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)

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

Oxidation of NADH by rat erythrocyte plasma membrane was stimulated by about 50-fold on addition of decavanadate, but not other forms of vanadate like orthovanadate, metavanadate aad vanadyl sulphate. The vanadate-stimulated activity was observed only in phosphate buffer while other buffers like Tris, acetate, borate and Hepes were ineffective. Oxygen was consumed during the oxidation of NADH and the products were found to be NAD+ and hydrogen peroxide. The reaction had a stoichiometry of one mole of oxygen consumption and one mole of H2O2 production for every mole of NADH that was oxidized. Superoxide dismutase and manganous inhibited the activity indicating the involvement of superoxide anions. Electron spin resonance in the presence of a spin trap, 5, 5'-dimethyl pyrroline N-oxide, indicated the presence of superoxide radicals. Electron spin resonance studies also showed the appearance of VIV species by reduction of VV of decavanadate indicating thereby participation of vanadate in the redox reaction. Under the conditions of the assay, vanadate did not stimulate lipid peroxidation in erythrocyte membranes. Extracts from lipid-free preparations of the erythrocyte membrane showed full activity. This ruled out the possibility of oxygen uptake through lipid peroxidation. The vanadate-stimulated NADH oxidation activity could be partially solubilized by treating erythrocyte membranes either with Triton X-100 or sodium cholate. Partially purified enzyme obtained by extraction with cholate and fractionation by ammonium sulphate and DEAE-Sephadex was found to be unstable.
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PMID:A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide. 608 22

NADH-ubiquinone (Q) reductase isolated from beef heart mitochondria exhibited, upon reduction by NADH, a prominent EPR signal at room temperature attributable to stable ubisemiquinone radical(s). The concentration of the ubisemiquinone radical reached as high as 40% of the total Q content in the reductase. The radical was virtually abolished by adding rotenone, whereas rotenone had no effect on the reduction of FMN by NADH. The radical showed an EPR signal of g = 2.0042 at approximately 9.5 GHz with no resolved hyperfine structure and had a line width of 6.8 Gauss at 23 degrees C. The Q-band EPR spectra at 35 GHz showed well resolved g-anisotropy and had a field separation between derivative extrema of 24 Gauss. These results substantiate the fact that this radical was bound to a protein; we call it ubiquinone protein-N (QP-N). The pH dependence of the EPR signals demonstrated that the species of the ubisemiquinone radical(s) consisted of not only an anionic form but also a neutral form. Only about half of the QP-N radical formed by NADH reduction was abolished by p-chloromercuric sulfonate. The microwave power saturation curve of the radical was biphasic; the first phase leveled off at about 5 milliwatts and then at about 20 milliwatts. These results suggested that the ubisemiquinone radical from QP-N was heterogenous, consisting of at least two populations of stable ubisemiquinone radical(s). It is suggested that two kinds of QP-N exist in NADH-Q reductase. Each mole of protein may bind two mol of Q.
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PMID:Evidence of an ubisemiquinone radical(s) from the NADH-ubiquinone reductase of the mitochondrial respiratory chain. 629 5

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme, which is a tetramer both in the mitochondrial inner membrane and as the purified enzyme reconstituted with phospholipid. For the active enzyme-phospholipid complex in the absence of ligands, we previously found that reaction with N-ethylmaleimide (at 5 mol/mol of enzyme subunit) resulted in progressive loss of enzymic activity with an inactivation stoichiometry of 1 equiv of sulfhydryl derivatized per mole of enzyme and a maximum derivatization of 2 equiv [Latruffe, N., Brenner, S. C., & Fleischer, S. (1980) Biochemistry 19, 5285-5290]. We now find, in the presence of nucleotide or substrate, that the rate of inactivation is significantly reduced, which indicates that these ligands afford protection of the essential sulfhydryl. Further, in the presence of ligands, the inactivation stoichiometry is 0.5, consistent with half-of-the-site reactivity of the essential sulfhydryl. Thus, at a low ratio of N-ethylmaleimide to enzyme, nucleotide or substrate affords essentially complete protection of the nonessential sulfhydryl from derivatization. The binding characteristics of NADH to both the native and N-ethylmaleimide-derivatized enzyme have been compared by fluorescence spectroscopy. Quenching of intrinsic tryptophan fluorescence of the protein shows that the enzyme, derivatized with N-ethylmaleimide either in the absence or in the presence of NAD+, binds NADH but with a reduced Kd (approximately 50 microM as compared with approximately 20 microM for native enzyme). However, a critical change has occurred in that resonance energy transfer from protein to bound NADH, observed in the native enzyme, is abolished in the N-ethylmaleimide-derivatized enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Half-site reactivity of an essential thiol group of D-beta-hydroxybutyrate dehydrogenase. 650 17

