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)

D-Erythrulose reductase of beef liver was crystallized from ammonium sulfate solution at pH 8.17. The crystals are needle-shaped. The enzyme protein contains 851 amino acid residues per mole of the enzyme: Lys28, His11, Arg52, Asp79, Thr58, Ser56, Glu68, Pro20, Gly80, Ala107, Val112, Met24, Ile31, Leu88, Tyr7, Phe22, Trp4, and Cys4. The enzyme is inactivated by exposure to temperatures below 12degrees. The inactivation is accelerated by increasing the salt concentration and decreasing the enzyme concentration. The pH of the medium also has a pronounced effect, the maximum stability of the enzyme is obtained at pH 8.5. NADP+ protected the enzyme from cold inactivation at all stages of the process and also afforded protection against inactivation by heat and pH. The cold inactivation of the enzyme is accompanied by dissociation of the enzyme protein to subunits.
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PMID:Studies on D-tetrose metabolism. VI. Crystallization and some properties of D-erythrulose reducatase from beef liver. 0 10

Structural and conformational organization of chicken liver fatty acid synthetase has been probed using its fluorescent coenzyme, NADPH. Three NADPH binding sites per mole of the enzyme complex, of apparently identical dissociation constant (KD = 0.6 muM) can be titrated at temperatures above 12 degrees. These results are in disagreement with the earlier studies of Hsu and Wagner (Hsu, R. Y., and Wagner, B. J. (1970) Biochemistry, 9, 245-251) in which four such sites could be titrated. At 12 degrees, the composite sites split into two subsets: a pair of sites with a KD of 0.3 muM and a third site with a Kd of 1.1 muM. At lower temperatures (5 degrees or 2 degrees), the site with weak affinity disappears, leaving a pair of sites with a Kd of 0.5 muM. Similar observations were made when the enzyme was modified with phenylmethylsulfonyl fluoride, a specific and selective inhibitor of fatty acyl-CoA deacylase (s) of the pigeon liver enzyme complex (Kumar, S. (1975) J. Biol. Chem. 250, 5150-5158). Partial modification with phenylmethylsulfonyl fluoride elicits a NADPH binding response similar to the binding observed at 12 degrees, i.e. two sets of binding sites with nonidentical dissociation constants. Further modification corresponding to the complete loss of deacylase function results in a set of two apparently identical binding sites, and the third site is not available for titration. The modified enzyme retains the two reductase functions as measured by the model substrates, acetoacetyl-N-acetylcysteamine and crotonyl-CoA. Furthermore, the addition of acetyl- and malonyl-CoA (100 muM each) to the modified enzyme lowers the NADPH binding affinity by a factor of 3. Other observations show that the quantum yield, as measured by the ratio of fluorescence intensity of bound and free NADPH, changes with temperature and ionic strength. Lowering the temperature from 30 degrees to 2 degrees increases the enhancement ratio by 50%, whereas increase in ionic strength from 0.05 to 0.2 M potassium phosphate lowers it to 50% of the original level. Measurement of NADPH binding in the presence of NADP+, NADH, NAD+ and adenosine-2'-monophospho-5'-diphosphoribose demonstrates that NADP+ shows competitive behavior for NADPH sites (KD = 10.6 muM), whereas NADH and NAD+ show noncompetitive (KD (apparent) = nearly 600 muM) and rather complicated interactions implicating nonspecific conformational alteration of the enzyme complex. The behavior of adenosine 2'-monophospho-5'-diphosphoribose is intermediate between NADP+ and NADH. These data are discussed in terms of substrate-mediated conformational changes and the moles of each of the reductase enzymes per mole of the enzyme complex, the polarity of the NADPH binding region, and the probable structure of the nicotinamide moiety when bound to the enzyme.
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PMID:Reduced nicotinamide adenine dinucleotide phosphate, a structural and conformational probe of chicken liver fatty acid synthetase. 0 63

