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)

The Alcaligenes eutrophus genes for beta-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase and poly(beta-hydroxybutyric acid) synthase (PHB synthase) which comprise the three-step PHB-biosynthetic pathway, were cloned. Molecular studies revealed that these genes are organized in a single operon. The A. eutrophus PHB-biosynthetic genes are readily expressed in other bacteria, and DNA fragments harbouring the operon can be used as a cartridge to confer to other bacteria the ability to synthesize PHB from acetyl-CoA. The biochemical and physiological capabilities of A. eutrophus for the synthesis of a wide variety of polyhydroxyalkanoates are discussed.
Mol Microbiol 1991 Mar
PMID:Physiology and molecular genetics of poly(beta-hydroxy-alkanoic acid) synthesis in Alcaligenes eutrophus. 204 47

25-Hydroxycholecalciferol (25-OHD3) is converted to 8 alpha,25-dihydroxy-3-oxoneocholecalciferol [8,25-(OH)2-3-oxoneo-D3] by liver microsomes, alveolar macrophages and myeloid leukemia cells. The characteristics of this reaction in liver microsomes have been determined. Omission of an NADPH-generating system or NADH resulted in a greater than 75% reduction in the production of 8,25-(OH)2-3-oxoneo-D3. In the absence of the cytosolic fraction, 25-OHD3 was converted to products that comigrated with 8,25-(OH)2-3-oxoneo-D3 on a silica column developed with hexane-isopropanol, thereby preventing quantitation. Production of 8,25-(OH)2-3-oxoneo-D3 was unaffected by EDTA and was stimulated by N,N'-diphenyl-p-phenylenediamine. Both progesterone and pregnenolone inhibited production of 8,25-(OH)2-3-oxoneo-D3; inhibition by progesterone was greater than that by pregnenolone. 8,25-(OH)2-3-Oxoneo-D3 did not bind the thymus receptor for 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] at concentrations 10-fold higher than that of 1,25-(OH)2D3. The lack of affinity of 8,25-(OH)2-3-oxoneo-D3 for the 1,25-(OH)2D3 receptor suggests that this metabolite is a degradative product of 25-OHD3, which might be produced when 25-OHD3 concentrations in the liver are excessive. Synthesis of this metabolite in the liver may be catalyzed by enzymes that also metabolize other steroids.
J Steroid Biochem Mol Biol 1991 Jun
PMID:Synthesis and function of 8 alpha,25-dihydroxy-3-oxoneocholecalciferol in liver. 206 90

Pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) has also 3 alpha- and 3 beta-HSD (3 alpha/beta-HSD) activities. The purified 20 beta-HSD preparation from neonatal pig testes could catalyze the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) in the presence of beta-NADPH to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol at the ratio of 4:3, and the specific 3 alpha/beta-HSD activity of 20 beta-HSD for 5 alpha-DHT was about 10 or 15 times larger than the 20 beta-HSD activities for 17 alpha-hydroxypregn-4-ene-3,20-dione (17 alpha-hydroxyprogesterone) or progesterone, respectively. The result indicates that the testicular 20 beta-HSD has high 3 alpha(axial, 3R)- and 3 beta(equatorial, 3S)-HSD activity. The testicular 20 beta-HSD could catalyze the reversible conversion of various 5 alpha- or 5 beta-dihydrosteroids which have a 3-carbonyl or 3-hydroxyl group with beta-NADP(H) as the preferred cofactor. The enzyme transferred the 4-proS hydrogen of NADPH to the 5 alpha-DHT for both 3 alpha- and 3 beta-hydroxylation and it was the same as the 20 beta-hydroxylation of 17 alpha-hydroxyprogesterone. Although the 3 alpha/beta-HSD activity has been known to be present in 3 alpha,20 beta-HSD of Streptomyces hydrogenans, the enzymological properties for 3 alpha/beta-HSD activity catalyzed by testicular 20 beta-HSD were different from the properties for 3 alpha/beta-HSD activity catalyzed by prokaryotic 3 alpha, 20 beta-HSD with respect to the specificity of the catalytic reaction and the cofactor requirement.
J Steroid Biochem Mol Biol 1991 Jun
PMID:20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: 3 alpha/beta-hydroxysteroid dehydrogenase activities catalyzed by highly purified enzyme. 206 95