A transmembrane electron transport system has been studied in HeLa cells using an external impermeable oxidant, ferricyanide. Reduction of ferricyanide by HeLa cells shows biphasic kinetics with a rate up to 500 nmoles/min/g w.w. (wet weight) for the fast phase and half of this rate for the slow phase. The apparent Km is 0.125 mM for the fast rate and 0.24 mM for the slow rate. The rate of reduction is proportional to cell concentration. Inhibition of the rate by glycolysis inhibitors indicates the reduction is dependent on glycolysis, which contributes the cytoplasmic electron donor NADH. Ferricyanide reduction is shown to take place on the outside of cells for it is affected by external pH and agents which react with the external surface. Ferricyanide reduction is accompanied by proton release from the cells. For each mole of ferricyanide reduced, 2.3 moles of protons are released. It is, therefore, concluded that a transmembrane redox system in HeLa cells is coupled to proton gradient generation across the membrane. We propose that this redox system may be an energy source for control of membrane function in HeLa cells. The promotion of cell growth by ferricyanide (0.33-0.1 mM), which can partially replace serum as a growth factor, strongly supports this hypothesis.
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PMID:Properties of a transplasma membrane electron transport system in HeLa cells. 653 37

We have examined the interaction of hepatic phenylalanine hydroxylase with the phenylalanine analogs, tryptophan and the diastereomers of 3-phenylserine (beta-hydroxyphenylalanine). Both isomers of phenylserine are substrates for native phenylalanine hydroxylase at pH 6.8 and 25 degrees C, when activity is measured with the use of the dihydropteridine reductase assay coupled with NADH in the presence of the synthetic cofactor, 6-methyl-5,6,7,8-tetrahydropterin. However, while erythro-phenylserine exhibits simple Michaelis-Menten kinetics (Km = 1.2 mM, Vmax = 1.2 mumol/min X min) under these conditions, the threo isomer exhibits strong positive cooperativity (S0.5 = 4.8 mM Vmax = 1.4 mumol/min X mg, nH = 3). Tryptophan also exhibits cooperativity under these conditions (S0.5 = 5 mM, Vmax = 1 mumol/min X mg, nH = 3). The presence of 1 mM lysolecithin results in a hyperbolic response of phenylalanine hydroxylase to tryptophan (Km = 4 mM, Vmax = 1 mumol/min X mg) and threo-phenylserine (Km = 2 mM, Vmax = 1.4 mumol/min X mg). erythro-Phenylserine is a substrate for native phenylalanine hydroxylase in the presence of the natural cofactor, L-erythro-tetrahydrobiopterin (BH4) (Km = 2 mM, Vmax 0.05 mumol/min X mg, nH = 2). Preincubation of phenylalanine hydroxylase with erythro-phenylserine results in a 26-fold increase in activity upon subsequent assay with BH4 and erythro-phenylserine, and hyperbolic kinetic plots are observed. In contrast, both threo-phenylserine and tryptophan exhibit negligible activity in the presence of BH4 unless the enzyme has been activated. The product of the reaction of phenylalanine hydroxylase with either isomer of phenylserine was identified as the corresponding p-hydroxyphenylserine by reaction with sodium periodate and nitrosonaphthol. With erythro-phenylserine, the hydroxylation reaction is tightly coupled (i.e. 1 mol of hydroxyphenylserine is formed for every mole of tetrahydropterin cofactor consumed), while with threo-phenylserine and tryptophan the reaction is largely uncoupled (i.e. more cofactor consumed than product formed). Erythro-phenylserine is a good activator, when preincubated with phenylalanine hydroxylase (A0.5 = 0.2 mM), with a potency about one-third that of phenylalanine (A0.5 = 0.06 mM), while threo-phenylserine (A0.5 = 6 mM) and tryptophan (A0.5 approximately 10 mM) are very poor activators. Addition of 4 mM tryptophan or threo-phenylserine or 0.2 mM erythro-phenylserine to assay mixtures containing BH4 and phenylalanine results in a dramatic increase in the hydroxylation at low concentrations of phenylalanine.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The interaction of aromatic amino acids with rat liver phenylalanine hydroxylase. 670 37

