LIGAND AFFINITY CHROMATOGRAPHIC PURIFICATION OF RAT-LIVER GOLGI ENDOMANNOSIDASE

In order to achieve isolation of endo-alpha-D-mannosidase, a GoIgi-loc ated processing enzyme that accomplishes deglucosylation of glycoprote ins with N-linked carbohydrate units by cleaving the linkage between t he glucose-substituted mannose residue and the remainder of the oligos accharide, we have prepared an affinity matrix (Glc alpha 1-->3Man-O-( CH2)8CONH-Affi-Gel 102) containing the derivative of the characteristi c disaccharide product of this enzyme. Chromatography of a Triton extr act of rat liver Golgi membranes on a column of this gel in the presen ce of castanospermine to prevent binding of alpha-glucosidases permitt ed a rapid purification of the endomannosidase (70,000-fold over the h omogenate) with a 12% yield. This purified enzyme was free of other pr ocessing glycosidases and was completely inhibited by Glc alpha 1-->3( 1-deoxy)mannojirimycin. Examination of the endomannosidase by SDS-poly acrylamide gel electrophoresis revealed a doublet (M(r) 60,000 and 56, 000) with the bands being of approximately equal density. Gel permeati on high performance liquid chromatography indicated that in its native form the enzyme has an oligomeric structure (M(r) similar to 560,000) consisting of eight to ten subunits.