Product inhibition of steroid-17 alpha-monooxygenase by endogenous 17 alpha-hydroxyprogesterone in microsomes and isolated Leydig cells from rat testis

Use of integrated rate equations for analysis of progesterone metabolism by isolated Leydig cells and microsomes from rat testis in presence of several progesterone concentrations within several periods reveals competitive product inhibition by endogenously formed 17 alpha-hydroxyprogesterone (Kpm = 0.1 microM) of steroid-17 alpha-monooxygenase activity (Ksm = 0.8 microM). The discrepancy between this very low interaction constant of endogenous 17 alpha-hydroxyprogesterone with the steroid-17 alpha-monooxygenase, and the respective values (from the literature) for exogenous 17 alpha-hydroxyprogesterone which are about 50-fold higher, may be explained by accumulation of endogenous 17 alpha-hydroxyprogesterone at the catalytic site of the steroid-17 alpha-monooxygenase. This mechanism may be important for intratesticular regulation of androgen biosynthesis from precursor steroids.     

Cofactor requirements of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities in cell extracts of Clostridium scindens

Two neutral steroid-transforming activities were demonstrated in cell extracts of Clostridium scindens. Steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase were found to be inducible in cells cultured in the presence of cortisol. Both activities required manganese ions and NAD+ or NADH for activity. Cortisol, cortisone and 11-desoxycortisol were substrates as well as inducers of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. 17 alpha-Hydroxyprogesterone was an effective inducer but did not serve as a substrate for either enzyme activity. C. scindens is the first bacterial species of the normal human intestinal flora reported to elaborate inducible steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. The results of cofactor, substrate specificity and induction studies suggest that these two activities may reside in the same enzyme complex.     

Preferential synthesis of diacyl and alkenylacyl ethanolamine and choline glycerophospholipids in rabbit platelet membranes

In rabbit platelet membranes, the contents of alkenylacyl phospholipids (plasmalogen) were 56% of phosphatidylethanolamine and 3% of phosphatidylcholine. This uneven distribution of plasmalogens in each phospholipid class could be attributed to the different substrate specificity of ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2). The properties of the enzymes were studied, using endogenous diglycerides and CDP-[3H]ethanolamine or CDP-[14C]choline as substrates. The newly formed phospholipids were mainly diacyl and alkenylacyl and only rarely alkylacyl type. The ratios of the labeled alkenylacyl to diacyl type of phospholipids clearly varied with the concentrations of CDP-ethanolamine or CDP-choline. When 1, 10, and 30 microM CDP-[3H]ethanolamine were used, the labeled phospholipids contained 53, 37, and 27% of the alkenylacyl type, respectively. The apparent Km for CDP-ethanolamine to synthesize alkenylacyl and diacyl types were 2.2 and 8.1 microM. On the other hand, when 1, 10, and 30 microM CDP-[14C]choline were used, the labeled lipids contained 10, 17, and 24% alkenylacyl type, respectively. The apparent Km for CDP-choline to synthesize alkenylacyl and diacyl types were 24 and 4.3 microM. Further, the syntheses of diacyl type of phosphatidylethanolamine and the alkenylacyl type of phosphatidylcholine were markedly inhibited by unlabeled CDP-choline and CDP-ethanolamine, respectively. The two enzymes had opposite substrate specificities, and ethanolaminephosphotransferase showed a high preference to plasmalogen synthesis, especially in the presence of CDP-choline.     

Divergent interactions involving the oxidosqualene cyclase and the steroid-3-ketoreductase in the sterol biosynthetic pathway of mammals and yeasts

In mammals and yeasts, oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol, the first cyclic intermediate in sterol biosynthesis. We used a murine myeloma cell line (NS0), deficient in the 17β-hydroxysteroid dehydrogenase type 7 (HSD17B7), as a model to study the potential interaction of the HSD17B7 with the OSC in mammals. HSD17B7 is the orthologue of the yeast steroid-3-ketoreductase (ERG27), an enzyme of ergosterol biosynthesis that plays a protective role towards OSC. Tracer experiments with NS0 cells showed that OSC is fully active in these mammalian cells, suggesting that in mammals the ketosteroid reductase is not required for OSC activity. Mouse and human HSD17B7 were overexpressed in ERG27-deletant yeast cells, and recombinant strains were tested for (i) the ability to grow on different media, (ii) steroid-3-ketoreductase activity, and (iii) OSC activity. Recombinant strains grew more slowly than the control yeast ERG27-overexpressing strain on sterol-deficient media, whereas the growth rate was normal on media supplemented with a 3-ketoreductase substrate. The full enzymatic functionality of mammalian steroid-3-ketoreductase expressed in yeast along with the lack of (yeast) OSC activity point to an inability of the mammalian reductase to assist yeast OSC. Results demonstrate that in mammals, unlike in yeast, OSC and steroid-3-ketoreductase are non-interacting proteins.     

