Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A full-length cDNA clone encoding barley dihydroflavonol-4-reductase was isolated from a kernel-specific cDNA library by screening with the cDNA of the structural gene (A1) for this enzyme from maize. Subsequently, the gene corresponding to the barley dihydroflavonol-4-reductase cDNA was cloned and sequenced. The gene contains three introns at the same positions as in the Zea mays gene, corresponding to the positions of the first three of the five introns present in the genes of Petunia hybrida and Antirrhinum majus. In vitro transcription and translation of the Hordeum vulgare cDNA clone yielded a protein which converts dihydroquercetin into 2,3-trans-3,4-cis-leucocyanidin with NADPH as cofactor. The protein has a deduced amino acid sequence of 354 residues and a molecular weight of 38,400 daltons. Dihydroflavonol reductases of barley, maize, petunia and snapdragon are highly polymorphic in the NH2- and C-terminal parts of the polypeptide chain while a central region of 324 residues contains 51% identical amino acids. This identity increases to 81% when only the barley and maize enzymes are compared. Recessive mutants in the Ant18 gene tested so far lack dihydroflavonol-4-reductase activity and accumulate small amounts of dihydroquercetin but have retained activity for at least two other enzymes in the flavonoid pathway. In testa-pericarp tissue of mutants ant18-159, ant18-162 and ant18-164, wild-type levels of steady state mRNA for dihydroflavonol reductase have been measured, while mRNA for this enzyme is not transcribed in mutant ant18-161. These data are consistent with the proposal that the Ant18 locus carries the structural gene for dihydroflavonol-4-reductase of barley.
Mol Gen Genet 1991 Nov
PMID:Structure of the Hordeum vulgare gene encoding dihydroflavonol-4-reductase and molecular analysis of ant18 mutants blocked in flavonoid synthesis. 172 Aug 64

The present studies on initial velocity of testosterone reduction by hepatic 5 beta-reductase (4-en-3-oxosteroid 5 beta-reductase) of chicken and mode of inhibition of the 5 beta-reduction by 5 beta-dihydrotestosterone and NADP+ indicated that the reduction of testosterone occurred after the 5 beta-reductase bound firstly to NADPH and then to testosterone, forming a ternary complex. After 5 beta-reduction, 5 beta-dihydrotestosterone and then NADP+ were liberated from the complex, following a mechanism of "ordered Bi-Bi". Effect of (4R)-5,10-seco-19-norpregna-4,5-diene-3,10,20-trione (a steroidal 5 alpha-reductase-inhibitor or Secosteroid), diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (the other steroidal 5 alpha-reductase-inhibitor or 4-MA), and glycyrrhetinic acid (3 beta-hydroxy-11-oxoolean-12-en-30-oic acid, a 5 beta-reductase-inhibitor) was examined upon the 5 beta-reductase activity by double reciprocal plots. The mode of inhibition against testosterone by 4-MA and glycyrrhetinic acid was found to be competitive, while that by Secosteroid was non-competitive.
J Steroid Biochem Mol Biol 1992 Jan
PMID:Kinetic mechanism of reduction of testosterone by hepatic 5 beta-reductase of chicken and inhibition of the reductase activity by a secosteroid, an azasteroid and glycyrrhetinic acid. 173 34

Progesterone 5 alpha-reductase activity and 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) enzymic activities (NADH-linked and NADPH-linked) were measured in anterior pituitaries (AP) from aged female rats during three stages of reproductive senescence (constant estrus: CE; repeated pseudopregnancies: PSP; and anestrus: AN). To assess ovarian influence on these enzymes during these stages of reproductive aging, we also determined enzyme levels from ovariectomized rats from each stage treated with estrogen or vehicle. Progesterone 5 alpha-reductase and NADH-linked 3 alpha-HSOR activities were 2-fold higher in pituitaries of CE rats as compared to those of PSP and AN rats. NADPH-linked 3 alpha-HSOR levels did not differ among the three stages. All three enzyme levels were elevated 2- to 5-fold as compared to the corresponding enzyme levels from young cycling rats. After ovariectomy (10 days), 5 alpha-reductase activity in PSP and AN rats was elevated 3- to 4-fold relative to mean levels in intact PSP and AN rats. Ovariectomy had no effect on 5 alpha-reductase levels in CE rats. Under similar conditions, young cycling rats exhibit a 10-12-fold increase. Treatment of ovariectomized PSP and AN rats for 3 days with estradiol benzoate (10 micrograms/day) restored 5 alpha-reductase levels. Ovariectomy had no effect on the NADPH-linked 3 alpha-HSOR levels in CE, PSP or AN animals which is similar to that observed with young rats. Ovariectomy also had no effect on the NADH-linked 3 alpha-HSOR levels except for the CE group. The ovariectomized CE rats exhibited reduced pituitary NADH-linked 3 alpha-HSOR levels (30%). In contrast, young rats exhibit elevated pituitary NADH-linked 3 alpha-HSOR levels after ovariectomy (4- to 5-fold). These changes suggest the possibility that altered processing of progesterone and its 5 alpha- and 3 alpha-reduced products may be one means by which the effectiveness of progesterone is reduced during aging. The results also suggest an altered ovarian role in the regulation of these enzymes.
J Steroid Biochem Mol Biol 1992 Jan
PMID:Pituitary progestin-metabolizing enzyme activities in the aged female rat. 173 37

