Rice alpha-mannosidase digesting the high mannose glycopeptide of glutelin

alpha -Mannosidase (EC 3.2,1.24) from rice dry seeds was purified to homoge neity, Optimum pH and K-m for pNP-alpha -Man hydrolysis were pH 4.3-4.5 and 1.04 mM, respectively. The enzyme digested mannobioses such as Man alpha - 1,2Man, Man alpha -1,6Man, Man alpha -1,3Man but Man alpha -1,4Man. Zn2+ io n was required for the activity, whereas EDTA and swainsonine inhibited the activity by 80 and 96%, respectively. The rice storage protein, glutelin w as prepared and its basic subunits were shown to have high mannose-type sug ar chains by two-dimensional mapping using NH2-P and C-ix silica columns. T hey were Man(9)GlcNAc(2), Man(8)GlcNAc(2), Man(7)GLcNAc(2), Man(6)GlcNAc(2) and Man(5)GlcNAc(2). All these oligosaccharides were digested by the purif ied alpha -mannosidase, and ManGlcNAc(2) and mannose were formed. Glycopept ides, having these high mannose-type sugar chains, could also be digested b y the alpha -mannosidase. Subunits were prepared from glutelin basic subuni t and the richest subunit among them, subunit 2 (isoform 2), was digested b y the alpha -mannosidase. Isoform 2 was digested by V8 protease only partia lly and slowly. However, isoform 2, pre-treated with the alpha -mannosidase , was rapidly and completely digested by V8 protease.