Studies on the Enzymatic Reduction of C-Nitroso Compounds1

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Abstract
The subcellular distribution of NADH-p-nitrosophenol (p-NSP) reductase activity at pH 6.0 in porcine liver was studied by spectrophotometric assay. About two-thirds of the activity was found in the cytosol fraction and the pH optimum of this fraction was about 5.5. The activity at pH 5.8 of cytosol fractions from various tissues of rats, quails, frogs, carp, and scallops was also studied. All these fractions showed more or less NADH-p-NSP reductase activity but the activity of NADH-aldehyde reductase (alcohol dehydrogenase [EC 1.1.1.1]) was detected only in those from liver and a few other tissues. Supernatants from sonicated cells of Bacillus subtilis and Escherichia coli also showed C-nitrosoreductase activity but were devoid of aldehyde reductase activity. The major C-nitrosoreductase of porcine liver cytosol was purified 20- to 30-fold by frac-tionation with ammonium sulfate, gel filtration, and ion-exchange chromatography. The pH optimum of this preparation was 5.5 and activity was strongly inhibited by p-chloromercuri-benzoate (p-CMB). The enzyme preparation was stable at 5°C for at least a week in the presence of NADH at pH 8.4. High concentrations of ammonium sulfate also stabilized the enzyme. An equilibrium between monomeric and dimeric forms of the enzyme was found and the molecular weight was estimated to be about 83,000 and 160,000 daltons for the monomeric and dimeric forms, respectively. The enzyme utilized NADH almost specifically and 2 mol of NADH were consumed per mol of p-NSP reduced to p-aminophenol. Nitrosobenzene and aldehydes could also serve as the electron acceptor. The aldehyde reductase activity became concentrated roughly in parallel with the C-nitrosoreductase activity during the course of the purification and these two activities could not be separated even after further purification by 5′-AMP-Sepharose affinity chromatography. N-Nitroso compounds were not affected by this enzyme.

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