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 PAGE-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 pI 4.97. 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 K(m) for N-acetyl-beta-D-glucosamine and -gqalactosomine 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 K(i) values of 3.6 and 8.5 mM respectively. Acetate acted as a competitive inhibitor.