Cobalt protoporphyrin (CoPP) administered subcutaneously to adult male rats caused a marked reduction in the conversion of 5 alpha-androstane-3 beta-17 beta-diol (3 beta-adiol) to its main triol derivative (6 alpha-atriol) by homogenates of the pituitary but not of the prostate or brain (ventromedial hypothalamus and cortex). No effect in the brain was observed when this heme analogue was infused intracerebroventricularly. 3 beta-adiol hydroxylase, the enzyme responsible for the reaction and whose main function is thought to be the elimination of dihydrotestosterone and its metabolites from target tissues, was also inhibited by CoPP and SKF-525A added in vitro. The reaction was microsomal and dependent on NADPH. It is proposed that the lack of reciprocal elevation of luteinizing hormone in the face of the low testosterone levels observed following treatment with CoPP may be due, in part, to increased levels of androstanediols. These metabolites accumulate because of increased production from testosterone and decreased conversion to their triol derivatives in the pituitary.
J Steroid Biochem Mol Biol 1991 Dec
PMID:Suppression of 5 alpha-androstane-3 beta,17 beta-diol hydroxylase activity in rat pituitary by cobalt protoporphyrin: implications for testosterone homeostasis. 175 95

19-Hydroxyandrost-4-ene-3,6,17-trione (19-OHAT), its 19-oxo derivative (19-oxo AT) and 4 beta, 5 beta-epoxyandrostane-3,6,17-trione (5) were synthesized as possible intermediates involved in a mechanism-based inactivation of aromatase caused by androst-4-ene-3,6,17-trione (AT). These compounds, inhibited the enzyme in a competitive manner with Ki's of 0.61, 7.5 and 5.1 microM for 19-OHAT, 19-oxo AT, and compound 5. The two 19-oxygenated steroids showed a time-dependent, pseudo-first order rate of inactivation of aromatase with kinact's of 0.222 and 0.076 min-1 for 19-OHAT and 19-oxo AT, respectively, while compound 5 did not. NADPH and oxygen were required for the inactivation. Androstenedione blocked the inactivation, while L-cysteine partially prevented that of 19-OHAT and almost completely that of 19-oxo AT. When the 19-oxygenated steroids were separately subjected to reaction with N-acetyl-L-cysteine, these rapidly disappeared from the reaction mixture with t1/2 of 25 min (19-OHAT) and 20 s (19-oxo AT). This finding indicates that L-cysteine prevents inactivation by a chemical dependent elimination of the inhibitors from the incubate. These results suggest that the 19-oxygenation rather than the 4,5-epoxidation may be involved in the time-dependent inactivation by AT.
J Steroid Biochem Mol Biol 1991 Dec
PMID:A time-dependent inactivation of aromatase by 19-oxygenated androst-4-ene-3,6,17-triones. 175 96

