Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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5 alpha-Dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase [3 alpha(beta)-HSDH] [EC 1.1.1.50/EC 1.1.1.51] which catalyses the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to both 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol was purified to an apparent homogeneous state using cytosol of three human hyperplastic prostates by a 4-step purification procedure. After each purification step 3 alpha-HSDH activity was coincident with 3 beta-HSDH activity. On average, specific 3 alpha-HSDH activity was enriched 856-fold, specific 3 beta-HSDH activity 749-fold compared to human prostatic cytosol using anion exchange, hydrophobic interaction, gel filtration and affinity chromatography. Examination of the purified enzyme by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed a single protein band with silver staining. The molecular weight of the enzyme was estimated as 33 kDa by SDS-polyacrylamide gel electrophoresis and as 28 kDa by Sephacryl S-200 gel filtration indicating that the native 3 alpha(beta)-HSDH is a monomer. In the presence of the preferred co-factor, NADPH, the purified enzyme had a mean apparent Km for 5 alpha-DHT of 3.9 microM and a Vmax of 93.3 nmol (mg protein)-1 h-1 with regard to 3 alpha-HSDH activity, and a Km of 6.3 microM and a Vmax of 20.6 nmol (mg protein)-1 h-1 with regard to 3 beta-HSDH activity.
J Steroid Biochem Mol Biol 1992 May
PMID:Purification and properties of the 5 alpha-dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase from human prostatic cytosol. 160 44

Metabolism of 11-deoxycorticosterone (DOC) by hamster adrenal mitochondria gives 19-hydroxy-DOC and corticosterone (via 11-hydroxylation) in approximately equal yields. The ratio of 19- to 11-hydroxylation was invariant with changes in concentration of substrate or a competitive inhibitor. It is most likely, therefore, that a single 11,19-hydroxylase catalyzes both oxidations. Both primary products are further oxidized to the corresponding carbonyl analogs, 19-oxo-DOC and 11-dehydrocorticosterone, at rates that are approx. 20% of their rates of formation. The oxidation of 11-dehydrocorticosterone is catalyzed by a dehydrogenase utilizing either NAD or NADP while the oxidation of 19-hydroxy-DOC is catalyzed by an oxidase requiring NADPH. The 11-dehydrocorticosterone is stable in this enzyme preparation while 19-oxo-DOC is metabolized to two additional products, which are tentatively identified as 19-oic-DOC and 19-norcorticosterone. 19-nor-DOC was found to be hydroxylated at a rate that is 20% faster than the rate for DOC under the same conditions. It is therefore possible that 19-norcorticosterone can arise from 19-oic-DOC via decarboxylation to 19-nor-DOC and subsequent 11-hydroxylation, but the kinetics of its formation suggest that it may actually be formed directly from 19-oxo-DOC without free intermediates. 4-Androstene-3,17-dione and 17-hydroxy-DOC were also substrates for this 11,19-hydroxylase, but 18-hydroxy-DOC was not. Maintenance of hamsters on a low sodium diet had no effect on the metabolism of DOC by the isolated adrenal mitochondria.
J Steroid Biochem Mol Biol 1992 May
PMID:Metabolism of 11-deoxycorticosterone by hamster adrenal mitochondria. 160 48

Studies were done to determine the role of the 17 alpha-hydroxylase in the conversion of 7 alpha-thiospironolactone (7 alpha-thio-SL) to a reactive metabolite causing the degradation of testicular cytochrome P-450. Incubation of guinea pig testicular microsomes with 7 alpha-thio-SL plus NADPH resulted in an approx. 70% decline in cytochrome P-450 content and even greater loss of 17 alpha-hydroxylase activity. Addition of the 17 alpha-hydroxylase inhibitor, SU-10'603, to the incubation medium prevented the degradation of P-450 by 7 alpha-thio-SL. Similarly, preincubation of testicular microsomes with anti-P-45017 alpha,lyase IgG to inhibit 17 alpha-hydroxylation, diminished the subsequent loss of P-450 caused by 7 alpha-thio-SL. The results indicate that the 17 alpha-hydroxylase catalyzes the conversion of 7 alpha-thio-SL to the reactive metabolite responsible for P-450 destruction. The accompanying loss of 17 alpha-hydroxylase activity supports the hypothesis that suicide inhibition is the mechanism involved.
J Steroid Biochem Mol Biol 1992 May
PMID:Destruction of testicular cytochrome P-450 by 7 alpha-thiospironolactone is catalyzed by the 17 alpha-hydroxylase. 160 53

