Purification and characterization of trimming glucosidase I from pig liver

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Abstract
Trimming glucosidase I has been purified about 400-fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enyzme has an apparent molecular mass of 85 kDa, and is an N-glycoprotein as shown by its binding to concanavalin A .sbd. Sepharose and its susceptibility to endo-.beta.-N-acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non-specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N-linked high-mannose sugar chain. Purified pig liver glucosidase I hydrolyzes specifically the terminal .alpha.1.sbd.2-linked glucose residue from natural Glc3-Man9-GlcNAc2, but is inactive towards Glc2-Man9-GlcNAc2 or nitrophenyl-/methyl-umbelliferyl-.alpha.-glucosides. The enzyme displays a pH optimum close to 6.4, does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (Ki .apprxeq. 2.1 .mu.M), N,N-dimethyl-1-deoxynojirimycin (Ki .apprxeq. 0.5 .mu.M) and N-(5-carboxypentyl)-1-deoxynojirimycin (Ki .apprxeq. 0.45 .mu.M), thus closely resembling calf liver and yeast glucosidase I. Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in the crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to crossreact with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross-reactivity.