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African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, trypanothione reductase, which acts on a cyclic derivative of glutathione, trypanothione. The high degree of sequence similarity between trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/K for oxidized trypanothione has been determined for trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 +/- 0.09 and 9.45 +/- 0.07, respectively, are deprotonated. V/K for oxidized trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pK values of 8.74 +/- 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2',3'-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O (i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV. Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense trypanothione reductase. 163 54

Nitric oxide reductase was purified from Paracoccus denitrificans very nearly to homogeneity by a simple method that involved the use of octyl glucoside to solubilize the enzyme from membranes and required a single hydroxyapatite column. The enzyme had specific activities of about 10 mumol NO reduced x min-1 x mg-1 at pH 6.5 in an amperometric assay system using phenazine methosulfate/ascorbate as the reducing agent and about 22 mumol NO reduced x min-1 x mg-1 at pH 5.0, which is the optimum pH. These values are based on average rates over kinetically complex progress curves and would be about three times greater if based on maximum rate values. The enzyme appeared to be reversibly inhibited by NOaq and to have a Km too low (probably less than or equal to 1 microM) to measure reliably by the amperometric method. The effective second-order rate constant of the enzyme lay within 1 to 2 orders of magnitude of the diffusion controlled limit. The enzyme was composed of a tight complex of two cytochromes: a cytochrome c (Mr = 17,500) and a cytochrome b (Mr = 38,000). The mole ratios of cytochrome c to cytochrome b and Mr 17,500 peptide to Mr 38,000 peptide were both about 1.7, and the heme content was about 3 mol/73,000 g (38,000 + 2(17,500)). Each subunit therefore contained only one heme group. The Mr 38,000 peptide aggregated when heated in the sample buffer used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the ascorbate-based activity, the enzyme showed a little NADH-NO oxidoreductase activity which was not inhibited by antimycin A. The enzyme lost activity with a half-life of about 2 days at 4 degrees C but could be preserved at -20 degrees C and in liquid nitrogen. It seemed not to be inactivated by aerobic solutions. These observations, and the recent ones by Carr and Ferguson (Carr, G.J., and Ferguson, S.J. (1990) Biochem. J. 269, 423-429) with a partially purified preparation of nitric oxide reductase, establish that the enzyme from Pa. denitrificans is a cytochrome bc complex which resembles that from Pseudomonas stutzeri (Heiss, B., Frunzke, K., and Zumft, W.G. (1989) J. Bacteriol. 171, 3288-3297). There would appear to be no functional relationship between nitric oxide reductase and a Mr = 34,000 peptide of Pa. denitrificans membranes reported previously to be present in purified preparations of a nitric oxide reductase (Hoglen, J., and Hollocher, T.C. (1989) J. Biol. Chem. 264, 7556-7563).
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PMID:Nitric oxide reductase. Purification from Paracoccus denitrificans with use of a single column and some characteristics. 164 15

A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase [NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)] mRNA was expressed in Escherichia coli. The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR). Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction. The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites. E. coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor. The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M. After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase. The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis. It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase. The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm. Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme. Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid. The remaining 19 amino acid residues at the NH2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol.
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PMID:Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein. 170 98

The reaction of ozone with a number of biological molecules was found to produce singlet oxygen in high yield. At pH 7.0, the reaction of ozone with an equimolar amount of biological molecule produced the following singlet oxygen yields (mole of singlet oxygen/mole of ozone): cysteine, 0.49 +/- 0.02; methionine, 1.13 +/- 0.11; reduced glutathione, 0.33 +/- 0.02; albumin, 1.00 +/- 0.05; uric acid, 0.64 +/- 0.09; ascorbic acid, 0.96 +/- 0.007; NADPH, 1.07 +/- 0.07; NADH, 0.95 +/- 0.01. Thus, singlet oxygen may be an important intermediate in the biochemical damage caused by ozone.
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PMID:Singlet oxygen production from the reactions of ozone with biological molecules. 202 12

