Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat hepatic cytochrome P-450 form RLM2 is a testosterone 15 alpha-hydroxylase reported to be male-specific on the basis of purification studies (Jansson, I., Mole, J., and Schenkman, J. B. (1985) J. Biol. Chem. 260, 7084-7093). The sex dependence, developmental regulation, xenobiotic induction, and hormonal control of P-450 RLM2 expression were studied using P-450 form-specific immunochemical and catalytic assays. Polyclonal antibodies raised to rat hepatic P-450 3 (P-450 gene IIA1) were found to cross-react strongly with P-450 RLM2, but not with 10 other rat P-450 forms, suggesting that P-450 3 and P-450 RLM2 are highly conserved in primary structure. Western blotting of liver microsomes under conditions where P-450s 3 and RLM2 are resolved electrophoretically revealed that P-450 RLM2 is markedly induced at puberty in male rats, with no protein detected (less than or equal to 5% of adult male levels) in adult females or immature animals of either sex. A similar developmental dependence was observed for hepatic microsomal testosterone 15 alpha-hydroxylase activity, which was found to be catalyzed primarily by P-450 RLM2. P-450 RLM2 was resistant to induction by several xenobiotics and in the case of phenobarbital and beta-naphthoflavone, was suppressed by 50-60%. Studies on the steroid hormonal regulation of P-450 RLM2 revealed that its adult male-specific expression is imprinted (programmed) in response to neonatal testosterone exposure. Ovariectomy studies demonstrated that suppression by estrogen does not contribute significantly to the absence of P-450 RLM2 in adult female rats. Although the male-specific developmental induction of P-450 RLM2 in response to neonatal testosterone is strikingly similar to that of P-450 2c (testosterone 2 alpha/16 alpha-hydroxylase; gene IIC11), P-450 RLM2 expression is not dependent on the pulsatile pituitary growth hormone secretion required for P-450 2c synthesis. Rather, hypophysectomy of adult male rats increased P-450 RLM2 and its associated testosterone 15 alpha-hydroxylase activity by 50-100%.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Adult male-specific and neonatally programmed rat hepatic P-450 forms RLM2 and 2a are not dependent on pulsatile plasma growth hormone for expression. 340 35

The affinity alkylating progesterone analogue 17-(bromoacetoxy)progesterone has been used to label the active site of a microsomal cytochrome P-450 enzyme from neonatal pig testis. The enzyme causes removal of the C20 and C21 side chains from the substrates progesterone and pregnenolone by catalyzing both 17-hydroxylase and C17,20-lyase reactions, which produce the corresponding C19 steroidal precursors of testosterone. The progesterone analogue causes simultaneous inactivation of the two catalytic activities of the enzyme by a first-order kinetic process that obeys saturation kinetics. Progesterone and 17-hydroxyprogesterone each protect the enzyme against inactivation. The progesterone and analogue is a competitive inhibitor of the enzyme with Ki values of 8.4 microM and 7.8 microM for progesterone and 17-hydroxyprogesterone, respectively. The enzyme inactivation and kinetic data are consistent with a theory proposing that the analogue and the two substrates compete for the same active site. The radioactive analogue 17-[( 14C]bromoacetoxy)progesterone causes inactivation of the enzyme with incorporation of 1.5-2.2 mol of the analogue per mole of inactivated enzyme. When this experiment is carried out in the presence of a substrate, then 0.9-1.2 mol of radioactive analogue is incorporated per mole of inactivated enzyme. The data suggest that the analogue can bind to two different sites, one of which is related to the catalytic site. Radiolabeled enzyme samples, from reactions of the 14C-labeled analogue with the enzyme alone or with enzyme in the presence of a substrate, were subjected to amino acid analysis and also to tryptic digestion and peptide mapping.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Affinity alkylation of the active site of C21 steroid side-chain cleavage cytochrome P-450 from neonatal porcine testis: a unique cysteine residue alkylated by 17-(bromoacetoxy)progesterone. 349 7

Aromatase cytochrome P-450 has been purified from human placenta to homogeneity, as demonstrated by electrophoresis on polyacrylamide gels with SDS, and by double diffusion against an antibody raised in rabbits. The enzyme converts androstenedione to estrone (Vmax 13.3 n moles/min/n mole P-450; Km 30 microM) and testosterone to estradiol. Aromatase activity requires P-450, P-450 reductase and NADPH. Enzyme activity is inhibited by anti-aromatase antibodies and by 4-hydroxyandrostenedione. The enzyme shows a molecular weight of 55,000, is extremely unstable and spontaneously forms P-420 with a half-life of 2.5 days.
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PMID:Purification to homogeneity of aromatase from human placenta. 394 46

