Two forms of N-acetylglucosaminidase were purified to homogeneity by ion exchange (TSK DEAE-3SW, Aquapore CX-300) and gel filtration (TSK 64000 SW) HPLC of Candida albicans ATCC 10261 culture filtrates. Synthesis and secretion of N-acetylglucosaminidase were induced by incubating starved yeast cells at 37 "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 O/ O total carbohydrate, M, 85 000 subunit) was isolated from cells grown in glucose/salts/biotin medium, and Nacetylglucosaminidase B (56 YO carbohydrate, M, 132 000 subunit) was isolated from cells grown in yeast extract/peptone/dextrose. The estimated relative molecular masses of the native enzymes, based on Sephacryl 5-300 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, centred at -I00000 (A) and -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, -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, (s-l) and K,,, (mM) of N-acetylglucosaminidase A using the following substrates were, respectively: pNPGlcNAc, 740,0077; 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.