(Z)-4',5'-Didehydro-5'-deoxy-5'-fluoroadenosine (I), 5'-deoxy-5'-difluoroadenosine (II), and 4',5'-didehydro-5'-deoxy-5'-fluoroarabinosyl-adenosine (III) are inhibitors of rat liver S-adenosyl-L-homocysteine hydrolase. Compounds I and II are time-dependent and irreversible inhibitors of the enzyme. Both I and II are oxidized by E.NAD to produce E.NADH, and fluoride anion is formed in the inactivation reaction (0.7 to 1.0 mole fluoride/mole of enzyme subunit, and 1.7 moles fluoride/mole of enzyme subunit from I and II, respectively). The enzyme is stoichiometrically labeled with [8-3H]-I, but the label is lost upon denaturation of the protein either with or without treatment of the labeled complex with sodium borohydride. The compound III, the arabino derivative of I, is a competitive inhibitor of the enzyme. The mechanism of the inhibition of S-adenosyl-L-homocysteine hydrolase by these inhibitors is discussed.
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PMID:The mechanism of inhibition of S-adenosyl-L-homocysteine hydrolase by fluorine-containing adenosine analogs. 209 66

We describe an assay for light microscopic visualization of specific glycosyltransferases on tissue sections or on cells. The assay uses a sequence of enzyme reactions that yields two moles of NADH for each mole of the uridine-5'-diphosphate (UDP) released during transfer of a monosaccharide from a UDP sugar to an acceptor. When diaphorase and tetrazolium salts are present in the incubation mixture, the tetrazolium salts are reduced to colored diformazans, which precipitate at the sites of glycosyltransferase activity. The validity of the assay was established by applying the technique to spermatozoa and liver, in which some glycosyltransferases have previously been localized. When suspensions of mouse spermatozoa were assayed for galactosyltransferase (GalTase) activity, diformazan precipitates appeared on the plasma membranes overlying the anterior heads of the spermatozoa, in agreement with immunochemical localizations. In mouse liver slices assayed with bilirubin as acceptor for glucuronyltransferase (GluTase) activity, dense diformazan deposits appeared on the hepatocytes but not on endothelial cells, also in agreement with immunochemical data. In the absence of acceptor or UDP sugar donor, diformazan deposits were minimal and random in all tissues tested. The assay's versatility was tested by incubating tissues with different sugar donors and acceptors to localize other sites of transferase activity. In mouse frozen liver sections, GalTase activity occurred in both hepatocytes and endothelial cells; in sections of rat submaxillary glands, GalTase activity was detected in mast cells. In liver sections, GlcuTase activity with o-aminophenol as acceptor was located primarily on the endothelial cells. With the appropriate sugar donor and acceptor, this assay should detect any transferase, other than the glucosyltransferases, that utilizes UDP sugars.
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PMID:Light microscopic localization of glycosyltransferase activities in cells and tissues. 210 33

Activated bleomycin appears to have two more oxidizing equivalents than the Fe(III).bleomycin to which it spontaneously decays. Activated bleomycin reacts with NADH and thio-NADH, two-electron reductants, and with KI, a one-electron reductant, to yield Fe(III).bleomycin. The observed stoichiometries were 0.85 +/- 0.07 eq of thio-NADH oxidized or 1.5 +/- 0.25 eq of KI oxidized per mole of activated bleomycin. None of these reactions requires the presence of a redox mediator, as does the reduction of Fe(III).bleomycin by NADH or thio-NADH. The oxidations of both pyridine nucleotide coenzymes and of KI are inhibited by DNA, the usual bleomycin target.
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PMID:The redox state of activated bleomycin. 241 20

