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)

About an eightfold increase in protamine kinase activity was detected following extraction of highly purified microsomes from bovine kidney with 1% Triton X-100. Relative to the soluble fraction, the microsomes contained about 30% protamine kinase activity. The microsomal protamine kinase was purified to apparent homogeneity. The purified enzyme exhibited an apparent M(r) approximately 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-200. Relative to protamine, the purified kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5, 8, and less than 0.1% activity with casein, histone H2B, and histone H1, respectively. The purified kinase phosphorylated several 40 S ribosome polypeptides. One of these polypeptides was identified as ribosomal protein S6 by N-terminal sequencing. About 2.5 mol of phosphoryl groups was incorporated per mole of ribosomal protein S6 following incubation of the 40 S ribosomes with the purified kinase. Following incubation with protein phosphatase 2A2, purified preparations of the protamine kinase were inactivated. These properties were identical to those of purified preparations of a protamine kinase from extracts of bovine kidney cytosol (Z. Damuni, G.D. Amick, and T.R. Sneed, 1989, J. Biol. Chem. 264, 6412-6418). Near identical peptide patterns were obtained following incubation of purified preparations of the microsomal and cytosolic protamine kinases with Staphylococcus aureus V8 proteinase. The results indicate that a form of the cytosolic protamine kinase is present in microsomes.
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PMID:Purification and properties of a protamine kinase from bovine kidney microsomes. 132 15

Microsomal P450s catalyze the monooxygenation of a large variety of hydrophobic compounds, including drugs, steroids, carcinogens, and fatty acids. The interaction of microsomal P450s with their electron transfer partner, NADPH-P450 reductase, during the transfer of electrons from NADPH to P450, for oxygen activation, may be important in regulating this enzyme system. Highly purified Bacillus megaterium P450BM-3 is catalytically self-sufficient and contains both the reductase and P450 domains on a single polypeptide chain of approximately 120,000 Da. The two domains of P450BM-3 appear to be analogous in their function and homologous in their sequence to the microsomal P450 system components. FAD, FMN, and heme residues are present in equimolar amounts in purified P450BM-3 and, therefore, this protein could potentially accept five electron equivalents per mole of enzyme during a reductive titration. The titration of P450BM-3 with sodium dithionite under a carbon monoxide atmosphere was complete with the addition of the expected five electron equivalents. The intermediate spectra indicate that the heme iron is reduced first, followed by the flavin residues. Titration of the protein with the physiological reductant, NADPH, also required approximately five electron equivalents when the reaction was performed under an atmosphere of carbon monoxide. Under an atmosphere of argon and in the absence of carbon monoxide, one of the flavin groups was reduced prior to the reduction of the heme group. The titration behavior of P450BM-3 with NADPH was surprising because no spectral changes characteristic of flavin semiquinone intermediates were observed. The results of the titration with NADPH can only be explained if (a) there was "rapid" intermolecular electron transfer between P450BM-3 molecules, (b) there is no kinetic barrier to the reduction of P450 by the one-electron-reduced form of the reductase, and (c) the "air-stable semiquinone" form of the reductase does not accumulate in this complex multidomain enzyme.
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PMID:P450BM-3: reduction by NADPH and sodium dithionite. 156 20

Chlorophyllin (CHL), a copper/sodium salt of chlorophyll used in the treatment of geriatric patients, is an anti-mutagen that has been demonstrated to inhibit carcinogen--DNA binding in vivo. To study the mechanism of inhibition, the microsomal metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and the kinetics of IQ--DNA binding were investigated in the presence and absence of CHL. In time-course studies, CHL produced greater than 80% inhibition of IQ--DNA binding and blocked the metabolism of IQ, such that 80% of the initial dose of carcinogen was recovered unmetabolized from the incubations after 1 h. Kinetic constants were determined for the in vitro DNA binding reaction, with the reaction rate measured as 'pmol IQ bound/mg DNA/min/mg microsomal protein'. Without altering V(max), the Km of the IQ--DNA binding reaction was increased by CHL, and the replot of Km/V(max) versus CHL concentration yielded a straight line with an inhibitor constant of 58.3 microM CHL. Spectrophotometric studies provided evidence in vitro for the formation of a non-covalent complex between CHL and IQ. The CHL--IQ complex had a stoichiometric ratio of 2:1 (mole ratio method) and an apparent dissociation constant from the Benesi-Hilderbrand plot of 1.41 x 10(-4)M at pH 7.4. These results are discussed in the context of a CHL inhibitory mechanism involving enzyme inhibition and molecular complex formation.
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PMID:Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA binding by chlorophyllin: studies of enzyme inhibition and molecular complex formation. 163 77

Human hepatic microsomal enzymes catalyzed the NADPH-dependent anaerobic reductive activation of [1-14C]metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] and [4,5-14C]ronidazole [(1-methyl-5-nitroimidazole-2-yl)methyl carbamate] to species that became covalently bound to proteins. Due to the low efficiency of the enzyme-catalyzed covalent binding of metronidazole, the stoichiometry of anaerobic reductive activation was studied with dithionite as the reductant. Two moles of dithionite was consumed per mole of [1-14C]metronidazole for maximal covalent binding to either DNA or immobilized sulfhydryl groups, demonstrating that four electrons are required for the reductive activation of metronidazole. These data implicate the involvement of a hydroxylamine in covalent binding. Maximal covalent binding of [4,5-14C]ronidazole to DNA also required four-electron reduction, consistent with previous studies of the covalent binding of this agent to immobilized sulfhydryl groups [Kedderis et al. (1988) Arch. Biochem. Biophys. 262, 40-48]. Studies of the covalent binding of variously radiolabeled ronidazole molecules to DNA suggested that the imidazole ring was intact while greater than 80% of the 2-carbamoyl group and the C4 proton were not present in the DNA adduct. Studies of both the chemical and human hepatic microsomal reduction of [4-3H]metronidazole demonstrated that covalent binding occurred with the stoichiometric loss of this label, implicating binding at the C4 position. These results suggest that the reductive activation of 5-nitroimidazoles generally proceeds via four-electron reduction to form hydroxylamines followed by nucleophilic attack at C4.
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PMID:Covalent interaction of 5-nitroimidazoles with DNA and protein in vitro: mechanism of reductive activation. 251 18

