Purification of glucosylceramidase by affinity chromatography

Abstract

Glucosylceramide:β-glucosidase (glucocerebrosidase, EC 3.2.1.45) has been purified 12 900-fold from human placenta using a specific affinity column. The ligand, glucosyl sphingosine, prepared from glucocerebroside by alkaline hydrolysis, was attached to epoxy-activated Sepharose 6B. The enzyme was applied to the column in citrate–butanol or citrate – ethylene glycol solution at its pH optimum (5.6). No enzyme was bound in the presence of detergent. Glucocerebrosidase was eluted with citrate–taurocholate buffer at low pH or with citrate-taurocholate buffer containing D-gluconolactone at the pH optimum. Citrate–taurocholate solution alone at the pH optimum would not elute the enzyme. The enzyme hydrolyzed both the natural substrate, glucocerebroside, and the artificial substrate, 4-methylumbelliferyl glucopyranoside. Glucocerebrosidase migrated as a single band on 10% sodium dodecyl sulfate–polyacrylamide tube and (or) slab gels, corresponding to a molecular weight of 75 000. It also ran as a single zone of enzyme activity or protein on native gels, composed of 2.2% polyacrylamide – 0.4% agarose containing sodium taurocholate. This is the first reported use of this gel system for the examination of glucocerebrosidase. Overall recovery is 30%. The procedure represents a more rapid and specific technique for purification of glucocerebrosidase than those previously reported.