3-Hydroxybutyrate dehydrogenase (BDH) is a lecithin-requiring mitochondrial enzyme which catalyzes the interconversion of 3-hydroxybutyrate and acetoacetate with NAD(H) as coenzyme. The purified enzyme devoid of lipid (i.e., the apodehydrogenase or apoBDH) can be reactivated with soluble lecithin or by insertion into phospholipid vesicles containing lecithin. Two different models have been proposed to explain the sigmoidal lipid activation curves. For both models, activation of BDH is assumed to require the binding of two lecithin molecules per functional unit. Activation of soluble enzyme (dimeric form) by short-chain (soluble) lecithin is consistent with a model in which lecithin binding is noncooperative, whereas activation of the membrane-bound enzyme (tetrameric form) indicates cooperativity between the lecithin binding sites. A new comprehensive model is presented in which lecithin is considered to be an essential allosteric activator that shifts the equilibrium between conformational states of the enzyme. Resonance energy transfer data, reflecting NADH binding to membrane-bound and soluble apoBDH, are consistent with such a lecithin-induced conformational change. Apparent dissociation constants for binding of NADH to BDH are approximately 10 microM and approximately 37 microM for BDH activated by bilayer and soluble lecithin, respectively. The maximal fluorescence resonance energy transfer (delta F max) increases with higher mole fraction of lecithin in the bilayer. The largest changes occur between mole fractions 0 and 0.13, thereby correlating with enzymic function. Essentially no binding of NADH is observed in the absence of lecithin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cooperativity in lipid activation of 3-hydroxybutyrate dehydrogenase: role of lecithin as an essential allosteric activator. 274 24

The protein ATPase inhibitor entraps about five nucleotides in pig heart mitochondrial F1, one at least being a triphosphate [Di Pietro, A., Penin, F., Julliard, J.H., Godinot, C., & Gautheron, D.C. (1988) Biochem. Biophys. Res. Commun. 152, 1319-1325]. The fate of these nucleotides was studied during ATP synthesis driven by NADH oxidation in reconstituted inverted submitochondrial particles. Iodinated F1, containing 0.7 mol of endogenous nucleotides/mol, was first loaded with tritiated adenine nucleotides in the presence or absence of the protein inhibitor and then reassociated with F1-depleted submitochondrial particles (ASU particles) to reconstitute an efficient NADH-driven ATP synthesis. In the absence of the protein inhibitor, 1.7 mol of labeled nucleotides remained bound per mole of reassociated F1, 0.8-0.9 mol being rapidly exchangeable against medium ADP or ATP, as measured after rapid filtration through nitrocellulose filters. In the presence of the protein inhibitor, as many as 3.25 mol of labeled nucleotides remained bound per mole of reassociated F1. Under hydrolysis conditions where ATPase activity was highly inhibited, no release of tritiated nucleotide occurred. In contrast, under ATP synthesis conditions where the protonmotive force was generated by NADH oxidation, the progressive reversal of inhibition by the protein inhibitor was correlated to a concomitant release of tritiated nucleotide. When ATP synthesis became fully active, about one nucleotide was completely exchanged whereas more than three nucleotides remained tightly bound and did not appear to be directly involved in ATP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fate of nucleotides bound to reconstituted Fo-F1 during adenosine 5'-triphosphate synthesis activation or hydrolysis: role of protein inhibitor and hysteretic inhibition. 290 4

The chloroperoxidase-catalyzed reactions of NAD(P)H with H2O2 in the presence of Cl- or Br- have been characterized. With 1 mol H2O2 per mol of NADH, one atom of 36Cl was incorporated into the 264-nm-absorbing intermediate product. This species was oxidized enzymatically by a second mole of H2O2 to a species distinct from NAD+, which retained one Cl atom. Spectroscopically identical species were also produced by reaction of NADH with one and two molar ratios of HOCl, respectively. These data indicate that, with respect to halogenation activities, chloroperoxidase functions similarly to myeloperoxidase, i.e., produces HOCl as the first product of Cl- oxidation by H2O2. Moreover, rapid chlorination of NAD(P)H followed by oxidation may be an important and highly lethal microbicidal effect of HOCl produced by myeloperoxidase in activated neutrophils.
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PMID:Chlorination of NADH: similarities of the HOCl-supported and chloroperoxidase-catalyzed reactions. 298 46


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