The purification and properties of two soluble reduced nicotinamide: acceptor oxidoreductases from Trichomonas vaginalis.
The occurrence of soluble reduced nicotinamide nucleotide:acceptor oxidoreductases has been reported in a number of strains of the oxygen-tolerant anaerobe Trichomonas vaginalis and other trichomonad species. The quantitatively more important enzyme in most strains of T. vaginalis is an NADH oxidase which produces water from the reduction of oxygen. This enzyme has been purified by a combination of gel filtration, chromatofocusing, Cibacron Blue chromatography and high pressure gel permeation chromatography. It is a monomeric protein with an estimated molecular mass from sodium dodecyl sulphate gel electrophoresis of 98 kDa; an isoelectric point of approximately pH 5.5 and a Km for NADH of 5.4 microM. The purified NADH oxidase is significantly inactivated during turnover under air (t1/2 3.65 min) and rapidly inactivated by microM levels of hydrogen peroxide. The NADPH-dependent minor activity requires a flavin. It has been partially purified by gel filtration and chromatofocusing. The apparent molecular mass of this enzyme is 36 kDa by gel filtration; it has an isoelectric point of approximately pH 5.2 and Km values for NADPH and FMN of 16.6 microM and 6.1 microM respectively. The product of oxygen reduction by this enzyme, using FMN as acceptor is hydrogen peroxide. The possible role of these two enzymes in the cell and their affinity with related enzymes from other organisms is discussed.
Linstead
D J
DJ
Department of Biochemical Microbiology, Wellcome Research Laboratories, Beckenham, Kent, U.K.
Bradley
S
S
D000818:Animals; D002850:Chromatography, Gel; D007526:Isoelectric Point; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D019255:NADPH Oxidases; D010084:Oxidation-Reduction; D014246:Trichomonas vaginalis / Q000201:enzymology*
D009097:Multienzyme Complexes; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases