Switch to
Predicate | Object |
---|---|
rdf:type | |
lifeskim:mentions | |
pubmed:dateCreated |
1994-10-6
|
pubmed:abstractText |
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.
|
pubmed:language |
eng
|
pubmed:journal | |
pubmed:citationSubset |
IM
|
pubmed:chemical | |
pubmed:status |
MEDLINE
|
pubmed:month |
Jul
|
pubmed:issn |
1350-0872
|
pubmed:author | |
pubmed:issnType |
Print
|
pubmed:volume |
140 ( Pt 7)
|
pubmed:owner |
NLM
|
pubmed:authorsComplete |
Y
|
pubmed:pagination |
1543-53
|
pubmed:dateRevised |
2008-11-21
|
pubmed:meshHeading |
pubmed-meshheading:8075797-Acetylglucosaminidase,
pubmed-meshheading:8075797-Amino Acid Sequence,
pubmed-meshheading:8075797-Amino Acids,
pubmed-meshheading:8075797-Candida albicans,
pubmed-meshheading:8075797-Enzyme Stability,
pubmed-meshheading:8075797-Genetic Variation,
pubmed-meshheading:8075797-Glycoproteins,
pubmed-meshheading:8075797-Glycosylation,
pubmed-meshheading:8075797-Kinetics,
pubmed-meshheading:8075797-Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase,
pubmed-meshheading:8075797-Molecular Sequence Data,
pubmed-meshheading:8075797-Protein Conformation,
pubmed-meshheading:8075797-Protein Processing, Post-Translational
|
pubmed:year |
1994
|
pubmed:articleTitle |
Purification and characterization of two forms of N-acetylglucosaminidase from Candida albicans showing widely different outer chain glycosylation.
|
pubmed:affiliation |
Lincoln University, Canterbury, New Zealand.
|
pubmed:publicationType |
Journal Article,
Comparative Study
|