The assay of glucocerebrosidase activity using the natural substrata

We examined eight published methods for the assay of glucocerebrosidase using the natural substrate glucocerebroside and tabulated the variable components and conditions while comparing these methods to ours. In each case assays were performed using our 8000-fold, partially purified human placental enzyme, having a pH optimum of 5.4-5.6 and a Km of approximately 0.60 mmol per liter. Results were obtained by following the release of radioactive ceramide or of unlabelled glucose. In many cases published results had been based on a one- or two-hour incubation time. Apparent specific activities varied over a 70-fold difference between the various procedures. When we measured activities from the linear (15-30 min) portion of rate curves the values increased by 1.4 to 3 times but still ranged from 6 X 10(3) - 180 X 10(3) nmol . mg-1 protein . h-1. We obtained maximum velocity using 1.2 mmol glucocerebroside, 0.5% (w/v)crude taurocholate and 2 microgram enzyme protein/ml. Specific activities reported from different laboratories are not directly comparable. Conditions for assay should be optimized for the enzyme preparation to be studied.