SUMMARY: Two distinct NADH oxidases, corresponding to HO-forming and HO-forming enzymes were purified to homogeneity from and their basic properties determined. The HO-forming enzyme was a tetramer with a subunit molecular mass of about 56 kDa and required flavin adenine dinucleotide (FAD) for full activity. The enzyme had an isoelectric point of 6.6 and exhibited optimal activity at pH 6.0 The HO-forming enzyme was a monomer with a molecular mass of 50 kDa and activity independent of exogenously added flavin. The enzyme had an isoelectric point of 4.8 and exhibited optimal activity between pH 7.0 and 7.5 Both enzymes oxidized NADH (K 0.05 and 0.025 mM for the HO- and HO-forming enzyme, respectively) but not NADPH and contained 1 mol of FAD per monomer. Spectra of the oxidized enzymes exhibited maxima at 271, 383 and 449 nm for the HO-forming enzyme and 271, 375 and 447 nm for the HO-forming enzyme. Antibodies raised against the HO-forming enzyme or the HO-forming enzyme reacted with their corresponding antigen, but did not cross-react. The amino-terminal regions of the two enzymes had completely different amino acid sequences.


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