Properties and Substrate Specificities of two Neuraminidases fromTrichomonas fetus

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Abstract
T. fetus, a flagellate protozoan, causes vaginal infections in cows, leading to sterility or abortion in early stage of pregnancy. Two T. fetus neuraminidases isolated from the culture medium were purified by gel chromatography, ion exchange chromatography and affinity chromatography on N-(4-nitrophenyl)-oxamic acid-Sepharose 4B. The MW of the 2 neuraminidases were determined as 320,000 (enzyme I) and 38,000 (enzyme II), respectively. Enzyme I seems to consist of 2 isoenzymes containing 4 subunits of almost equal MW. The pH optima of both enzymes depend on the substrates and range from pH 4.7 to 5.5. Due to the type of substrate, the Km varies between 5.0 .times. 10-2 M and 6.6 .times. 10-3 M for enzyme I and between 1.4 .times. 10-2 M and 4.9 .times. 10-3 M for enzyme II. Among the different groups of NeuAc[N-acetylneuraminic acid]-containing substrates (i.e., glycoproteins, glycolipids, oligosaccharides and synthetic ketosides), enzyme I preferably cleaves high MW glycoprotein type substrates, whereas enzyme II shows higher affinities to low-MW oligosaccharides. The ganglioside II3NeuAc-GgOse4Cer is susceptible to both enzymes only after removal of the lipophilic ceramide residue. Both enzymes show differences in the specificity towards .alpha.2 .fwdarw. 3, .alpha.2 .fwdarw. 6, and .alpha.2 .fwdarw. 8 glycosidic linkages of NeuAc. Taking the rate of cleavage of the .alpha.2 .fwdarw. 3 linkage in II3NeuAc-Lac as 100, enzyme I has a cleavage rate of 65 for the .alpha.2 .fwdarw. 6 linkage in II6NeuAc-Lac, and 15 for the .alpha.2 .fwdarw. 8 linkage in II3(.rarw. 2.alpha.NeuAc8)2-Lac, whereas enzyme II exhibits values around 50 for both the .alpha.2 .fwdarw. 6- and the .alpha.2 .fwdarw. 8-linked substrates. The activity of neuraminidase I and II is not influenced by Ca2+ but is inhibited by Cu2+, Hg2+, and 4-hydroxymercurisulfonic acid. The inhibition by Hg2+ and by the latter is reversible with enzyme I by addition of dithioerythritol.

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