Redox inactivation of glutathione reductase involves metal cations, since chelators protected against NADPH-inactivation, 3 microM EDTA or 10 microM DETAPAC yielding full protection. Ag+, Zn2+ and Cd2+ potentiated the redox inactivation promoted by NADPH alone, while Cr3+, Fe2+, Fe3+, Cu+, and Cu2+ protected the enzyme. The Zn2+ and Cd2+ effect was time-dependent, unlike conventional inhibition. Glutathione reductase interconversion did not require dioxygen, excluding participation of active oxygen species produced by NADPH and metal cations. One Zn2+ ion was required per enzyme subunit to yield full NADPH-inactivation, the enzyme being reactivated by EDTA. Redox inactivation of glutathione reductase could arise from the blocking of the dithiol formed at the active site of the reduced enzyme by metal cations, like Zn2+ or Cd2+. The glutathione reductase activity of yeast cell-free extracts was rapidly inactivated by low NADPH or moderate NADH concentrations; NADP+ also promoted rapid inactivation in fresh extracts, probably after reduction to NADPH. Full inactivation was obtained in cell-free extracts incubated with glucose-6-phosphate or 6-phosphogluconate; the inactivating efficiency of several oxidizable substrates was directly proportional to the specific activities of the corresponding dehydrogenases, confirming that redox inactivation derives from NADPH formed in vitro.
Mol Cell Biochem 1991 Mar 13
PMID:Metals are directly involved in the redox interconversion of Saccharomyces cerevisiae glutathione reductase. 186 75

Previous investigations have established that spironolactone (SL) is converted to a reactive metabolite by adrenal microsomal enzymes, resulting in the degradation of cytochrome P-450 (P-450). Deacetylation of SL to 7 alpha-thiospironolactone (7 alpha-thio-SL) is the first step in the activation pathway, but further NADPH-dependent metabolism of 7 alpha-thio-SL is required for P-450 destruction. Studies were done to evaluate the role of the steroid 17 alpha-hydroxylase in the activation of 7 alpha-thio-SL by adrenal microsomes. Incubation of guinea pig adrenal microsomes with 7 alpha-thio-SL in the presence of NADPH effected greater than 50% declines in P-450 content and in 17 alpha-hydroxylase activity but no change in the rate of 21-hydroxylation. Preincubation of the microsomes with antisera to the 17 alpha-hydroxylase P-450 isozyme (P-450(17 alpha,lyase)) decreased 17 alpha-hydroxylase but not 21-hydroxylase activity and prevented the degradation of P-450 by 7 alpha-thio-SL. Control IgG had no effect on 17 alpha-hydroxylase activity or on the 7 alpha-thio-SL-mediated destruction of P-450. When added to a purified P-450(17 alpha,lyase) preparation, 7 alpha-thio-SL and the endogenous substrate progesterone caused typical type I spectral changes, but SL did not. Incubation of a purified and reconstituted 17 alpha-hydroxylase system, consisting of P-450(17 alpha,lyase), NADPH-P-450 reductase, cytochrome b5, and dilauroylphosphatidylcholine, with 7 alpha-thio-SL plus NADPH effected the complete degradation of the P-450(17 alpha,lyase). Neither progesterone nor SL caused P-450 destruction with the reconstituted enzyme preparation. The results provide direct evidence for the activation of 7 alpha-thio-SL by the 17 alpha-hydroxylase and support the hypothesis that a mechanism-based inhibition of the enzyme occurs. The data also provide additional evidence that 7 alpha-thio-SL is an obligatory intermediate in the degradation of P-450 by SL.
Mol Pharmacol 1991 Aug
PMID:Role of the steroid 17 alpha-hydroxylase in spironolactone-mediated destruction of adrenal cytochrome P-450. 187 14

17 Beta-Hydroxysteroid dehydrogenase (17 beta-HSD) is present in multiple forms in human breast tissue. One soluble form, with a molecular weight of approximately 35 kDa, was purified to near homogeneity from whole normal breast tissue. This form catalysed the oxidation of oestradiol and the reduction of oestrone, with NADP+ and NADPH as the preferred coenzymes. Three other soluble forms with higher molecular weights (in the range 50-80 kDa) were isolated. They catalysed the oxidation of oestradiol but not the reduction of oestrone, and all of them had properties very different from those of the low molecular weight enzyme. Activities of 17 beta-HSD were measured in particulate and soluble fractions from normal breast adipose and non-adipose tissues, and from breast tumours obtained from post-menopausal women, in the oxidative direction with NAD+ and NADP+ as coenzymes and in the reductive direction with NADH and NADPH as coenzymes. Particulate fractions from tumours had much higher oxidative and reductive activities than those from normal tissues. Soluble fractions from tumours had higher oxidative activities than those from the normal tissues but similar reductive activities. The major soluble form of 17 beta-HSD in adipose tissue was the 35 kDa enzyme which had both oxidative and reductive activities. In contrast, the majority of the soluble activity in non-adipose tissue was due to enzymes, with molecular weights in the range 50-80 kDa, which had oxidative activity only. The soluble fractions of tumours, like those of non-adipose tissue, contained enzymes with molecular weights in the range 50-80 kDa. In addition, they contained a 35 kDa enzyme with properties different from those of the enzyme with the same molecular weight present in adipose tissue.
J Mol Endocrinol 1991 Aug
PMID:17 Beta-hydroxysteroid dehydrogenases in human breast tissues: purification and characterization of soluble enzymes and the distribution of particulate and soluble forms in adipose, non-adipose and tumour tissues. 189 41

