Conditions affecting the activity of glucocerebrosidase purified from spleens of control subjects and patients with type 1 Gaucher disease.

Glucocerebrosidase was purified to homogeneity from spleens of control subjects and Type 1 Gaucher disease patients by immunoaffinity chromatography. Activation of the enzyme by taurocholate, phosphatidylserine and sphingolipid activator protein 2 (saposin C; SAP-2) was investigated by titration of combinations of various effectors in the absence and presence of Triton X-100. The specific activity of Type 1 Gaucher glucocerebrosidase was found to be less than 20% of the corresponding control value under most conditions. However, in the presence of optimal amounts of activator protein SAP-2 and phosphatidylserine (and in the absence of Triton X-100 and/or taurocholate), the specific activity of mutant enzyme towards artificial and natural lipid substrates was close to normal when measured at pH 5.0-5.5. At pH values below 5.0, the specific activity of mutant enzyme decreased more rapidly compared to that of control enzyme. The activity of Type 1 Gaucher glucocerebrosidase in the intact cell might, in a comparable manner, be highly dependent on the extent of activation by endogenous activators and on the intralysosomal pH. Values for residual glucocerebrosidase activity, as measured in vitro in extracts of cells and tissues from Type 1 Gaucher disease patients, are indeed highly dependent on the assay conditions employed. Consequently such measurements are of little value in the assessment of the actual capacity for glucosylceramide hydrolysis and for prediction of the clinical severity of the disease.