Inhibition of pyridine-nucleotide-dependent enzymes by pyrazoles. Synthesis and enzymology of a novel A-ring pyrazole steroid

A novel A-ring pyrazole steroid, 2,3-bisaza-A-nor-1,5(10)-estradien-17 beta-ol (3), was synthesized as a potential inhibitor of steroidal NAD(P)H-dependent oxidoreductases. Compound 3 proved to be a potent inhibitor of 3(17)beta-hydroxysteroid dehydrogenase (from P. testosteroni) exhibiting a Ki of 90 +/- 20 nM. The activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase (from S. hydrogenans), steroid-5 alpha-reductase (from rat prostate), and 3 alpha-hydroxysteroid dehydrogenase (from rat liver) were unaffected by pyrazole 3. Dead end inhibition studies indicate an ordered binding of cofactor prior to substrate or pyrazole inhibitor.     

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of 110-130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of 70-100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c-src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Is steroid-16α-hydroxylase supported by more than one monooxygenase?

Steroid-16α-hydroxylase activities have been measured in normal and induced rat livers using four different substrates. The male/female activity ratio as well as the induction factor vary with the substrate indicating that steroid-16α-hydroxylase activity is a heterogenous enzyme. Experiments using specific inhibitors led to the conclusion that steroid-16α-hydroxylase is supported by at least two cytochrome P-450 forms, different from the cytochrome P-448.     

Existence of endogenous substrate proteins for Ca2+/calmodulin-dependent and Ca2+/phospholipid-dependent protein kinases in rat adrenal glomerulosa cells

In rat adrenal glomerulosa cells, endogenous substrate proteins for Ca2+/calmodulin (CaM)-dependent protein kinase (glomerulosa CaM kinase) and Ca2+/phospholipid-dependent protein kinase (protein kinase C) were investigated. In a 105,000 g-supernatant fraction (cytosol), the Mr 100,000 protein was phosphorylated in the presence of calcium (calculated free Ca2+ concentration, 460 microM) alone or calcium and CaM, and the phosphorylation of this protein was completely inhibited by the CaM antagonists pimozide (500 microM) and melittin (5 microM) in the presence of calcium alone, respectively. These results indicate that the Mr 100,000 protein is a major substrate for glomerulosa CaM kinase, and considerable amounts of endogenous CaM might be present in the cytosol. In the presence of phospholipids (the micelles of 8 micrograms of phosphatidyl serine and 1 microgram of diacylglycerol), at least twelve proteins of Mr 127,000, 80,000, 70,000, 36,000, 35,000, 33,000, 32,000, 30,000, 27,000, 22,000, 19,000 and 17,000 were phosphorylated, and the phosphorylation of these proteins was enhanced by the addition of calcium, indicating that these proteins are substrates for protein kinase C. No endogenous protein phosphorylation was found in a 105,000 g-particulate fraction. Thus, these findings demonstrate that adrenal glomerulosa cells have specific substrate proteins for glomerulosa CaM kinase and protein kinase C, respectively.     

Phosphatidylethanolamine N-methyltransferase in human red blood cell membrane preparations. Kinetic mechanism

