PURIFICATION AND CHARACTERIZATION OF 2 FORMS OF N-ACETYLGLUCOSAMINIDASE FROM CANDIDA-ALBICANS SHOWING WIDELY DIFFERENT OUTER CHAIN GLYCOSYLATION

Two forms of N-acetylglucosaminidase were purified to homogeneity by ion exchange (TSK DEAE-3SW, Aquapore CX-300) and gel filtration (TSK G4000 SW) HPLC of Candida albicans ATCC 10261 culture filtrates. Synthesis and secretion of N-acetylglucosaminidase were induced by incubating starved yeast cells at 37 degrees C in medium containing N-acetylglucosamine (GlcNAc). The form of the enzyme depended on the cell growth and starvation conditions before GlcNAc induction. N-Acetylglucosaminidase A (32% total carbohydrate, M(r) 85000 subunit) was isolated from cells grown in glucose/salts/biotin medium, and N-acetylglucosaminidase B (56% carbohydrate, M(r) 132000 subunit) was isolated from cells grown in yeast extract/peptone/dextrose. The estimated relative molecular masses of the native enzymes, based on Sephacryl S-300 gel filtration were: A form. 350000; B form, 600000; A and B forms after endoglycosidase H (endo H) treatment, 180000. The purified enzymes migrated on SDS polyacrylamide gels as heterogeneous glycoproteins of M(r) centred at similar to 100000 (A) and similar to 150000 (B) but were reduced to a single 58000 band after denaturation with SDS and cleavage of asparagine-linked sidechains by endo H. When the native glycoproteins were treated with endo H, both enzyme forms had three oligosaccharide sidechains of M(r) similar to 3000 that were endo H resistant. Therefore the difference in the size of N-acetylglucosaminidase A and B was due to variations in outer chain glycosylation of endo H-sensitive inner core structures. N-Acetylglucosaminidase was active and stable over a broad ph range with maximum activity against both p-nitrophenylGlcNAc (pNPGlcNAc) and pNPGalNAc at ph 4.0. The kinetic parameters k(cat) (s(-1)) and K-m (mM) of N-acetylglucosaminidase A using the following substrates were, respectively: pNPGlcNAc, 740, 0.77; pNPGalNAc, 910, 1.26; N,N'-diacetylchitobiose 620, 0.20; and N,N',N''-triacetylchitotriose, 170, 0.044. The enzyme showed substrate inhibition with all substrates above 0.5 mM except for pNPGalNAc.