The success rate of liver transplantation has improved markedly during the last few years and, although this patient population receives multiple drug therapies, the effect of liver transplantation on drug metabolism has been studied very little. Therefore, the purpose of this study was to assess the metabolism of model drug substrates after liver transplantation in the rat. Rat livers were stored for 4 hr in cold Euro-Collins solution, transplanted orthotopically, and then perfused 2 hr later with oxygenated Krebs-Henseleit buffer, using a nonrecirculating system. Rates of monooxygenation of the model compound p-nitroanisole, conjugation of p-nitrophenol, and uptake of oxygen were measured. All parameters studied were elevated significantly, by nearly 2-fold, by transplantation. Specifically, monooxygenation was increased from 2.9 +/- 0.2 to 5.1 +/- 0.4 mumol/g/hr, conjugation was elevated from 3.3 +/- 0.6 to 7.7 +/- 0.1 mumol/g/hr, and O2 uptake was stimulated from basal values of 114 to 197 mumol/g/hr. Transplantation did not, however, alter rates of monooxygenation and conjugation in isolated microsomes supplemented with excess cofactor. When donor rats were pretreated with the Kupffer cell toxicant gadolinium chloride (10 mg/kg, intravenously) 30 hr before liver storage, the elevation after transplantation in all parameters studied was prevented. Depletion of carbohydrate reserves by fasting of donor rats did not prevent stimulation of monooxygenation and conjugation. On the other hand, urea synthesis from ammonium chloride, a process dependent on mitochondrial NADPH, was increased and monooxygenation was diminished after transplantation, suggesting the involvement of mitochondria in this phenomenon. Indeed, mitochondria isolated 2 hr postoperatively exhibited significantly elevated respiratory control ratios and higher state 3 rates of respiration. Taken together, these data support the hypothesis that Kupffer cells, activated by transplantation, release mediators that stimulate mitochondria in parenchymal cells and enhance drug metabolism by increasing cofactor supply (e.g., NADPH for monooxygenation and UDP-glucuronic acid for glucuronidation).
Mol Pharmacol 1992 Jun
PMID:Stimulation of monooxygenation and conjugation after liver transplantation in the rat: involvement of Kupffer cells. 161 13

Proton nuclear magnetic resonance spectroscopy has been used to detect two water molecules bound to residues in the active site of the Lactobacillus casei dihydrofolate reductase (DHFR). Their presence was detected by measuring nuclear Overhauser effects between NH protons in protein residues and protons in the individual bound water molecules in two-dimensional nuclear Overhauser effect spectroscopy (NOESY), in nuclear Overhauser effect spectroscopy in the rotating frame (ROESY) and three-dimensional 1H-15N ROESY-heteronuclear multiple quantum coherence spectra recorded on samples containing appropriately 15N-labelled DHFR. For the DHFR-methotrexate-NADPH complex, two bound molecules were found, one close to the Trp5 amide NH proton and the other near to the Trp21 indole HE1 proton: these correspond to two of the water molecules (Wat201 and Wat253) detected in the crystal structure studies described by Bolin and co-workers. However, the nuclear magnetic resonance experiments did not detect any of the other bound water molecules observed in the X-ray studies. The nuclear magnetic resonance results indicate that the two bound water molecules that were detected have lifetimes in the solution state that are longer than approximately two nanoseconds. This is of considerable interest, since one of these water molecules (Wat253) has been implicated as the likely proton donor in the catalytic reduction of dihydrofolate to tetrahydrofolate.
J Mol Biol 1992 Jul 20
PMID:Nuclear magnetic resonance detection of bound water molecules in the active site of Lactobacillus casei dihydrofolate reductase in aqueous solution. 164 Apr 65