The successive methylations of phosphatidylethanolamine to form phosphatidylcholine were measured using exogenously added intermediates and membrane preparations from human red blood cells. The addition of phosphatidylethanolamine resulted in no increase in methylation rate over that with endogenous substrate; however, the addition of monomethylphosphatidylethanolamine (PME) and dimethylphosphatidylethanolamine (PDE) markedly increased the reaction rate and allowed studies into the kinetic mechanism for the second and third methylation reactions. The data are consistent with catalysis of the last two methylations being by a single enzyme with a random Bi-Bi sequential mechanism. Analysis of PDE:phosphatidylcholine product ratios indicates that the enzyme can conduct multiple methylations of enzyme-bound phospholipid. The nature of the acyl chain (16:0 versus 18:1) of the phospholipid had only a small effect on the value of the kinetic constants. The maximal velocities obtained with the 18:1 substrate were less than 5% lower than those obtained with the 16:0 substrate. The Km values for the two phospholipids were 20-45 and 10-14 microM for the methylation of PME and PDE, respectively. The Km for S-adenosylmethionine (AdoMet) was 5-9 microM with PME and 4 microM with PDE as substrates. Depending on the acyl chain and the phospholipid, the Ki(AdoMet) varied from 8 to 19 microM, the Ki(PME) from 41 to 82 microM, and the Ki(PDE) from 35 to 61 microM. The Ki for S-adenosylhomocysteine (AdoHcy) was between 1.0 and 1.4 microM depending upon the variable substrate. The endogenous concentrations of PME and PDE in red blood cell membranes were estimated to be 0.49 and 0.24 mumol/liter packed cells, respectively. The product from the utilization of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a competitive inhibitor of its precursor, AdoMet, and a noncompetitive inhibitor of the two phospholipid substrates.     

18q deletion syndrome in a child with steroid-17,20-lyase deficiency

The del (18q) syndrome is characterised by poor growth, variable mental retardation, facial dysmorphism, and abnormalities of the genitalia. In genetic males, genital abnormalities vary from testicular ectopia, and microphallus to severe hypospadias. Genetic females frequently have hypoplasia of the labia minora. We describe a child with del (18q) syndrome and severe ambiguous genitalia. Serum testosterone after 4 doses of hCG (5000 IU/m2/dose) was only 50 ng/dL (expected greater than 300 ng/dL). When testicular tissue was incubated with [1,2-3H]progesterone and 17-hydroxy-[4-14C]progesterone, there was synthesis of 17-hydroxy-[1,2-3H]progesterone but no further metabolism of 17-hydroxyprogesterone to androgens. These data suggested the presence of steroid-17,20-lyase deficiency. In order to determine if steroid-17,20-lyase deficiency was a common feature in del (18q) syndrome we examined 6 other patients (3 girls; 3 boys) with a deletion of the long arm of chromosome 18 distal to band q21. All 6 had dehydroepiandrosterone sulfate (DHEA-S) levels which were lower than those of age-matched controls. Four had delayed puberty. Serum testosterone levels were also low in 2 of the 3 affected boys. These results together with the findings in the index case suggest that a structural or regulatory gene for steroid-17,20-lyase may be located on the long arm of chromosome 18, distal to band q21.     

Effects of fatty acids on activity of cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

Effects of fatty acids, prostaglandins, and phospholipids on the activity of purified cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver were investigated. Prostaglandins A2, E1, E2, F1 alpha, and F2 alpha, thromboxane B2, and most phospholipids were without effect; lysophosphatidylcholine was a potent inhibitor. Several saturated fatty acids (carbon chain length 14-24), at concentrations up to 1 mM, had little or no effect on hydrolysis of 0.5 microM [3H]cGMP or 0.5 microM [3H]cAMP with or without 1 microM cGMP. In general, unsaturated fatty acids were inhibitory, except for myristoleic and palmitoleic acids which increased hydrolysis of 0.5 microM [3H]cAMP. The extent of inhibition by cis-isomers correlated with the number of double bonds. Increasing concentrations of palmitoleic acid from 10 to 100 microM increased hydrolysis of [3H]cAMP with maximal activation (60%) at 100 microM; higher concentrations were inhibitory. Palmitoleic acid inhibited cGMP hydrolysis and cGMP-stimulated cAMP hydrolysis with IC50 values of 110 and 75 microM, respectively. Inhibitory effects of palmitoleic acid were completely or partially prevented by equimolar alpha-tocopherol. Palmitelaidic acid, the trans isomer, had only slightly inhibitory effects. The effects of palmitoleic acid (100 microM) were dependent on substrate concentration. Activation was maximal with 1 microM [3H]cAMP and was reduced with increasing substrate; with greater than 10 microM cAMP, palmitoleic had no effect. Inhibition of cGMP hydrolysis was maximal at 2.5 microM cGMP and was reduced with increasing cGMP; at greater than 100 microM cGMP palmitoleic acid increased hydrolysis slightly. Palmitoleic acid did not affect apparent Km or Vmax for cAMP hydrolysis, but increased the apparent Km (from 17 to 60 microM) and Vmax for cGMP hydrolysis with little or no effect on the Hill coefficient for either substrate. These results suggest that certain hydrophobic domains play an important role in modifying the catalytic specificity of the cGMP-stimulated phosphodiesterase for cAMP and cGMP.     

