PURIFICATION AND BASIC CHARACTERISTICS OF SIALIDASE FROM TRITRICHOMONAS-MOBILENSIS

Sialidase, produced by Tritrichomonas mobilensis, a colonic parasite o f squirrel monkeys, was purified by adsorption on human RBCs in ice-co ld culture supernatant followed by release in PBS at 37 degrees C. The enzyme was purified by size-exclusion chromatography of RBC-eluted ma terial giving a single peak with sialidase activity of molecular weigh t approx. 630 kDa, representing a quadrimer of the complex of three su bunits. SDS-PAGE under reducing conditions showed three bands of 56, 6 1, and 66 kDa, identical with the molecular weight of the three subuni ts of T. mobilensis sialic acid-specific lectin (Babal et al., 1994). The enzyme treatment changed the agglutination of human RBCs by other lectins: created agglutinability with galactose-specific peanut agglut inin, but did not change agglutination with alpha 2,6 sialic acid link age-specific Sambucus nigra lectin and linkage nonspecific TML. A 4-fo ld decrease of the agglutination with alpha 2,3 sialic acid linkage-sp ecific Maackia amurensis lectin was observed. Histochemistry of kidney glomeruli after T. mobilensis sialidase treatment showed peanut agglu tinin positivity on membranes of podocytes indicating selectivity for alpha 2,3 linked sialic acid. The sialidase did riot hydrolyze colomin ic acid and was inhibited by 2,3-dehydro-2-deoxy-NeuAc. Divalent catio ns were not required for activity. The enzyme activity was optimal at pH 6.5-7 with RBCs as substrate and could be stored for 1 year at 4 de grees C.