Partial purification and characterization of an α- d-mannosidase from mature seeds of Prunus serotina Ehrh.

Abstract

An α- d-mannosidase (EC 3.2.1.24) has been purified approximately 90-fold from mature black cherry ( Prunus serotina Ehrh.) seeds in 35% yield using O-(diethylaminoethyl)-cellulose chromatography and Sephacryl S-200 gel filtration. The purified enzyme had a molecular weight of 150 000, as determined by Sephacryl S-200 gel filtration, and an isoelectric point at 4.8–5.2. The enzyme showed tight binding to concanavalin A-Sepharose 4B (Con A-Sepharose) with less than 25% of the applied activity being eluted by 1.0 M α-methyl- d-mannoside. The black cherry α- d-mannosidase showed high activity towards the synthetic substrates p- nitrophenyl-α- d - mannoside (K m = 2.8 mM) and 4-methyl-umbelliferyl-α- d-mannoside ( K m = 2.2 mM) at pH 4.0. The enzyme did not exhibit a metal ion requirement. However, silver nitrate (1 mM) inhibited activity by 70%. Metal chelators and thiol reagents had no effect on enzyme activity. α- d-Mannosidase activity was potently inhibited by d-mannono-(1,5)-lactone and the indolizidine alkaloid swainsonine, which caused 50% inhibition at approximately 73 μM and 0.4 μM, respectively. The β-glucosidase inhibitor castanospermine had no effect on this enzyme. Prolonged incubation of α- d-mannosidase with black cherry mandelonitrile lyase at 30°C did not change the molecular weight of the lyase. Furthermore, the elution profile of mandelonitrile lyase upon Con A-Sepharose 4B chromatography was unaltered by prior incubation of this glycoprotein with black cherry α- d-mannosidase for 8 h.