When isolated rat pancreatic islets are exposed to L-leucine (20 mM), the rate of NH4 production is close to the summed rates of L-[1-14C] leucine decarboxylation and alpha-ketoisocarproate production, whereas the rates of acetoacetate production and L-[U-14C]-leucine oxidation are compatible with conversion of each mole of the amino acid to one mole of acetoacetate and three moles of CO2. ATP content, ATP/ADP ratio, and adenylate charge are maintained at normal values by L-leucine, whereas the NADH/NAD+ ratio (but not the NADPH/NADP+ ratio) is significantly increased. The release of insulin evoked by L-leucine is potentiated by 2-ketoisovalerate, unaffected by L-valine, and inhibited by menadione. L-leucine mimicks the effect of D-glucose on 86Rb+ and 45Ca2+ handling by the islets. However, relative to its rate of oxidation, the insulinotropic effect of L-leucine is less marked than that of D-glucose. This may be due, in part at least, to a decrease in the oxidation of endogenous nutrients. It is concluded that the metabolic, cationic, and secretory effects of L-leucine in isolated islets are not incompatible with the fuel hypothesis for insulin release.
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PMID:The stimulus-secretion coupling of amino acid-induced insulin release: metabolism and cationic effects of leucine. 676 28

The 7 alpha-dehydroxylation of primary bile acids by Eubacterium sp. V.P.I. 12708 required a cell extract prepared from a cholic acid-induced culture and NAD+. NADH (0.5 mM) inhibited bile acid 7-dehydroxylase activity more than 50% when added to reaction mixtures containing NAD+ (0.5 mM). Saturation kinetics and double reciprocal plots of NADH inhibition were consistent with negative cooperativity. 7-Dehydroxylase activity was modulated by the molar ratio of NAD+-NADH with maximal activity at a NAD+ mole fraction of 0.75 to 0.85. NADH stimulated 7-dehydroxylase activity (30% to 50%) at low concentration (less than 0.15 mM) and inhibited at higher concentrations. Reduction of the proposed delta 6-intermediate (3 alpha-hydroxy-5 beta-6-cholen-24-oic acid) to lithocholic acid required a cell extract from a cholic acid-induced culture and was stimulated by the addition of NAD+. Reduced flavin nucleotides stimulated (32% to 62%) and NADH (0.5 mM) inhibited (78%) the reduction of the delta 6-intermediate to lithocholic acid. 7-Dehydroxylase was highly specific for bile acid substrates and required a free C-24 carboxyl group and an unhindered 7 alpha- or 7 beta-hydroxy group on the B-ring of the steroid nucleus for activity. Bile acid 7 alpha- and 7 beta-dehydroxylase and delta 6-reductase activities all co-eluted from an anaerobic high performance liquid chromatography gel filtration column. However, approximately 80% to 96% of the total units of activity were lost. A substantial portion (20% to 30%) of the total activity was recovered when material from low molecular weight (8,000 to 14,000 Mr) eluting fractions was added back to fractions containing enzyme activity. These studies show that 7-dehydroxylase is highly specific for substrates and its activity may be regulated by the NAD+-NADH ratio in the bacterial cell.
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PMID:Regulation of bile acid 7-dehydroxylase activity by NAD+ and NADH in cell extracts of Eubacterium species V.P.I. 12708. 683 78

Alloxan behaves as a substrate for NADH:ferricytochrome b5 oxidoreductase (EC 1.6.2.2). The apparent Km for alloxan was 10 mM in liver microsomes and 20 mM with the enzyme prepared by lysosomal digestion. The apparent Km for NADH was the same with microsomes and the isolated enzyme (30 microM). The maximum turnover rate was calculated as 426 moles electrons/min X mole enzyme. Cytochrome b5 was shown to reduce alloxan nonenzymatically.
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PMID:Reduction of alloxan by microsomal electron transport proteins. 688 44

The respiratory chain-linked external NADH dehydrogenase has been isolated from Candida utilis in highly purified form. The enzyme is soluble and has a molecular weight of approx. 1.5 x 10(6). The enzyme contains two moles of FMN per mole of enzyme and is composed of two large subunits of mol. wt. 270 000 and eight smaller subunits of mol. wt. 135 000. Iron and copper are present in the preparations, but appear to be contaminants. The enzyme catalyzes the oxidation of NADH and NADPH at nearly equal rates and reacts readily with 2,6-dichlorophenolindophenol, CoQ6 and CoQ1 derivatives as acceptors. Rotenone (10(-5) M) and seconal (10(-3) M) do not inhibit enzymatic activity.
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PMID:Electron transport systems of Candida utilis: purification and properties of the respiratory chain-linked external NADH dehydrogenase. 719 Apr 38


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