Purification and characterization of glucosidase I involved in N-linked glycoprotein processing in bovine mammary gland.
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.
Shailubhai
K
K
Department of Animal Sciences, University of Maryland, College Park 20742.
Pratta
M A
MA
Vijay
I K
IK
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D006023:Glycoproteins; D009844:Oligosaccharides; C042059:glucosidase I; D000520:alpha-Glucosidases