Glucocerebrosidase Activator Protein of Bovine Spleen.

Glucocerebrosidase stimulating proteins ("co-(beta)-glucosidase") from bovine spleen were purified and characterized. The nature of the stimulation was investigated. The purification involved acidification of homogenized spleen, heat denaturation, and chromatography with DEAE-Sephacel, Sephadex G-75, hydroxylapatite, and decyl agarose columns. Gel electrophoresis of the product revealed a trace of inert protein, a major diffuse b and , and a minor, faster moving b and . The latter two b and s were shown to stimulate glucosidase. The coglucosidase M(,r) was 4,900 as measured with a Sephacryl S-200 column containing guanidine hydrochloride. A pI of 4.3-4.4 was indicated by isoelectric focusing. Coglucosidase was destroyed by Pronase. Coglucosidase at 0.15 (mu)g/ml stimulated glucosidase 50%. It raised the V(,max)'s and lowered the K(,m)'s when both glucosyl ceramide and methylumbelliferyl-(beta)-glucoside were used. The distribution of coglucosidase among particulate fractions obtained by subcellular fractionation paralleled that of glucosidase, but more stimulating activity was found in the cytosolic fraction. A complex between coglucosidase and radioactive glucosyl ceramide and methylumbelliferyl-(beta)-glucoside could not be detected under equilibrium and non-equilibrium binding conditions. Complex formation between coglucosidase and glucosidase was shown by the binding of the enzyme to an affintiy column containing coglucosidase. This binding could be blocked by phosphatidyl serine. This lipid and other acidic lipids also stimulated the enzyme. Coglucosidase and phosphatidyl serine protected glucosidase against inactivation by sulfhydryl reagents. Extracting lipids from glucosidase resulted in a loss of enzyme activity and of sensitivity to activation by coglucosidase. Adding back the lipids or phosphatidyl serine increased the sensitivity of the delipidated enzyme to coglucosidase. It is proposed that coglucosidase is a significant component of the glucocerebroside hydrolysis system, and that lipids act on glucosidase to produce an enzyme conformation which allows coglucosidase to bind to and stimulate glucosidase.PhDBiochemistryUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/158400/1/8125067.pd