Purification of glucosylceramidase by affinity chromatography.
Glucosylceramide: beta-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.
D002846:Chromatography, Affinity; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005959:Glucosidases / Q000302:isolation & purification*; D005962:Glucosylceramidase / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D008722:Methods; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy
D005959:Glucosidases; D005962:Glucosylceramidase