Oxidative stress induced by cumene hydroperoxide was studied in cultured neonatal rat myocytes. A progressive increase of irreversible cell injury as determined by leakage of the cytoplastic enzyme alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) from the cells was noted at concentrations ranging from 25-100 microM cumene hydroperoxide (incubation time 90 min). Cumene hydroperoxide-induced damage was reduced or prevented by several compounds: the application of Trolox C, a water-soluble vitamin E analogue, and of phospholipase A2 inhibitors chlorpromazine and (to a lesser extent) quinacrine prevented alpha-HBDH release. ICRF-159, a chelator of divalent cations, ascorbic acid, a potent antioxidant, and the cysteine protease inhibitor leupeptin did not reduce the cumene hydroperoxide-induced cytotoxicity. Detoxification of hydroperoxides by the glutathione peroxidase system results in an increased flux through the pentose phosphate shunt and loss of NADPH. Glucose inhibited the cumene hydroperoxide-induced alpha-HBDH release, probably by replenishing NADPH. These results indicate that cumene hydroperoxide, after exhaustion of the glutathione system, induces irreversible injury in cultured myocytes by a mechanism that depends to a large extent on deterioration of cellular membranes caused by lipid peroxidation and phospholipase activation.
J Mol Cell Cardiol 1990 Oct
PMID:Prevention of cumene hydroperoxide induced oxidative stress in cultured neonatal rat myocytes by scavengers and enzyme inhibitors. 209 37

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 microM vs 22 microM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 microM and 43 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Recognit
PMID:Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography. 209 89

Nifurtimox (Nfx) (4(5-nitrofurfurylidene)amino)-3-methylthiomorpholine-1, 1-dioxide) is a drug used against Chagas' disease, a parasitic sickness afflicting several million Latin Americans. Nfx administration to Sprague-Dawley male rats (220-250 g) at a dose of 100 mg/kg caused pronounced alterations in the adrenal cortex involving the fasciculata and reticularis zones but which were not evident in the glomerulosa. Alterations observed involved mitochondria, nuclei, Golgi apparatus, and the endoplasmic reticulum but were more intense in the mitochondria. There is Nfx nitroreductase activity in the adrenal microsomal, mitochondrial, and cytosolic-rich fractions but most of it is in the mitochondrial-rich fraction. Activity in the first two fractions requires NADPH and that in the cytosol is only observed in the presence of hypoxanthine as substrate. Enzymatic activity in all fractions is inhibited by oxygen. CO does not inhibit mitochondrial Nfx nitroreductase and inhibits only 10% of the microsomal enzyme activity. Hypoxanthine-dependent cytosolic activity is inhibited by allopurinol. Present results suggest that Nfx is activated to damage-producing reactive metabolites by nitroreductive biotransformation in rat adrenal organelles. Mitochondrial and microsomal bioactivation would occur at the level of the flavoenzyme P-450 reductase rather than at P-450 itself, and cytosolic bioactivation would be mediated by xanthine oxidase. Epidemiological studies on adrenal function in patients undergoing Nfx treatment would be necessary to establish the potential toxicological relevance of these findings.
Exp Mol Pathol 1990 Feb
PMID:Ultrastructural effects of Nifurtimox on rat adrenal cortex related to reductive biotransformation. 210 46

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.
Mol Cell Biochem 1990 Feb 09
PMID:Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450. 210 21

In previously described activation systems [Clive D, Spector JFS (1975): Mutat Res 31:17-29] for the mouse lymphoma mutation assay the cofactor isocitrate is rapidly exhausted and the resultant loss of NADPH can halt metabolic processes. Presented here are data obtained with a non-toxic balance of NADP (1.4 mg/ml), isocitrate (6.0 mg/ml), and S9 (less than or equal to 4%) in Fischer's medium which produces a more stable supply of the required cofactors. By spectrophotometric analysis, the molar concentration of NADPH remains at greater than or equal to 50% or more of the maximum over the usual 4-hr treatment period. Accompanying this increase in NADPH duration was increased toxicity and mutant frequency at most doses among cells treated with the reference mutagens 3-methylcholanthrene (MCA), 2-acetylaminofluorene (AAF), benzo(a)pyrene (BAP), 9,10-dimethyl-1,2-benzanthracene (DMBA), or cyclophosphamide (CPA), but not with dimethylnitrosamine (DMN)-possibly a reflection of the single enzyme mediated step in the metabolism of this chemical. These observations also suggest that results attributed to varying the amounts of S9 in an activation mixture may be due to suboptimal cofactor levels and further emphasize the need to maintain sufficient NADPH exposure to evaluate the effects of metabolic enzyme levels or compare the relative activities of analogous chemicals.
Environ Mol Mutagen 1990
PMID:Development of an optimal S9 activation mixture for the L5178Y TK+/- mouse lymphoma mutation assay. 212 88