Methylene hydroxylation by cytochrome P-450(cam) (cytochrome m) can be resolved into four distinct steps: substrate addition, m(o) --> m(os); reduction, m(os) --> m(rs); dioxygen addition, m(rs) --> m(O2) (rs); followed by a second putidaredoxin (Pseudomonas putida ferredoxin)-mediated reduction and product formation. The isolated ferrous oxy-substrate complex exhibits first-order decay kinetics with the relatively slow rate constant of k [unk] 0.01 sec(-1), at 25 degrees , without product release. Putidaredoxin addition accelerates the decomposition with second-order kinetics, k [unk] 51,000 M(-1) sec(-1), and initiation of product formation. Cytochrome m forms a complex with putidaredoxin with dissociation constant of K(D) = 3 muM. In the complete three-protein hydroxylase system, consisting of cytochrome m, putidaredoxin, and the reductase (a DPNH-specific flavo-protein), camphor hydroxylation occurs with a stoichiometry of 1 mole each of DPNH and O(2) used per mole of product formed; the K(M) for putidaredoxin is about 4.2 muM.Putidaredoxin, on treatment with carboxypeptidase A, loses one molecule each of tryptophan and glutamine sequentially from the carboxy terminus to expose a terminal arginine. The tryptophan-free product has been separated from native putidaredoxin and other impurities, and retains the visible and electron paramagnetic resonance spectra and the redox potential of the active center of native putidaredoxin. This modified redoxin binds less tightly to cytochrome m, K(D) [unk] 150 muM, and is 50 times less effective in stimulation of the m(O2) (rs) decay rate. A similar decrease in specific activity is observed in the complete hydroxylase system.
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PMID:A role of the putidaredoxin COOH-terminus in P-450cam (cytochrome m) hydroxylations. 453 Feb 69

The addition of activators like flavone and hexobarbital to hepatic microsomes markedly stimulates H2O2 formation. The similar increase observed with flavone of microsomal hydroxylation of benzo(a)pyrene and its inhibition by catalase and methanol suggests but does not prove a necessary interaction of microsomal H2O2 production with benzo(a)pyrene hydroxylation. Hexobarbital and flavone-stimulated H2O2 formation is optimal at a stoichiometric relationship of these activators and NADPH. This implies either their direct participation as electron donors or their indirect involvement in electron transport by facilitation of stoichiometric substrate cytochrome P-450/NADPH flavoprotein interactions. Steady state kinetics data are consistent with a scheme in which the formation in microsomes of a complex of 1 mole of NADPH with NADPH-cytochrome P-450 reductase and 1 mole hexobarbital with cytochrome P-450 regulates H2O2 formation.
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PMID:Studies on the mechanism of stimulation of microsomal H2O2 formation and benzo(a)pyrene hydroxylation by substrates and flavone. 628 11

The rate of transfer of spin-labeled phospholipid from donor vesicles of sonicated 1-acyl-2-(10-doxylstearoyl)-sn-glycero-3-phosphocholine to other vesicle was determined as a function of content of cytochrome P-450 and the phosphatidylcholine/phosphatidylethanolamine ratio in the acceptor vesicles. The transfer rate was measured as an increase in intensity that resulted from a decrease in the line width in the EPR spectrum of the spin-labeled phospholipids as they was transferred to the nonspin-labeled acceptor vesicles. A lower transfer rate was observed for acceptor vesicles of pure egg phosphatidylcholine vesicles than for vesicles for a mixture of phosphatidylcholine and phosphatidylethanolamine. The presence of cytochrome P-450 in the acceptor vesicles further increased the transfer rate. Those alterations in the mole ratios of the protein and the two phospholipids that made the bilayer of the reconstituted vesicles more like the membrane of the endoplasmic reticulum resulted in an increase in phospholipid-transfer rate. The mole ratios of components that produce high phospholipid-transfer rates were similar to those that in an earlier study produced a 31P-NMR spectrum characteristic of a nonbilayer phase. These findings suggest that, in the membrane of the endoplasmic reticulum, phospholipid exchange may be an important element in function and interaction with other intracellular organelles.
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PMID:Phospholipid transfer between vesicles. Dependence on presence of cytochrome P-450 and phosphatidylcholine-phosphatidylethanolamine ratio. 628 73

