Properties of the isoenzyme forms A-1, A-2 and B of N-acetyl-beta-D-glucosaminidase purified from baboon kidneys.
Three forms of N-acetyl-beta-D-glucosaminidase (NAG: A, B and I) were separated from baboon kidney using Con A-Sepharose and DEAE-Trisacryl chromatography. 2. The A form was further purified into two forms A-1 and A-2 using hydroxylapatite chromatography and anodic PAGE. Both were homogeneous on SDS-PAGE and anodic PAGE but microheterogeneous on PAG-IEF, which could be eliminated by prior treatment with endoglycosidase H or glycopeptidase F. 3. The carbohydrate content accounted for some of this microheterogeneity since it varied from 31 for A-1 to 17% for A-2 and the sialic acid was 6 and 1%. Deamidation may also contribute since the acidic amino acids (29 mol%) and ammonia were high following acid hydrolysis. 4. The mol. wt for A-1, determined by SDS-PAGE, was 52.1 K. 5. The pH optimum was 4.55 and the pI4.97. 6. The optimum temperature for NAG A and B was 50 degrees and 42 degrees C, but B retained more activity above 55 degrees C. 7. The Km for N-acetyl-beta-D-glucosamine and -galactosamine for both isoforms was 0.497 and 0.627 mM respectively. 8. Several ions were found to be uncompetitive inhibitors. Ag+ and Pb2+ were the most potent having Ki values of 3.6 and 8.5 mM respectively. Acetate acted as a competitive inhibitor.
Beukes
H A
HA
Department of Surgery, University of Witwatersrand, Johannesburg, South Africa.
Oosthuizen
M M
MM
Lambrechts
H
H
Myburgh
J A
JA
Litthauer
D
D
Naude
R J
RJ
D000118:Acetylglucosaminidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D008970:Molecular Weight; D010215:Papio; D013379:Substrate Specificity
D000596:Amino Acids; D002241:Carbohydrates; D007527:Isoenzymes; D000118:Acetylglucosaminidase