A variety of cardiovascular drugs were treated with 10 equivalent amounts of nitrite in acidic solutions. Among 18 drugs, a preparation of bamethan [1-(4-hydroxyphenyl)-1-hydroxy-2-butylaminoethane] showed strikingly high mutagenicity by this treatment toward Salmonella typhimurium TA98 and TA100 strains. Treatment of bamethan with an equivalent amount of nitrite gave N-nitrosobamethan I which was not mutagenic. However, treatment of bamethan with 4 equivalent amounts of nitrite afforded a highly mutagenic compound II, which was identified as 3-diazo-N-nitrosobamethan by its physicochemical analysis and chemical properties. Specific mutagenic activity of II was 9200 His+ revertants/mu mole toward TA98 and 8060 His+ revertants/mu mole toward TA100. Addition of microsomal system little affected the activity. Bamethan is administrated orally during long period for treatment of cardiovascular diseases. It is noted that this drug can produce the highly mutagenic diazo compound by reaction with nitrite which is present in digestive tracts.
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PMID:Formation of a highly mutagenic diazo compound from the bamethan-nitrite reaction. 288 Dec 2

Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP. Pf was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75 mM inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3 +/- 0.1 sec (SEM, seven preparations, 23 degrees C), corresponding to a Pf of 5 x 10(-4) cm/sec; the activation energy (Ea) for Pf was 17.6 +/- 0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised approximately 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6 +/- 2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membranes) and 0.190 (microsomes), and were unaffected by VP. We conclude: (1) granules have among the lowest water permeabilities of biological membranes, (2) granule water permeability is not altered by bladder pretreatment with VP, (3) granule membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.
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PMID:Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules. 314 39

The specificity of phosphatidylethanolamine (PE) N-methyltransferase for molecular species of PE has been investigated. Phosphatidylcholine (PC), synthesized by incubation of [methyl-3H]S-adenosyl-L-methionine with microsomes or pure enzyme (Ridgway, N. D., and Vance, D. E. (1987) J. Biol. Chem. 262, 17231-17239) plus microsomal PE, had a distribution of methyl label in molecular species similar to the mole percent distribution of molecular species in the precursor PE. A similar lack of specificity was observed with PE that was synthesized from egg PC by transphosphatidylation with phospholipase D. Phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME), both with the acyl composition of egg PC, were methylated by the pure enzyme and showed a distribution of labeled molecular species in PDME and PC, respectively, similar to the mole percent distribution of egg PC. Results with synthetic PEs and pure methyltransferase showed higher rates of methylation with more unsaturated species. Long chain saturated PEs (e.g. dipalmitoyl-PE) were not methylated by the enzyme. Maximal methylation rates were obtained with two or more double bonds in the substrate PE. Rates of methylation of the saturated and monoenoic PEs could be enhanced when 40 mol % polyunsaturated-rich microsomal PC was included in the mixed micelles. PC isolated from primary cultures of rat hepatocytes pulsed with [methyl-3H]methionine was analyzed by high performance liquid chromatography. Initially, the labeling pattern of PC molecular species varied slightly from that of total hepatocyte PE and hepatocyte microsomal PE. 1-Palmitoyl-2-docosahexaenoyl-PC had the highest specific activity at the end of the pulse and was preferentially labeled relative to the mole percent distribution of hepatocyte PE molecular species. During the 24-h chase period both the percent distribution of label and specific activity of this species of PC declined. In the same time period, there was a corresponding increase in specific activity and percent distribution of label in 1-palmitoyl and 1-stearoyl species with linoleate and arachidonate in the sn-2 position.
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PMID:Specificity of rat hepatic phosphatidylethanolamine N-methyltransferase for molecular species of diacyl phosphatidylethanolamine. 318 18

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

Oligodendroglia were isolated from bovine brain, and a "crude" microsomal fraction obtained from cell homogenates was subfractionated into myelin (MP), plasma membranes (PM), Golgi (GF), smooth (SER) and rough (RER) endoplasmic membranes using discontinuous-sucrose gradient centrifugation. The submicrosomal fractions were characterized by ultrastructural examination and analysis of the specific organelle markers. The myelin and plasma membrane rich fractions contained characteristically the highest amounts of the lipid with lower mole percentages of total phospholipids and phosphatidylcholine, and higher concentrations of phosphatidylethanolamine (+ plasmalogens), cholesterol and galactolipids. Considerable amounts of the typical myelin galactolipids (galacto-cerebrosides, sulfatides and monogalactosyl diglycerides) were also found in the Golgi fraction (GF). The GF fraction had the greatest enrichment of glycolipid-forming galactosyltransferases, and the distribution of these enzymes correlated well with that of the Golgi marker enzymes. The results give evidence that intracellular Golgi apparatus of oligodendroglia is rich in the myelin-specific lipids, and suggest its involvement in the synthesis and processing of myelin lipids.
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PMID:Studies on the submicrosomal fractions of bovine oligodendroglia: lipid composition and glycolipid biosynthesis. 340 84

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


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