One molecular form of cytochrome P-450IIA from liver microsomes of guinea pigs treated with 3-methylcholanthrene was purified to a specific content of 17.4 nmoles/mg of protein. The difference spectrum of reduced hemoprotein-carbon monoxide complex of this cytochrome exhibits an absorption maximum at 448 nm. The absolute absorption spectrum of the oxidized form of this hemoprotein suggests a high-spin state of heme iron. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein shows a single band of polypeptide stained with Coomassie brilliant blue at the position corresponding to Mr 54,000. On the other hand, the other two forms of cytochrome P-450, cytochrome P-450I and P-450IIB, were also separated and purified to specific contents of 8.7 and 5.2 nmoles/mg of protein, respectively. Both cytochrome P-450I and P-450IIB exhibit absorption maxima at 450 nm in the difference spectrum of reduced hemoprotein-carbon monoxide complex, and a low-spin state of ferric iron in the heme. The spectrophotometrical property of cytochrome P-450I and P-450IIB was clearly different from that of cytochrome P-450IIA. Molecular activities of a reconstituted aryl hydrocarbon hydroxylase (EC 1.14.14.1) containing, respectively, cytochrome P-450I, P-450IIA, and P-450IIB were 0.224, 0.250, and 0.395 (moles per minute per mole of cytochrome P-450), and were estimated to be one-tenth that of cytochrome P-448 induced in rat liver by 3-methylcholanthrene, indicating the presence of the low inducibility by 3-methylcholanthrene of aryl hydrocarbon hydroxylase in liver microsomes of guinea pigs.
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PMID:Purification and characterization of three forms of microsomal cytochrome P-450 in liver from 3-methylcholanthrene-treated guinea pigs. 630 24

The yeast Saccharomyces cerevisiae, produces a cytochrome P-450 enzyme with a Soret peak in the reduced-CO difference spectrum at 448 nm. The enzyme purified to homogeneity (88-97% pure on a specific content basis) has a molecular wt. of 55 500 as determined by SDS-PAGE. Amino acid analysis of yeast cytochrome P-448 revealed 407 amino acid residues per molecule with a 43% complement of hydrophobic residues. Although the number of residues is smaller than cytochrome P-448 enzymes from mammalian sources, the percentage of hydrophobic residues is almost identical. Estimation of the haem content of yeast cytochrome P-448 showed that one haem group was present per molecule. Phospholipid was present at very low levels. The molecular wt. of the polypeptide chain plus an estimated 5-6 units of hexose and of hexosamine is in good agreement with the molecular wt. value obtained from SDS-PAGE. A reconstituted system of purified cytochrome P-448, purified NADPH-cytochrome P-450 (c) reductase and phospholipid showed aryl hydrocarbon hydroxylase activity towards benzo[a]pyrene. Both protein components, NADPH and dilauroyl phosphatidylcholine (or emulgen 911) were necessary for full activity. The NADPH requirement could be replaced by cumene hydroperoxide or H2O2 generated in situ from a glucose oxidase system; in each case Vmax is increased, but the apparent affinity for benzo[a]pyrene, as measured by an increased Km, is lowered. The spin state of purified yeast cytochrome P-448 was 94% low spin (22 degrees C) as determined from the temperature-dependent spin-state equilibrium. The addition of benzo[a]pyrene to this enzyme resulted in a change to higher spin state (18% high spin at 22 degrees C). Equilibrium gel filtration analysis of the number of benzo[a]pyrene binding sites per mole of enzyme monomer showed a value of 1 for purified yeast cytochrome P-448 and 6 for this enzyme in microsomal form. The corresponding values for purified and microsomal cytochrome P-450 from phenobarbital-pretreated rats are 1 and 6, respectively. However, purified cytochrome P-448 from beta-naphthoflavone-induced rats gave a value of 6 benzo[a]pyrene binding sites. Type I binding spectra with purified yeast cytochrome P-448 were observed with benzo[a]pyrene, lanosterol, ethylmorphine, dimethylnitrosamine, sodium phenobarbitone and perhydrofluorene. Type II spectral changes were observed with imidazole, aniline and benzphetamine. Cytochrome P-448 from Saccharomyces cerevisiae is identified as a distinct enzyme of the P-450 family. This enzyme however has many properties in common with cytochrome P-448 from mammalian sources.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Studies on the properties of highly purified cytochrome P-448 and its dependent activity benzo[a]pyrene hydroxylase, from Saccharomyces cerevisiae. 632 93

