-hydroxy-5-androsten-17-one (dehydroepiandrosterone) and 3 beta-hydroxy-5-pregnen-20-one (pregnenolone) are 15nM and 40nM respectively, which are orders of magnitude lower than found in earlier studies. The purpose of this study was to investigate the substrate and nucleotide specificity of the 3 beta-hydroxysteroid dehydrogenase, and the ability of various steroids to inhibit the reaction at these lower steroid concentrations. Each steroid inhibited the metabolism of the other competitively, and the Ki values obtained were not significantly different from their respective Km values. The ability of various steroids to inhibit the reaction at concentrations of 100nM was usually less than that found at micromolar concentrations. However, certain steroids showed marked inhibition. For example, estrone and estradiol-17 beta inhibit the oxidation of both substrates competitively with Ki values of between 15 and 24nM. The Km values of dehydroepiandrosterone and pregnenolone with NADP+ as cofactor are higher than those with NAD+ as cofactor and the V values are much lower. These data indicate that in human placental microsomes a single 3 beta-hydroxysteroid dehydrogenase, essentially NAD+ specific, metabolizes dehydroepiandrosterone and pregnenolone.     

Formaldehyde dehydrogenase from Pseudomonas putida. Purification and some properties

Formaldehyde dehydrogenase was isolated and purified in an overall yield of 12% from cell-free extract of Pseudomonas putida C-83 by chromatographies on columns of DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite. The purified enzyme was homogeneous as judged by disc gel electrophoresis and was most active at pH 7.8 using formaldehyde as a substrate. The enzyme was also active toward acetaldehyde, propionaldehyde, glyoxal, and pyruvaldehyde, though the reaction rates were low. The enzyme was NAD+-linked but did not require the external addition of glutathione, in contrast with the usual formaldehyde dehydrogenase from liver mitochondria, baker's yeast, and some bacteria. The enzyme was markedly inhibited by Ni2+, Pd2+, Hg2+, p-chloromercuribenzoate, and phenylmethanesulfonyl fluoride. The molecular weight of the enzyme was estimated to be 150,000 by the gel filtration method, and analysis by SDS-polyacrylamide gel electrophoresis indicated that the enzyme was composed of two subunit monomers. Kinetic analysis gave Km values of 67 microM for formaldehyde and 56 microM for NAD+, and suggested that the reaction proceeds by a "Ping-pong" mechanism. The enzyme catalyzed the oxidation of formaldehyde accompanied by the stoichiometric reduction of NAD+, but no reverse reaction was observed.     

Stereospecific removal of the 16 alpha-hydrogen in the biosynthesis of 5,16-androstadien-3 beta-ol from pregnenolone

[16 alpha-2H]Pregnenolone was synthesized by catalytic deuteriation of 3 beta-hydroxy-5,16-pregnadien-20-one followed by base-catalyzed back exchange of the 17 alpha-2H atom, and [16 beta-2H]pregnenolone by catalytic hydrogenation of 3 beta-hydroxy-5,16-[16-2H]pregnadien-20-one, which had been synthesized from [16,16-2H]dehydroepiandrosterone. The labelled pregnenolones were incubated separately with the microsomal fraction of boar testis. The metabolites were analyzed by gas chromatography-mass spectrometry, and the isotope compositions of the following six metabolites were determined: 17-hydroxypregnenolone, dehydroepiandrosterone, 5-androstene-3 beta,17 alpha-diol, 5-androstene-3 beta,17 beta-diol,16 alpha-hydroxypregnenolone and 5,16-androstadien-3 beta-ol. The first four metabolites derived either from [16 alpha-2H]- or from [16 beta-2H]pregnenolone showed essentially the same isotope compositions as those of their respective precursors. The 16 alpha-hydroxypregnenolone and the 5,16-androstadien-3 beta-ol biosynthesized from [16 alpha-2H]pregnenolone lost the 2H label, while the same metabolites biosynthesized from [16 beta-2H]pregnenolone retained the albel. The result shows that the 16 alpha-hydrogen is stereospecifically removed with the retention of the 16 beta-hydrogen in the biosynthesis of 5,16-androstadien-3 beta-ol.     

Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase

The inhibitory mechanisms of ribavirin 5'-monophosphate (RMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites ribavirin and tiazofurin, were investigated in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. The hepatoma IMP dehydrogenase has a tetrameric structure with a subunit molecular weight of 60,000. For the substrates IMP and NAD+, Km's were 23 and 65 microM, respectively. Product-inhibition patterns showed an ordered Bi-Bi mechanism for the enzyme reaction where IMP binds to the enzyme first, followed by NAD+; NADH dissociates from the ternary complex first and then XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme-XMP complex. RMP exerted the same inhibitory mechanisms as XMP, and the inhibition by TAD was similar to that by NADH. However, the Ki values for RMP (0.8 microM) and TAD (0.13 microM) were orders of magnitude lower than those of XMP (136 microM) and NADH (210 microM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural substrates and products, RMP with the IMP-XMP site and TAD with the NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from this inactivation by IMP or XMP. These results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin targeted against the two different ligand sites of IMP dehydrogenase.     

Pyruvate dehydrogenase complex of ascites tumour. Activation by AMP and other properties of potential significance in metabolic regulation.

