Splenic Glucocerebrosidase and Its Cytosolic Activator Protein: Effects on Substrate Hydrolysis and Covalent Inhibition by Conduritol B Epoxides

Abstract

β-Glucocerebrosidase, an enzyme associated with the lysosomal membrane, looses much of its activity during purification, especially if the procedure includes one or more delipidation steps (1). The activity could be restored by the additon of a cytosolic protein fraction from the same tissue (calf spleen). Activity loss and reactivation go largely unnoticed when the activity determinations are carried out in the presence of high concentrations (up to 10 mM) of taurocholate which has early been found to stimulate activity (2). These observations point to the presence of activating factors in the native environment of the enzyme; their chemical nature and their physiological significance is however, a still unsettled question. In part, this is due to the difficulties in obtaining reliable figures on the intracellular activity of the enzyme. The molar concentration of active enyzme in cells or crude preparations required for the calculation of intrinsic activities under different conditions is not known and activity measurements including those with the natural substrate glucocerebroside call for a disruption of the native environment by detergents. Acidic lipids like taurocholate, phosphatidylserine or dicetyl phosphate are added for maximal activity not only for measurements in micellar systems but also with enyzme and substrate incorporated into liposomes (3, 4).