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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+ + 12 = 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.  相似文献   

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1. Three aspecific ATPases were found in the sedimentible fractions of Trichomonas vaginalis. 2. One, with a pH optimum of 5.5, was equally activated by Ca2+ or Mg2+, moderately stable, preferred nucleotide diphosphates as substrates, and was inhibited by vanadate, oligomycin, nitrate and Na+. 3. A second, with a pH optimum of 7.5, was activated by Mg2+, preferred guanosine diphosphate as substrate, and was the least stable and most subject to inhibitors (vanadate, oligomycin, NEM, NBD-Cl, azide and Cl-). 4. The third, pH optimum 8.0, was activated by Ca2+, was latent and the most stable, reacted equally well with nucleotide tri- or diphosphates, and was the least susceptible to inhibitors (vanadate and NEM). 5. All exhibited proton-translocating ability.  相似文献   

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Background  

Trichomonas vaginalis is a human urogenital pathogen responsible for trichomonosis, the number-one, non-viral sexually transmitted disease (STD) worldwide, while T. tenax is a commensal of the human oral cavity, found particularly in patients with poor oral hygiene and advanced periodontal disease. The extent of genetic identity between T. vaginalis and its oral commensal counterpart is unknown.  相似文献   

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We have investigated the susceptibility of Trichomonas vaginalis to Mengo virus infection by comparing the outcome of Mengo virus or purified Mengo virus RNA infection in T. vaginalis and in CCL-1 mouse fibroblasts. While the adsorption and entry of Mengo virus into T. vaginalis occurred in the same manner as in fibroblasts, the uncoating was much slower. In addition, Mengo virus infection of T. vaginalis displayed no eclipse nor any subsequent production of infectious virus. Purified RNA failed to initiate productive infection in T. vaginalis, whereas it provoked viral replication in the fibroblast controls. It was shown by assessment of protein synthesis in T. vaginalis and mouse fibroblasts cell-free systems that the protozoan ribosomes were able to translate endogenous mRNA and poly-U, but not viral RNA.  相似文献   

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A genotyping method has been developed to distinguish each Trichomonas vaginalis isolate and has provided the first genome mapping studies of this protist with an estimated 180Mb genome. The technique was developed using high molecular weight DNA prepared from five laboratory isolates from Australia and USA and 20 clinical isolates from South Africa. Inhibition of the notorious T. vaginalis endogenous nucleases by addition of potent inhibitors was essential to the success of this study. Chromosomal DNA larger than 2.2Mb was macrorestricted to a minimum segment size of approximately 50kb, separated by pulsed field gel electrophoresis and hybridised with a variety of gene probes. Each isolate generated a unique pattern that was distinguished by each of the probes. Four single copy gene probes (fd, hmp35, ibp39 and pfoD) were identified but probes which identified several bands (pfoB and alpha-scs) per isolate were most informative for genotyping. The pyruvate:ferredoxin oxidoreductase B gene probe identified two to seven copies of pfoB (or its closely related homologue pfoA) per genome in different isolates and is an obvious candidate probe to identify epidemiological linkage between infections by this genotyping method. Cleavage of the genomes into smaller fragments failed to distinguish isolates from diverse locations indicating the proximal regions of genes are conserved.  相似文献   

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