Cyclic nucleotide phosphodiesterase in rat neostriatum: properties of the enzyme in crude homogenates

Homogenates of rat neostriatum hydrolysed cGMP faster than cAMP at both high (100 microM) and low (1 microM) substrate concentrations, although the hydrolysis of both nucleotides exhibited similar kinetic properties. Kinetic analysis of the effect of substrate concentration on the rate of cAMP and cGMP hydrolysis gave results characteristic of a negatively cooperative enzyme species, with two apparent Km's for each nucleotide. The ratio between the Vmax of the high Km form and the Vmax of the low Km form was similar in various subcellular fractions of neostriatal tissue, in a preparation of synaptic membranes from whole brain, and in homogenates of other brain regions, including both neural-rich and glial-rich tissues. In homogenates of neostriatum cAMP could almost completely block cGMP hydrolysis and vice versa. The kinetics of this inhibition were competitive at low (1 microM) substrate concentrations, and non-competitive at high (100 microM) substrate concentrations. Various phosphodiesterase inhibitors failed to preferentially inhibit the hydrolysis of either nucleotide at high or low nucleotide concentrations. Preliminary studies of the effect of a Ca(2+)-dependent endogenous activator preparation on the hydrolysis of cyclic nucleotides in homogenates of rat neostriatum showed a specific activation of cGMP hydrolysis at low nucleotide concentrations. The rate of cGMP hydrolysis at 1 microM substrate concentration was doubled in the presence of the activator preparation and 100 microM-CaCl2, while cGMP hydrolysis at 100 microM or cAMP hydrolysis at both 1 microM and 100 microM remained unaffected. These observations raise the possibility that cAMP and cGMP may be hydrolysed by the same enzyme in rat neostriatum, and that an endogenous activating factor may determine the relative affinities of the enzyme for the two nucleotides.     

Effect of substrate on Ca2(+)-concentration required for activity of the Ca2(+)-dependent proteinases, mu- and m-calpain

The Ca2+ concentrations required for half-maximal activity of mu- and m-calpain purified from bovine skeletal muscle were tested using four different protein substrates and three different synthetic peptide substrates. Hammersten casein, the commonly used substrate for measuring mu- and m-calpain activity, required 2.5 microM Ca2+ for half-maximal activity of mu-calpain and 290 microM Ca2+ for half-maximal activity of m-calpain. When Hammersten casein was dialyzed against 8 M urea and 10 mM EDTA to remove all endogenous Ca2+, it required 1.9 and 290 microM Ca2+ for half-maximal activity of mu- and m-calpain, respectively. Rabbit skeletal muscle myofibrils and rabbit skeletal muscle troponin required 65 microM and 24 microM Ca2+ for half-maximal activity of mu-calpain and 380 microM and 580 microM Ca2+ for half-maximal activity of m-calpain, respectively. The three synthetic substrates tested, Suc-Leu-Tyr-MCA, Boc-Leu-Thr-Arg-MCA, and Suc-Leu-Leu-Val-Tyr-MCA, required 1.6 microM to 3.7 microM Ca2+ for half-maximal activity of mu-calpain and 200 to 560 microM Ca2+ for half-maximal activity of m-calpain.     

Multidrug resistance protein 4 (MRP4/ABCC4) mediates efflux of bimane-glutathione

Multidrug resistance proteins (MRPs) are ATP-dependent export pumps that mediate the export of organic anions. ABCC1 (MRP1), ABCC2 (MRP2) and ABCC3 (MRP3) are all able to facilitate the efflux of anionic conjugates including glutathione (GSH), glucuronide and sulfate conjugates of xenobiotics and endogenous molecules. Earlier studies showed that ABCC4 functions as an ATP-driven export pump for cyclic AMP and cyclic GMP, as well as estradiol-17-beta-D-glucuronide. However, it was unclear if other conjugated metabolites can be transported by ABCC4. Hence in this study, a fluorescent substrate, bimane-glutathione (bimane-GS) was used to further examine the transport activity of ABCC4. Using cells stably overexpressing ABCC4, this study shows that ABCC4 can facilitate the efflux of the glutathione conjugate, bimane-glutathione. Bimane-glutathione efflux increased with time and >85% of the conjugate was exported after 15min. This transport was abolished in the presence of 2.5microM carbonylcyanide m-chlorophenylhydrasone (CCCP), an uncoupler of oxidative phosphorylation. Inhibition was also observed with known inhibitors of MRP transporters including benzbromarone, verapamil and indomethacin. In addition, 100microM methotrexate, an ABCC4 substrate or 100microM 6-thioguanine (6-TG), a compound whose monophosphate metabolite is an ABCC4 substrate, reduced efflux by >40%. A concentration-dependent inhibition of bimane-glutathione efflux was observed with 1-chloro-2,4-dinitrobenzene (CDNB) which is metabolized intracellularly to the glutathione conjugate, 2,4-dinitrophenyl-glutathione (DNP-GS). The determination that ABCC4 can mediate the transport of glucuronide and glutathione conjugates indicates that ABCC4 may play a role in the cellular extrusion of Phase II detoxification metabolites.     

