Purification and characterization of two 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) 85,000 subunit) was isolated from cells grown in glucose/salts/biotin medium, and N-acetylglucosaminidase B (56% carbohydrate, M(r) 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 S-300 gel filtration were: A form, 350,000; B form, 600,000; A and B forms after endoglycosidase H (endo H) treatment, 180,000. The purified enzymes migrated on SDS polyacrylamide gels as heterogeneous glycoproteins of M(r) centred at approximately 100,000 (A) and approximately 150,000 (B) but were reduced to a single 58,000 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) approximately 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 kcat (s-1) and Km (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.
Molloy
C
C
Lincoln University, Canterbury, New Zealand.
Cannon
R D
RD
Sullivan
P A
PA
Shepherd
M G
MG
D000118:Acetylglucosaminidase / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002176:Candida albicans / Q000201:enzymology* / Q000254:growth & development; D004795:Enzyme Stability; D014644:Genetic Variation; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational
D000596:Amino Acids; D006023:Glycoproteins; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase