NADH oxidase activity was detected in the 105,000g supernatant ("soluble") fraction of Trichomonas vaginalis and the enzyme was purified 50-fold by centrifugation, ammonium sulfate precipitation, Sephadex G-200, and DEAE-Sephadex A-25 chromatography. The ratio of oxygen uptake to NADH oxidation was approximately one-half. Addition of catalase did not affect the rate of oxygen uptake elicited by NADH. Since the purified fraction was free from interfering enzymes, the postulated reaction is as follows: NADH + H+ + 1 2 = NAD+ + H2O. Among numerous substances tested, only NADH was a functional substrate, whereas NADPH was not oxidized. The purified enzyme had a Vmax of 16.5 μmole of oxygen consumed/min/mg protein, and the apparent Km for NADH was 7.4 μM. Substrate inhibition was observed at 3.7 mM NADH. The purified NADH oxidase was competitively inhibited by NAD+ as well as by NADP+ with 50% inhibition at 1 and 5 mM, respectively. The enzyme was also markedly inhibited by p-chloromercuribenzoate, hydrogen peroxide, and transient metal-chelators such as bathophenanthroline or o-phenanthroline. A flavoprotein antagonist, atebrin was slightly less inhibitory. Various quinones, flavin nucleotides and artificial dyes, except for p-benzoquinone, ferricyanide and cytochrome c, did not function in accepting electrons from NADH oxidase. These three compounds, however, were still poor electron acceptors in the enzymatic reaction suggesting that the trichomonad NADH oxidase has little diaphorase activity. All of these findings indicate that T. vaginalis has an unique NADH oxidizing enzyme in that H2O seems to be the prdouct of oxygen reduction. This NADH oxidase appears important in the aerobic metabolism of this parasite.
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