The effects of two anticonvulsant drugs, phenytoin and sodium valproate, on the bioactivation of vitamin D3 have been studied with respect to the microsomal and mitochondrial cytochrome P-450-linked monooxygenase systems that contribute to 25-hydroxylation of vitamin D3 in rabbit liver, and the mitochondrial cytochrome P-450-linked monooxygenase system that catalyzes 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in rabbit kidney. These anticonvulsant drugs were found to inhibit the 25-hydroxylase activity on vitamin D3 in liver microsomes and mitochondria, respectively, but not to inhibit the 1 alpha-hydroxylation of 25-hydroxyvitamin D3, even over a wide concentration range. Moreover, the activities of the components of the cytochrome P-450-linked monooxygenase systems: NADPH-cytochrome P-450 reductase, NADPH-ferredoxin reductase and ferredoxin, were never inhibited by these drugs. It is possible that the inhibition of bioactivation of vitamin D3 by these anticonvulsant drugs causes rickets and osteomalacia, and the site of inhibition is expected to be the cytochrome P-450 mediated reactions in liver mitochondria.
J Steroid Biochem Mol Biol 1991 Oct
PMID:The effects of anticonvulsant drugs on vitamin D3-activating cytochrome P-450-linked monooxygenase systems. 165 98

NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent Kms for alpha-ketoglutarate, NADPH and NH4+ are 1.2 mM, 9.7 microM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 microM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn2+ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 microM) yet N-ethylmaleimide does not. In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.
Mol Cell Biochem 1991 Jun 26
PMID:Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum. 165 3

The use of clozapine, a unique antipsychotic drug, has been restricted due to a 1-2% incidence of drug-induced agranulocytosis. Metabolic activation of clozapine in neutrophils or stem cells could be the molecular mechanism underlying this side effect. Clozapine oxidation by human myeloperoxidase and horseradish peroxidase was evident from the disappearance of the UV absorbance at 290 nm. High performance liquid chromatography analysis revealed the formation of at least four radioactive peaks as a result of clozapine metabolism, including radioactivity coeluting with the protein. The tight association of radioactivity with the enzymatic protein was metabolism-dependent. This protein binding, which correlates with the total metabolism of clozapine, was reduced in the presence of glutathione and was absent in the presence of ascorbate. Similarly, in the presence of both reducing agents, the metabolite peaks in the high performance liquid chromatography radiogram, which are not associated with protein, disappeared. In contrast, in the presence of glutathione, two additional metabolites were found that could be isolated and identified by NMR and mass spectroscopy as clozapine glutathionyl adducts. Evidence for one-electron transfer reactions or the intermediate formation of a clozapine radical during the peroxidase-mediated metabolism of clozapine stems from the observation of thiyl and ascorbyl radicals in the presence of glutathione and ascorbate, respectively. The ascorbyl radical was detected by direct ESR spectroscopy in a peroxidase system. Its steady state concentration was significantly increased in the presence of clozapine. Glutathionyl radical formation was demonstrated by radical trapping with 5,5-dimethyl-1-pyrroline N-oxide in a peroxidase system. Again, the radical adduct concentration was significantly increased in the presence of clozapine. Similarly, when oxygen consumption was measured in peroxidase systems in the presence of glutathione or NADPH, the rate of oxygen uptake was markedly enhanced upon addition of clozapine. Thus, the data support the possibility of clozapine activation to free radical metabolites, which may cause oxidative stress or lead to adduct formation. Further, it can be concluded from these data that radical scavengers such as ascorbic acid, when coadministered with clozapine to patients, may reduce oxidative stress and protein adduct formation.
Mol Pharmacol 1991 Nov
PMID:Possible role of free radical formation in clozapine (clozaril)-induced agranulocytosis. 165 15