We have previously shown that human placental estradiol-17 beta dehydrogenase (EC 1.1.1.62; 17 beta-EDH) catalyzes the conversion of estradiol-17 beta to estrone and stereospecifically reduces NAD+ to [4-pro-S]NADH, [( 4-B]NADH). Subsequently, this enzyme was found to reduce the ketone function at C-20 of progesterone, and evidence indicates that both activities reside at the same active site. This study was done to further elucidate spatial arrangements of cofactor and the 21-carbon substrate as they bind at the active site. The cofactor, [4B-3H]NADPH, was generated with homogeneous 17 beta-EDH from term human placenta, utilizing [17 alpha-3H]estradiol-17 beta and NADP+. The resulting [4B-3H]NADPH was then purified by ion exchange chromatography and was separately incubated (24.4 microM) with a large molar excess of progesterone (150 microM) as substrate in the presence of the enzyme. Following incubation, the steroid reactants and products were extracted, separated by high-performance liquid chromatography and quantitated as to mass and tritium content. Oxidized and reduced cofactor were separated by ion-exchange chromatography and similarly quantitated. In all incubations, equimolar amounts of 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OHP) and NADP+ were obtained. Radioactivity was stoichiometrically transferred from [4B-3H]NADPH to the steroid product [( 3H]20 alpha-OHP). These results further substantiate a single active site for both 17 beta- and 20 alpha-dehydrogenation enzyme activities. In addition, the enzyme is B-side specific, catalyzing the transfer of the 4B-hydrogen from the dihydronicotinamide moiety of the cofactor, for both C-18 and C-21 steroid substrates. Since the 20 alpha-dehydrogenation by other enzyme sources has always been demonstrated to be an A-side specific reaction, this observation represents an important exception to the Alworth-Bentley rules of enzyme stereospecificity.
J Steroid Biochem Mol Biol 1990 Sep
PMID:Stereospecificity of hydrogen transfer between progesterone and cofactor by human placental estradiol-17 beta dehydrogenase. 214 72

Formation of catecholestrogens (CE) by rat hepatic microsomes was re-examined because as recently shown; (1) CE formation can be catalyzed by an NADPH-dependent estrogen-4-hydroxylase (E-4-H(NADPH)) and by a peroxidatic, organic hydroperoxide-dependent estrogen-2/4-hydroxylase (E-2/4-H(OHP)), in addition to the established NADPH-dependent estrogen 2-hydroxylase (E-2-H(NADPH)); and (2) the indirect radiometric and the COMT-coupled radioenzymatic assays, used in many previous studies, may fail to provide an accurate measure, in particular, of 4-OH-CE. Using a direct product isolation assay, hepatic microsomes of both male and female rats were shown to express E-2/4-H(OHP) activity with properties similar to those of peroxidatic activity in other tissues. The activities of E-2/4-H(OHP) and E-2-H(NADPH) were affected differently by 5 out of 7 inducers of cytochromes P-450 administered in vivo. Phenobarbital and dexamethasone caused a 4- and 2-3-fold increase in E-2-H(NADPH) activity, respectively, but only a 38 and 20% increase in E-2/4-H(OHP) activity. Ketoconazol and beta-naphtoflavone caused a modest increase in E-2-H(NADPH) activity but a decrease in OHP-dependent activity. Clofibrate decreased peroxidatic activity by 50% and NADPH-dependent activity by approximately 20%. Both activities were increased by ethanol but decreased by isoniazide, an agent which induces the same form of cytochromes P-450 as ethanol. Polyclonal antibody against P-450p, a form of P-450 induced by glucocorticoids, inhibited E-2-H(NADPH) but not E-2/4-H(OHP) activity of untreated and of dexamethasone- and phenobarbital-treated rats. This study establishes that CE formation may occur in liver via the peroxidatic pathway and indicates that this pathway depends on forms of P-450 different from those mediating E-2-H(NADPH) activity. It also confirms and extends previous observations of the involvement of multiple, constitutive and induced forms of cytochrome P-450 in NADPH-dependent 2-hydroxylation in liver.
J Steroid Biochem Mol Biol 1990 Oct
PMID:Hepatic catecholestrogen synthases: differential effect of sex, inducers of cytochromes P-450 and of antibody to the glucocorticoid inducible cytochrome P-450 on NADPH-dependent estrogen-2-hydroxylase and on organic hydroperoxide-dependent estrogen-2/4-hydroxylase activity of rat hepatic microsomes. 217 38


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