The alcohol-inducible CYP2E subfamily in rabbits contains two genes; CYP2E1 encodes the cytochrome earlier termed P-450 3a, and CYP2E2 encodes a cytochrome that is 97% identical in amino acid sequence to cytochrome P-450 (P-450) 2E1. In the present studies, the ontogenic expression of these two cytochromes was examined. In liver, P-450 2E2 mRNA is detectable immediately after birth and reaches slightly greater than the adult level at 2 weeks of age; in contrast, P-450 2E1 mRNA is not detectable until day 14 and increases rapidly to approximately twice the adult level at 5 weeks of age. P-450 2E protein is present in liver immediately after birth, coincident with the appearance of P-450 2E2 mRNA, peaks at 2 weeks, and then, despite the continued elevation in P-450 2E mRNA, decreases to the adult level at 5 weeks. In kidney, P-450 2E2 mRNA is not detectable at any age; P-450 2E1 mRNA, however, is present at 1 week, and the level increases to about half the adult level at 5 weeks of age. P-450 2E protein in this tissue is elevated at 2 weeks, relative to mRNA levels, and reaches approximately half the adult level at 5 weeks. The lack of close correlation between mRNA and protein levels in the liver and kidney of newborn rabbits indicates that the posttranscriptional control of P-450 2E enzyme levels that predominates in adult animals is also operative during the neonatal period. Monooxygenase activities with ethanol and p-nitrophenol as substrates reflect the developmental increase in P-450 2E protein, as well as the appearance and levels of spectrally detectable P-450, cytochrome b5, and NADPH-P-450 reductase in hepatic microsomes. The expression of P-450 2E2, but not P-450 2E1, in early neonates suggests that these two closely related cytochromes may have functional differences that are important during the first few weeks of life.
Mol Pharmacol 1991 Jul
PMID:Differences in the developmental expression of rabbit cytochromes P-450 2E1 and 2E2. 190 76

The role of protein residues in activating the substrate in the reaction catalyzed by the flavoprotein p-hydroxybenzoate hydroxylase was studied. X-ray crystallography (Schreuder, H. A., Prick, P.A.J., Wieringa, R.K., Vriend, G., Wilson, K.S., Hol, W.G. J., and Drenth, J. (1989) J. Mol. Biol. 208, 679-696) indicates that Tyr-201 and Tyr-385 form a hydrogen bond network with the 4-OH of p-hydroxybenzoate. Therefore, site directed mutants were constructed, converting each of these tyrosines into phenylalanines. Spectral (visible and fluorescence) properties, reduction potentials, and binding constants are very similar to those of wild type, indicating that there are no major structural changes in the mutants. In the absence of substrate, the mutants and wild type exhibit similar pH-dependent changes in the FAD spectrum. However, the enzyme-substrate complex of Tyr-201----Phe lacks an ionization observed in both wild type and Tyr-385----Phe, which preferentially bind the phenolate form of substrates. Tyr-201----Phe shows no preference, indicating that Tyr-201 is required to ionize the substrate. The mutants have less than 6% the activity of the wild type enzyme. The effects on catalysis were studied by stopped flow techniques. Reduction of FAD by NADPH is slower by 10-fold in Tyr-201----Phe and 100-fold in Tyr-385----Phe. When the reduced Tyr-201----Phe-p-hydroxybenzoate complex reacts with oxygen, a long-lived flavin-C(4a)-hydroperoxide is observed, which slowly eliminates H2O2 with very little hydroxylation. Thus, the role of Tyr-201 is to activate the substrate by stabilizing the phenolate. Tyr-385----Phe reacts with oxygen to form 25% oxidized enzyme, and 75% flavin hydroperoxide, which successfully hydroxylates the substrate. This mutant also hydroxylates the product (3, 4-dihydroxybenzoate) to form gallic acid.
 ...
PMID:Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants. 191 43


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