Ligand affinity chromatographic purification of rat liver Golgi endomannosidase.
In order to achieve isolation of endo-alpha-D-mannosidase, a Golgi-located processing enzyme that accomplishes deglucosylation of glycoproteins with N-linked carbohydrate units by cleaving the linkage between the glucose-substituted mannose residue and the remainder of the oligosaccharide, we have prepared an affinity matrix (Glc alpha 1-->3Man-O-(CH2)8CONH-Affi-Gel 102) containing the derivative of the characteristic disaccharide product of this enzyme. Chromatography of a Triton extract of rat liver Golgi membranes on a column of this gel in the presence of castanospermine to prevent binding of alpha-glucosidases permitted a rapid purification of the endomannosidase (70,000-fold over the homogenate) with a 12% yield. This purified enzyme was free of other processing glycosidases and was completely inhibited by Glc alpha 1-->3(1-deoxy)mannojirimycin. Examination of the endomannosidase by SDS-polyacrylamide gel electrophoresis revealed a doublet (M(r) 60,000 and 56,000) with the bands being of approximately equal density. Gel permeation high performance liquid chromatography indicated that in its native form the enzyme has an oligomeric structure (M(r) approximately 560,000) consisting of eight to ten subunits.
Hiraizumi
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Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02215.
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D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000138:chemical synthesis; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008361:Mannosidases / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D043323:alpha-Mannosidase
D004187:Disaccharides; D008024:Ligands; D046911:Macromolecular Substances; C085900:8-methoxycarbonyloctyl-3-O-glucopyranosyl-mannopyranoside; D008361:Mannosidases; D043323:alpha-Mannosidase