Mechanism of action of lipoprotein lipase on triolein particles: effect of apolipoprotein C-II

Triolein particles stabilized by a phosphatidylcholine monolayer were used to study the lipoprotein lipase (LpL) reaction. They were prepared in two different sizes and with triolein and phosphatidylcholine in the molar ratios of 0.9-1.2 : 1 (small particles) and 8-17 : 1 (large particles). The rate of hydrolysis by LpL of phosphatidylcholine on the surface of both lipid particles was only 1/20 as much as that of triolein, even if it was activated to the maximum by apolipoprotein C-II (apoC-II). Thus, the phospholipase activity of LpL was low enough to measure the initial rate of hydrolysis of triolein without causing a gross change of the surface of the lipid particle. When the hydrolysis of triolein by LpL was monitored, fatty acid was released at a constant rate until all of the triolein molecules were hydrolyzed. The enzyme required 220 +/- 17 and 66 +/- 9 nM apoC-II for its half-maximal activity (Km (apoC-II] with small and large particles as a substrate (1.15 mM triolein for small and 2.13 mM triolein for large particles), respectively, using various concentrations of LpL. The Km(apoC-II) values for these two substrates became similar when LpL activity was analyzed with respect to the density of apoC-II on the phosphatidylcholine monolayer at the surface of the particles (bound apoC-II/phosphatidylcholine). The concentration of substrate particles did not affect the Km(apoC-II) values. The presence of an adequate amount of apoC-II increased the maximal activity of LpL (Vmax(triolein)) from 0.48 +/- 0.21 to 6.81 +/- 0.45 and from 0.32 +/- 0.04 to 7.13 +/- 0.64 mmol/h/mg with a slight decrease in the apparent Michaelis constant (Km(triolein)) for small (from 90 to 54 microM triolein) and large (from 1.00 to 0.65 mM triolein) particles, respectively. Although the apparent Km for triolein in large particles was about ten times greater than that in small particles, the values became similar when they were corrected for the concentration of phosphatidylcholine (50-100 microM phosphatidylcholine), which corresponded to the surface area of the substrate particles. It was suggested that bound apoC-II molecules were transferred relatively slowly to other lipid particles while LpL molecules moved rapidly among the lipid particles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

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.     

Inhibitors of human and rat testes microsomal 17beta-hydroxysteroid dehydrogenase (17beta-HSD) as potential agents for prostatic cancer

In a screening programme for inhibitors of human testis 17beta-hydroxysteroid dehydrogenase (17beta-HSD type 3), as potential agents for the treatment of hormone-dependent prostatic cancer, we have used crude human testis microsomal 17beta-hydroxysteroid dehydrogenase as a convenient source of the enzyme. Crude human enzyme was shown to have a similar substrate profile to recombinant Type 3 17beta-HSD from the same source as determined by the low Km/Vmax ratio for the reduction of androstenedione compared to the oxidation of testosterone, and a low level of activity in reduction of oestrone. Screening of a wide range of compounds of different structural types as potential inhibitors of the microsomal enzyme in the reduction step revealed that certain p-benzoquinones and flavones/isoflavones were potent inhibitors of the enzyme, diphenyl-p-benzoquinone (2.7 microM), phenyl-p-benzoquinone (5.7 microM), 7-hydroxyflavone (9.0 microM), baicalein (9.3 microM) and biochanin A (10.8 microM). Some structure-activity relationships within the flavone/isoflavone series are discussed. Studies with rat testis microsomal 17beta-HSD showed that it differed from the human enzyme mainly in its greater ability to accept oestrone as substrate and the pH-optimum for oxidation of testosterone. It was found to be much less sensitive to inhibition by the compounds studied so negating it use as a more readily available tissue for the screening of potential inhibitors.