Properties of the isoenzyme forms A-1, A-2 and B of N-acetyl-beta-D-glucosaminidase purified from baboon kidneys
| Autor: | Hugo Lambrechts, Mathys M.J. Oosthuizen, Derek Litthauer, H. A. G. Beukes, J.A. Myburgh, Ryno J. Naudé |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Carbohydrates
Kidney Biochemistry Isozyme Substrate Specificity chemistry.chemical_compound Endoglycosidase H Acetylglucosaminidase Animals Amino Acids Deamidation Polyacrylamide gel electrophoresis Chromatography biology Isoelectric focusing Hydrogen-Ion Concentration Sialic acid Isoenzymes Molecular Weight chemistry Galactosamine biology.protein Acid hydrolysis Electrophoresis Polyacrylamide Gel Isoelectric Focusing Papio |
| Zdroj: | The International journal of biochemistry. 23(2) |
| ISSN: | 0020-711X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|