Reduction of 1-nitroso-2-naphthol by NADPH in the presence of liver microsomes.

1. The endogenous, NADPH-supported production of H2O2 and of O2-.-radicals in liver microsomes, was very strongly enhanced in the presence of 1-nitroso-2-naphthol. 2. A 30-fold induction by NON was the consequence of its direct reduction to NON-radicals, catalyzed by microsomal NADPH:cytochrome P450 reductase. 3. Nitroso radicals reduce molecular oxygen to superoxide anion radicals, which were readily dismutated by superoxide dismutase to hydrogen peroxide. 4. O2-.-radicals were the sole precursors of all NON-induced production of H2O2 in liver microsomes.

Bacteriocin-Producing Strain of Lactococcus lactis subsp. diacitilactis S50.

Lactococcus lactis subsp. diacitilactis S50 produces a bacteriocin, designated bacteriocin S50, which has a narrow antibacterial spectrum. It was active only against Lactococcus species, including a nisin producer exhibiting a bactericidal effect. The activity of bacteriocin S50 was sensitive to proteases. It retained antimicrobial activity after being heated to 100 degrees C for up to 60 min and in the pH range 2 to 11.

4-Chloroacetylpyridine adenine dinucleotide. A highly reactive and chromophoric affinity label of glyceraldehyde-3-phosphate dehydrogenase from sturgeon.

The analogue of NAD+, 4-chloroacetylpyridine-adenine dinucleotide (clac4PdAD+), inactivated the glyceraldehyde-3-phosphate dehydrogenase from sturgeon at a high rate. An affinity labeling was shown to occur with clac4PdAD+. The mononucleotide 4-chloroacetylpyridine 1-beta-D-ribose 5'-phosphate (clac4PdMN+) reacted with the enzyme in a second-order reaction whose rate was much smaller than that calculated for clac4PdAD+ taken as a second-order rate reagent. The rate of the reaction of clac4PdAD+ with the enzyme was determined by stopped flow, using as a probe the long-wavelength absorption maximum (430 nm) formed concomitantly with inactivation of the enzyme. Computer-assisted graphic simulation showed that the clac4PdAD+ analogue could bind to the active site of the enzyme from Bacillus stearothermophilus in a similar manner to that of NAD+, and that the reactive carbon and the reactive thiolate of Cys-149 were within bonding distance. The absorption at 430 nm was linearly proportional to the substoichiometric concentration of clac4PdAD+/mole subunit. Thiol titration suggested the modification of one thiol residue per subunit. The modified thiol was identified by degradation as Cys-149. In contrast to the absorption band generated during the reaction of the 3-chloroacetylpyridine-adenine dinucleotide (clac3PdAD+) with the same enzyme [Eur. J. Biochem. (1982) 127, 519-524; 129, 437-446], enzyme inactivated with clac4PdAD+ and clac4PdMN+ exhibited an absorption maximum at long wavelength which was still present after denaturation. The chromophore is proposed to be the enol form of the alpha-thioether ketone produced by alkylation of the thiolate of Cys-149 by the chloroacetyl group.

Reduction of aryl-nitroso compounds by pyridine and flavin coenzymes.

1. A systematic kinetic investigation of the reduction of aryl-nitroso compounds by pyridine and flavin coenzymes and their analogs, in enzymatic and nonenzymatic systems, has been reported. 2. Two main groups of nitroso compounds have been investigated, representatives nitroso-benzene and 1-nitroso-2-naphthol; in all enzymatic and nonenzymatic systems, the former was always reduced to phenyl-hydroxyl-amine and the latter to 1-amino-2-naphthol. 3. Pyridine compounds included NADH, APAD-4H2 and DBNA-4H2 in nonenzymatic systems, and liver alcohol dehydrogenase. Flavin compounds included 1,5-dihydrolumiflavin and various forms of reduced 5-ethyl-lumiflavin, in nonenzymatic systems, and the flavoenzymes glucose-oxidase and NADPH-cytochrome P450 reductase. 5. Pyridine coenzymes and their analogs reduced nitroso compounds by a direct hydride transfer, with a primary kinetic isotope of 9.5 +/- 2.2. 6. All flavin compounds (glucose-oxidase and its nonenzymatic analog 1,5-dihydrolumiflavin and NADPH-cytochrome P450 reductase and its analog 5-ethyl-1,5-dihydrolumiflavin) reduced aryl-nitroso compounds with high efficiency (k2 greater than 10(5)M(-1) min(-1)). 7. The flavin compounds have been shown to be much more efficient reductans of nitroso compounds, compared to pyridine coenzymes, both in enzymatic and nonenzymatic systems; the only exception to this rule presented the extremely efficient reduction of p-substituted aryl-nitroso compounds by liver alcohol dehydrogenase.

The oxidative part of the glucose-oxidase reaction.

1. Kinetic parameters of the oxidative part of glucose-oxidase reaction have been measured with 16 different electron-acceptors and glucose as a substrate. 2. In each case, the rate-limiting portion of the oxidative part of reaction was the formation of the E-FADH2.Acceptor-complex; this rate was pH-independent around the pH-optimum of the enzyme. 3. In each case, E-FADH2 acceptor-complex was undetectable in the steady-state kinetics, with the exception of cytochrome-c. 4. The rates of redox reactions between various forms of reduced 5-ethyl-lumiflavin and five different electron-acceptors have been examined with a conventional spectrophotometry. In each case, it was found that the reactions proceeded at high rates whenever thermodynamically feasible, and were totally prevented in the opposite case. 5. Molecular oxygen was able to oxidize only the neutral form of 5-ethyl-1,5-dihydrolumiflavin to its radical form, at a moderate rate; all other forms of reduced 5-ethyl-lumiflavin were not oxidized by O2. 6. By the comparison of enzymatic and model redox reactions, it was possible to establish the minimal mechanism of the oxidative part of the glucose-oxidase catalytic cycle.