PURIFICATION AND ENZYMATIC-PROPERTIES OF ENDO-ALPHA-1,2-MANNOSIDASE FROM PIG-LIVER INVOLVED IN OLIGOSACCHARIDE PROCESSING

An endo-alpha 1,2-mannosidase, which is involved in N-linked oligosacc haride processing, has been purified to homogeneity from crude pig liv er microsomes using conventional techniques. Two catalytically active polypeptides, of 48 kDa and 50 kDa, have been isolated which degrade [ C-14]Glc(3-1)-Man(9)-GlcNAc(2) to [C-14]Glc(3-1)-Man and a specific Ma n(8)-GlcNAc(2) isomer. They are not, however, active on synthetic alph a-mannosides. [C-14]Glc(1)-Man(9)-GlcNAc(2) was found to be approximat ely sevenfold more rapidly hydrolyzed than the [C-14]Glc(2)- and [C-14 ]Glc(3)-homologues. The 48 kDa and 50 kDa proteins are not N-glycosyla ted and ran on Superdex((TM)) 75 as monomers. Kinetic studies showed t hat these proteins had similar catalytic properties: (i) the pH optima were found to be close to 6.5; (ii) neither activity was metal ion de pendent; (iii) hydrolysis of [C-14]Glc(3)-Man(9)-GlcNAc(2) was inhibit ed strongly by Glc-alpha 1,3-Man (app. K-i approximate to 120 mu M), b ut not by 1-deoxymannojirimycin or swainsonine, Other evidence, includ ing immunological data, strongly suggests that the 48 kDa and 50 kDa p olypeptides are proteolytic degradation products of a single endo-alph a 1,2-mannosidase, rather than distinct subunits of an oligomeric comp lex, Possible functions of the endo-alpha 1,2-mannosidase in N-linked oligosaccharide processing are discussed.