Abstract
A rapid and effective method was devised for the reduction of activity of reduced nicotinamide adenine dinucleotide (NADH) oxidase in crude extracts of Bacillus cereus T. The addition of 25 μmoles of MnCl2 per mg of extract protein in tris(hydroxymethyl)aminomethane-hydrochloride buffer reduced NADH oxidase activity by 90% within 1 min, and this reduction was independent of pH between pH 7.0 and 8.5. Other divalent cations such as Mg2+, Ba2+, Ca2+, and Co2+ also reduced NADH oxidase activity, but monovalent cations such as Na+ and K+ were ineffective. The reduction of NADH oxidase activity by divalent cations was presumably due to the removal of an essential flavine cofactor, since the addition of riboflavine and flavine mononucleotide to treated extracts was shown to completely restore NADH oxidase activity. The specificity, convenience, and efficiency of the procedure were shown to be applicable to crude extracts of B. megaterium and B. subtilis and should facilitate spectrophotometric measurements of nicotinamide adenine dinucleotide-dependent dehydrogenases in these and other microorganisms.
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