1. AMP is an activator of the pyruvate dehydrogenase complex of the Ehrlich--Lettré ascites tumour, increasing its V up to 2-fold, with Ka of 40 microM at pH 7.4. This activation appears to be an allosteric effect on the decarboxylase subunit of the complex. 2. The pyruvate dehydrogenase complex has a Km for pyruvate within the range 17--36 microM depending on the pH, the optimum pH being approx. 7.4, with a V of approx. 0.1 unit/g of cells. The rate-limiting step is dependent on the transformation of the enzyme--substrate complex. The Km for CoA is 15 microM. The Km for NAD+ is 0.7 mM for both the complex and the lipoamide dehydrogenase. The complex is inhibited by acetyl-CoA competitively with CoA; the Ki is 60 microM. The lipoamide dehydrogenase is inhibited by NADH and NADPH competitively with NAD+, with Ki values of 80 and 90 microM respectively. In the reverse reaction the Km values for NADH and NADPH are essentially equal to their Ki values for the forward reaction, the V for the latter being 0.09 of that of the former. Hence the reaction rate of the complex in vivo is likely to be markedly affected by feedback isosteric inhibition by reduced nicotinamide nucleotides and possibly acetyl-CoA.     

Purification and properties of 16 alpha-hydroxyprogesterone dehydroxylase from Eubacterium sp. strain 144

Eubacterium sp. strain 144 converts 16 alpha-hydroxyprogesterone to 17-isoprogesterone. The first step of this reaction is catalyzed by 16 alpha-hydroxyprogesterone dehydroxylase (16 alpha-dehydroxylase). This enzyme was purified 40-70-fold and characterized. 16 alpha-Dehydroxylase was found to be active in two molecular weight forms of Mr 181 000 and 326 000. A subunit relative molecular weight of 42 400 was determined by sodium dodecyl sulfate gel electrophoresis of the purified enzyme. Although active with both 16 alpha-hydroxyprogesterone and 16 alpha-hydroxypregnenolone, the affinity of 16 alpha-dehydroxylase for the latter steroid was twice that of the former based on the apparent Km values. Evidence of possible substrate inhibition at high concentrations was seen with 16 alpha-hydroxypregnenolone. 16-Ketoprogesterone was found to be a competitive inhibitor of 16 alpha-dehydroxylase with respect to both steroid substrates. Although generally unaffected by low concentrations of non-ionic detergents, 16 alpha-dehydroxylase activity was stimulated 3-7-fold by sodium dodecyl sulfate and inhibited strongly by cetyltrimethylammonium bromide.     

IMP dehydrogenase and action of antimetabolites in human cultured blast cells

Tiazofurin was demonstrated to be an effective inhibitor of the growth of human cultured blast cells, and the high specific activities of IMP dehydrogenase (EC 1.1.1.205) were observed in all the cell extracts tested. IMP dehydrogenase has been purified to homogeneity from MOLT 4F human T-lymphoblast, and the Km values for IMP and NAD were 29 and 54 microM, respectively. The inhibitory mechanisms of thiazole-4-carboxamide adenine dinucleotide (TAD) and ribavirin 5'-monophosphate (RMP), the active forms of the antimetabolites tiazofurin and ribavirin, were investigated on the purified enzyme. RMP inhibits competitively with respect to IMP as well as XMP, and the inhibition by TAD was similar to that by NADH, which was uncompetitive with NAD. However, the Ki values of RMP (0.58 microM) and TAD (0.075 microM) were several orders of magnitude lower than those of XMP (85 microM) and NADH (94 microM). Thus, the drugs interact with the two distinct sites of IMP dehydrogenase with much higher affinities than the natural substrates and products. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner, and the enhancement was further increased by the addition of TAD. The combination of tiazofurin and ribavirin exerted a synergistic effect on the growth inhibition in MOLT 4F cells.     

Purification and characterization of histidinol dehydrogenase from cabbage

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms.     

Kinetic analysis of human placental, ovarian, and adrenal 3 beta-hydroxysteroid dehydrogenase inhibition by epostane in vitro

The effect of epostane [(2 alpha,4 alpha,5 alpha,17 beta)-4,5-epoxy-17-hydroxy-4,17-dimethyl-3-oxo- androstane-2-carbonitrile] on the conversion of pregnenolone to progesterone and of dehydroepiandrosterone (DHA) to androstenedione was studied in human term placental microsomes and in comparison with human ovarian and adrenal microsomes. Using pregnenolone as substrate, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the three tissues had a similar Km (3-6 microM) but Vmax ranged from 1.3 nmol/mg protein per min in ovary to 10 nmol/mg protein per min in placenta. Epostane inhibited 3 beta-HSD activity in all three tissues with the characteristics of a pure competitive inhibitor: mean Ki values were 1.7 microM for placenta, 0.5 microM for adrenal and 0.1 microM for ovary. Moreover, in placental microsomes epostane inhibited the conversion of DHA to androstenedione with a Ki of 0.6 microM. The mechanism of action of epostane explains its effectiveness in blocking progesterone synthesis during the luteal phase and in pregnancy in women, and its strong anti-steroidogenic effect in other endocrine tissues in vitro.