Purification and characterization of glucosidase II involved in N-linked glycoprotein processing in bovine mammary gland
| Autor: | K Shailubhai, Shailendra Saxena, B. Dong-Yu, Inder K. Vijay |
|---|---|
| Rok vydání: | 1987 |
| Předmět: |
Kojibiose
Size-exclusion chromatography Enzyme-Linked Immunosorbent Assay Cross Reactions Biochemistry Dithiothreitol Substrate Specificity chemistry.chemical_compound Mammary Glands Animal Affinity chromatography Acetylglucosaminidase Animals Immunoelectrophoresis Molecular Biology Glycoproteins chemistry.chemical_classification biology Molecular mass Chemistry alpha-Glucosidases Cell Biology Ligand (biochemistry) Molecular biology Molecular Weight Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase Enzyme Polyclonal antibodies biology.protein Cattle Electrophoresis Polyacrylamide Gel Female Research Article Chromatography Liquid |
| Zdroj: | Biochemical Journal. 247:563-570 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj2470563 |
| Popis: | Glucosidase I, the first enzyme involved in the post-translational processing of N-linked glycoproteins, was purified to homogeneity from the lactating bovine mammary tissue. The enzyme was extracted by differential treatment of the microsomal fraction with Triton X-100 and Lubrol PX. The solubilized enzyme was subjected to affinity chromatography on Affi-Gel 102 with N-5-carboxypentyldeoxynojirimycin as ligand and DEAE-Sepharose CL-6B chromatography. Purified glucosidase I shows a molecular mass of 320-330 kDa by gel filtration on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis under reducing conditions indicates a single band of approx. 85 kDa, indicating that the native enzyme is probably a tetrameric protein. Several criteria, including pH optimum of 6.6-7.0, specific hydrolytic action towards Glc3Man9GlcNAc2, to release the terminally alpha-1,2-linked glucosyl residue, and total lack of activity towards Glc1Man9GlcNAc2 and Glc2Man9GlcNAc2 saccharides, which are the biological substrates for processing glucosidase II, and 4-methylumbelliferyl alpha-D-glucopyranoside show the non-lysosomal origin and the processing-specific role of the purified enzyme. The enzyme does not require any metal ions for its activity. Hg2+, Ag+ and Cu2+ are potent inhibitors of the enzyme; this inhibition can be reversed by adding an excess of dithiothreitol. Among the saccharides tested, kojibiose (Glc alpha 1----2Glc) was inhibitory to the enzyme. Polyclonal antibodies raised against the enzyme in rabbit were found to be specific for glucosidase I, as revealed by Western-blot analysis and by immunoadsorption with Protein A-Sepharose. Anti-(glucosidase I) antibodies were cross-reactive towards a similar antigen in solubilized microsomal preparations from liver, mammary gland and heart from the bovine, guinea pig, rat and mouse. |
| Databáze: | OpenAIRE |
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