1887

Abstract

Two forms of -acetylglucosaminidase were purified to homogeneity by ion exchange (TSK DEAE-3SW, Aquapore CX-300) and gel filtration (TSK G4000 SW) HPLC of ATCC 10261 culture filtrates. Synthesis and secretion of -acetylglucosaminidase were induced by incubating starved yeast cells at 37 °C in medium containing -acetylglucosamine (GlcNAc). The form of the enzyme depended on the cell growth and starvation conditions before GlcNAc induction. -Acetylglucosaminidase A (32% total carbohydrate, 85000 subunit) was isolated from cells grown in glucose/salts/biotin medium, and -acetylglucosaminidase B (56% carbohydrate, 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, 180 000. The purified enzymes migrated on SDS polyacrylamide gels as heterogeneous glycoproteins of centred at ~ 100 000 (A) and ~ 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 ~ 3000 that were endo H resistant. Therefore the difference in the size of -acetylglucosaminidase A and B was due to variations in outer chain glycosylation of endo H-sensitive inner core structures. -Acetylglucosaminidase was active and stable over a broad pH range with maximum activity against both -nitrophenylGIcNAc (NPGIcNAc) and NPGalNAc at pH 4.0. The kinetic parameters (s) and (mM) of -acetylglucosaminidase A using the following substrates were, respectively: NPGIcNAc, 740, 0.77; NPGalNAc, 910, 1.26; '-diacetylchitobiose 620, 0.20; and -triacetylchitotriose, 170, 0.044. The enzyme showed substrate inhibition with all substrates above 0.5 mM except for NPGalNAc.

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/content/journal/micro/10.1099/13500872-140-7-1543
1994-07-01
2019-11-22
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http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-140-7-1543
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