The mechanism of stimulation of 17 beta-estradiol (E2) formation from estrone (E1) by 5 alpha-dihydrotestosterone (5 alpha-DHT) in placental villi was investigated by examining; (1) if dehydroepiandrosterone (DHA) was stimulatory, (2) if NAD(P)H-generating, non-steroidal substrates stimulated E2 formation, (3) the subcellular localization of the effect, (4) if NAD(P) or NAD(P)H was required and (5) rates of 5 alpha-DHT oxidation by villi and microsomes. Although 5 alpha-DHT and DHA both inhibited the E2 to E1 reaction in villi and microsomes, only 5 alpha-DHT stimulated the conversion of E1 to E2. Glucose and lactate were slightly stimulatory when compared with 5 alpha-DHT. Stimulation of E2 formation was observed with microsomes but not with cytosol, and NAD or NADP was required. The results indicate that neither inhibition of the back reaction, E2 to E1, nor NADH or NADPH formation via the 3 beta-hydroxysteroid dehydrogenase/5-ene-3-ketosteroid isomerase reaction can account for the stimulation. It is proposed that the mechanism of stimulation involves one or more forms of membrane-bound 17 beta-hydroxysteroid oxidoreductase with NADH or NADPH formed as a product of 5 alpha-DHT oxidation being used as the cofactor for E1 reduction. This may involve a direct transfer of reduced pyridine nucleotide between enzyme molecules without equilibration with intracellular coenzyme pools.
J Steroid Biochem Mol Biol 1991 Nov
PMID:Regulation of human placental 17 beta-hydroxysteroid oxidoreductase: mechanism of stimulation of 17 beta-estradiol formation from estrone by 5 alpha-dihydrotestosterone in homogenates and villi in vitro. 165 69

Cytochromes P450b and P450e (IIB1 and IIB2, respectively) are two remarkably similar microsomal hemoproteins whose inductions in rat liver are generally believed to be coordinately controlled by such xenobiotics as phenobarbital. To critically examine this assumption, we used a new system of primary cultures of adult rat hepatocytes on Matrigel to evaluate whether organochlorine pesticides, as "phenobarbital-like" agents, directly induce these cytochromes in parallel in the liver parenchymal cell. For 14 of the pesticides we tested, as well as for phenobarbital, P450b and P450e mRNAs, measured on Northern blots, rose in concert as much as 58- and 6-fold, respectively, over the amounts in incubated control cultures. Kepone (chlordecone) treatment of the cultures increased P450e mRNA in a dose-dependent manner that disclosed a 10-fold greater potency, compared with cultures exposed to phenobarbital. Kepone also resembled phenobarbital in these experiments, in that there were dose-dependent increases in the amounts of hepatocellular P450p, P450pcn2, P450PB-1, P450f, and NADPH-cytochrome P450 oxidoreductase mRNAs. However, in the same kepone-treated cells, P450b mRNA or P450b immunoreactive protein was induced only slightly, if at all. In contrast, additions to the medium of mirex, a structural analog of kepone, effectively induced both P450b and P450e mRNAs and their proteins. Selective induction of P450e by kepone in the hepatocyte cultures, the first pharmacologic dissociation of the induction of P450b and P450e mRNAs and proteins, was not apparent in kepone-treated rats, where both P450b and P450e mRNAs were increased to equivalent extents. We conclude that the P450b and P450e genes may be expressed independently by process(es), possibly involving extrahepatic factors, that can be defined with the present hepatocyte culture system.
Mol Pharmacol 1991 Aug
PMID:Selective induction of cytochrome P450e by kepone (chlordecone) in primary cultures of adult rat hepatocytes. 171 15


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