A flavoprotein catalyzing the reduction of cytochrome c by NADPH was solubilized and purified from microsomes of yeast grown anaerobically. The cytochrome c reductase had an apparent molecular weight of 70,000 daltons and contained one mole each of FAD and FMN per mole of enzyme. The reductase could reduce some redox dyes as well as cytochrome c, but could not catalyze the reduction of cytochrome b5. The reductase preparation also catalyzed the oxidation of NADPH with molecular oxygen in the presence of a catalytic amount of 2-methyl-1,4-naphthoquinone (menadione). The Michaelis constants of the reductase for NADPH and cytochrome c were determined to be 32.4 and 3.4 micron M, respectively, and the optimal pH for cytochrome c reduction was 7.8 to 8.0. It was concluded that yeast NADPH-cytochrome c reductase is in many respects similar to the liver microsomal reductase which acts as an NADPH-cytochrome P-450 reductase [EC 1.6.2.4].
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PMID:Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase. 1 31

The aerobic NADPH reduction of the Triton N-101 disintegrated cytochrome P-450LM system has been studied. At this organization level--the components being dispersed in solution--a first-order monophasic reaction is exhibited. Neither the complex formation of cytochrome and reductase, respectively, nor preceding diffusion is rate limiting. The initial rate follows the ratio reductase/P-450 (mole/mole) thus indicating a Michaelis-Menten type enzyme mechanism. A model treatment of the reaction fits the systems behaviour as a whole. A multiparameter equilibrium state and different specified time function equations were developed for the determination of individual step rate constants and other system parameters as well.
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PMID:Kinetics of elementary steps in the cytochrome P-450 reaction sequence. IV. Mechanism of the NADPH reduction reaction of cytochrome P-450LM. 4 46

NAD(P)H: FMN oxidoreductase (flavin reductase) couples in vitro to bacterial luciferase. This reductase, which is also postulated to supply reduced flavin mononucleotide in vivo as a substrate for the bioluminescent reaction, has been partially purified and characterized from two species of luminous bacterial. From Photobacterium fischeri the enzyme has a M. W. determined by Sephadex gel filtration, of 43,000 and may have a subunit structure. The turnover number at 20 degrees C, based on a purity estimate of 20 percent, is 1.7 times 10-4 moles of NADH oxidized per min per mole of reductase. The reductase isolated from Beneckea harveyi has an apparent molecular weight of 23,000; its purity was too low to permit estimation of specific activity. Using a spectrophotometric assay at 340 nm with the P. fischeri reductase, both NADH (Km, 8 times 10-5 M) and NADPH (Km, 4 times 10-4 M) were enzymatically oxidized, the Vmax with NADH being approximately twice that of NADPH. Of the flavins tested in this assay, only FMN (Km, 7.3 times 10-5 M) and FAD (Km, 1.4 times 10-4 M) were effective, FMN having a Vmax three times that of FAD. In the coupled assay, i.e., measuring the bioluminescence intensity of the reaction with added luciferase, the optimum FMN concentration was nearly 100 times less than in the spectrophotometric assay. The studies reported suggest the existence of a functional reductase-luciferase complex.
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PMID:Flavin mononucleotide reductase of luminous bacteria. 4 4

The interaction between cytochrome P-450 and NADPH-cytochrome c reductase during catalysis has been investigated with a reconstituted monooxygenase system composed of the two purified enzyme components and synthetic phospholipid. Steady state kinetic data are consistent with a scheme in which the formation of a binary complex between the two proteins precedes catalysis. The formation of this binary complex is described by a simple mass action equation. In agreement with this equation, the observed Vmax for benzphetamine N-demethylation was found to be directly proportional to the calculated concentration of the cytochrome P-450 . reductase complex. Furthermore, with appropriate reductase/cytochrome P-450 mole ratios, the Vmax could be shown to be linearly dependent on either the reductase or the cytochrome P-450 concentration alone. In contrast, the Km parameter is independent of the complex concentration, indicating that no change in the rate-limiting step has occurred. Thus a distinction should be made between a rate-limiting enzyme component and the rate-limiting step in this multienzyme system.
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PMID:Studies on the association of cytochrome P-450 and NADPH-cytochrome c reductase during catalysis in a reconstituted hydroxylating system. 10 41

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.
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PMID:Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes. 17 49

