[beta]-Glucosidase activator protein from bovine spleen ("coglucosidase")

Abstract

[beta]-Glucosidase-stimulating proteins ("co-[beta]-glucosidase") have been isolated from bovine spleen by acidification of homogenized spleen, heat denaturation, and chromatography with DEAE-Sephacel, Sephadex G-75, hydroxyapatite, and decyl agarose columns. Gel electrophoresis of the product revealed a trace of inert protein and two fast-moving bands, a major diffuse band and a minor, faster-moving band. The latter two bands could be eluted from the gel and shown to stimulate a glucosidase preparation from bovine spleen. They both stained with Stains All and fast green, but poorly with Coomassie blue. The bands could also be visualized by ultraviolet scanning. Periodate-Schiff stain was positive for the major band. The Mr of the coglucosidase was about 20,400 as measured with the gel permeation column, but 4900 as measured with a Sephacryl S-200 column containing guanidine hydrochloride and roughly 6200 as measured by gel electrophoresis with Na dodecyl sulfate. A pI of 4.3-4.4 was indicated by isoelectric focusing. Neutral sugar was found to be present, but no sialic acid. It was destroyed by Pronase, but not by lyophilization, N-ethylmaleimide, or alkaline phosphatase. Stimulation of the basal activity (1 nmol/h assayed with methylumbelliferyl glucoside) was 50% when 0.15 [mu]g/ml of coglucosidase was included in the incubation. The activating protein raised the V values and lowered the Km values when both glucosyl ceramide and the artificial substrate were used. In contrast, phosphatidyl serine raised both the V, and the Km for cerebroside hydrolysis. The activator protein was found to occur in the soluble part of spleen as well as in the mitochondrial and lysosomal fractions.