Purification of ±-Mannosidase from the Cotyledons of Pea Seeds ( Pisum sativum L.)

Abstract

Summary The predominant swainsonine-sensitive form of α-mannosidase (EC 3.2.1.24) has been purified from the cotyledons of developing pea seeds ( Pisum sativum L.). The purification sequence involved extraction with Tris. CI, pH 7.6; adjustment of pH to pH 4.9; selection of proteins precipitating between 50–70% of saturation with ammonium sulphate; gel filtration on Biogel P-200; affinity chromatography on concanavalin A-Sepharose 4B; anion exchange on DEAE-Sephacel; chromatofocusing, and gel filtration on Sephadex G-75. Substrate affinity could not be employed, because unlike the jack bean enzyme, the pea enzyme did not attach to yeast α-mannan-Sepharose, nor to p -aminophenyl-α-D-mannopyranoside-agarose. The pea enzyme is a glycoprotein of estimated M r 300,000 to 320,000 with a pI of 4.3.