Dihydropteridine reductase [EC 1.6.99.7] was purified from bovine liver in 50% yield and crystallized. The physicochemical properties of the purified enzyme were quite similar to those of sheep liver dihydropteridine reductase. During the course of purification, however, the enzyme was found to be separated into 2 major peaks together with minor peaks by column chromatography on CM-Sephadex, and one of the major peaks was identified as a binary complex of the enzyme with NADH. The reductase-NADH complex was also prepared in vitro and crystallized. Upon addition of quinonoid-dihydropterin to the complex, NADH was oxidized and released from the enzyme. The amount of bound NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH complex in bovine liver extract as a predominant form was in accord with the pyridine nucleotide specificity for NADH as a coenzyme. The results further support the view that NADH is the natural coenzyme of this reductase.
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PMID:Dihydropteridine reductase from bovine liver. Purification, crystallization, and isolation of a binary complex with NADH. 19 36

15-Hydroxyprostaglandin dehydrogenase was isolated from human term placenta up to a final purification of 380-fold. A spec. act. of 2000 mU/mg of protein was reached. The preparation was not homogeneous as judged by analytical disc electrophoresis. The enzyme could be stored in the presence of 50% glycerol and 10mM 2-mercaptoethanol without any loss of activity for at least one year. A distinct single protein band stained after discontinuous polyacrylamide gel electrophoresis was shown by enzymatic activity staining to correspond to 15-hydroxyprostaglandin dehydrogenase activity. Thus no evidence for the exitstence of isoenzymes was obtained. The protein in the final preparation steps showed neither alcohol dehydrogenase, NAD reductase, nor NADH oxidase activity, nor enzymatic conversion of prostaglandin or 15-oxoprostaglandin in the absence of NAD and NADH. No spontaneous reactions between NAD and prostaglandin or NADH and 15-oxoprostaglandin were detectable in the absence of the enzyme. Ethanol and glycerol slightly inhibited the reaction. Various buffers (Tris/HC1, potassium phosphate, HEPES, and triethanolamine) and salts (ammonium chloride, ammonium sulfate, potassium chloride, and sodium chloride) had different effects on the reaction rate. The pH profile of the reaction shows a plateau between pH 7.0 and 7.8 and a steep maximum at pH 9.5. A linear Arrhenius plot was obtained for the temperature dependence of the reaction from 20 to 37 degrees C. The molar activation enthalpy of the reaction was calculated to be 13.1 kcal/mole. The molecular weight of 15-hydroxyprostaglandin dehydrogenase was estimated to be 32000 -/+ 3000 by gel filtration on Sephadex G-150 in the presence of 10mM mercaptoethanol.
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PMID:[15-Hydroxyprostaglandin dehydrogenase from human placenta. 1. Isolation and characterization]. 24 91

The acetylation of spinach ferredoxin by acetic anhydride modified about four moles of amino groups. The absorption spectra, CD spectra, the fluorescence of sole tryptophan residue and the biological activity of acetylated ferredoxin were investigated. An equilibrium existed between two different states, D- and N-form, of the acetylated ferredoxin and was dependent on the cation concentration. D-form completely reverted to N-form upon the binding of one mole of cation, Na+ or Mg2+. Although the N-form was indistinguishable from native ferredoxin in every property tested, the D-form was significantly different from the N-form or native ferredoxin and was very unstable, especially at low salt concentrations. It is suggested that the amino groups might be important in maintaining the protein conformation by forming salt linkages, but may not be essential for the activity. Furthermore, since the D-form, unlike the N-form and native ferredoxin, was inactive in the ferredoxin-NADP+ reductase [EC 1.6.7.1] assay system and had no inhibitory effect in this system, it was considered to be incapable of forming a complex with ferredoxin-NADP+ reductase. On the other hand, the N-form of the modified ferredoxin was as active as native ferredoxin. It is suggested that amino groups of spinach ferredoxin are not essential for the redox reaction of ferredoxin or for complex formation with the reductase.
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PMID:Chemical modification of spinach ferredoxin. Properties of acetylated spinach ferredoxin. 84 28


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