Glutathione (GSH) is a strong nucleophile which reacts well with soft electrophiles, but poorly with both weak and strong electrophiles. Weak electrophiles have low reactivity with all nucleophiles while strong electrophiles react well with weak nucleophiles including superabundant H(2)O. There are enzymes, the GSH transferases, which catalyze GSH conjugation with all the types of electrophiles described above. In order to deal with the wide variety of potential substrates, a multiplicity of GSH transferases exists-each tissue having its own collection and each enzyme having a different substrate specificity. These enzymes are often very abundant, e.g., in the rat liver cytosol, their concentration is 0.2 mM. THE FOLLOWING SUBSTRATES ARE CONSIDERED IN SOME DETAIL: 1-chloro-2,4-dinitrobenzene, the electrophile derived metabolically from paracetamol N-acetyliminoquinone?), benzo(a)pyrene-4-5-oxide, cholesterol-5alpha,6alpha-oxide, benzo(a)pyrene-7,8-diol-9,10-oxide and the electrophiles derived metabolically from aflatoxin B(1) (the 2,3-oxide?). According to the substrate, optimal enzyme rates vary over seven orders of magnitude from 10(-5) to 10(-12) mole/min/mg. Despite the wide embrace of the GSH transferases, not all metabolically produced electrophiles are substrates. We know of the following examples: N-methylol-4-aminoazobenzene and its 4'-hydroxy derivative (these are soft electrophiles and react well with GSH noncatalytically), N-sulfonyloxy-N-methyl-4-aminoazobenzene, N-sulfonyloxy-N-acetyl-2-aminofluorene (these are strong electrophiles which do not react selectively with GSH) and N-hydroxy-2-aminofluorene which appears to react only slowly with GSH. It is of interest in the present context that all these compounds are derived from either arylamine or arylamide carcinogens. Whether the reaction be enzymic or nonenzymic, conjugation with GSH is a very important means of detoxication accounting in some cases for up to 60% of the biliary metabolites. As seen in the example of aflatoxin B(1), very low enzymic rates observed in vitro are sufficient to account for apparently high rates of biliary excretion of GSH conjugates.GSH transferases have evolved other functions apart from the catalysis of GSH conjugation. GSH transferase B participates in the hepatic uptake of bilirubin and the intracellular distribution of the heme prosthetic group. It also has GSH peroxidase activity which suggests that it might participate in the detoxication of by-products of oxygen utilization including those produced by the action of cytochrome P-450. It is shown that GSH transferase B inhibits lipid peroxidation in vitro.
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PMID:The role of glutathione in detoxication. 633 28

NADPH-cytochrome P-450 reductase and two purified isozymes of cytochrome P-450 have been incorporated into phospholipid vesicles by a cholate dialysis technique. The enzyme system reconstituted in this manner was catalytically active. The observed kinetics for substrate oxidation indicated that both enzymes were associated with the liposomal membranes, and were not simply entrapped in the interior of the vesicle. The N-demethylation of benzphetamine was measured in order to determine the effect of variations in the mole ratio between the two enzymes and between the lipid and the total enzyme on the observed steady-state kinetics. In addition, the kinetic isotope effects for the O-deethylation of 7-ethoxycoumarin were measured in order to compare these parameters to those previously observed in a reconstituted system [G. T. Miwa, and A. Y. H. Lu (1981) Arch. Biochem. Biophys. 211, 454-458]. The results were all consistent with the association of the two proteins by lateral diffusion in the vesicle membrane. Moreover, the observed reduction in catalytic activity, as the enzymes were diluted in the vesicle membrane, can only be explained by the formation of a transient P-450-reductase complex, and not by the existence of a stable complex between the two proteins. These results provide compelling evidence for a mass action model for the interaction of these two enzymes in liposomal membranes.
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PMID:The association of cytochrome P-450 and NADPH-cytochrome P-450 reductase in phospholipid membranes. 643 33


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