Characterization of the activation of rat liver β-glucosidase by sialosylgangliotetraosylceramide

We show that sialosylgangliotetraosylceramide (G(M1)) is a potent activator of delipidated (sodium cholate- and 1-butanol-extracted) lysosomal rat liver glucocerebroside:β-glucosidase. Stimulation of 4-methylumbelliferyl-β-D-glucopyranoside hydrolysis by the β-glucosidase was markedly dependent upon the concentration of G(M1) in the assay medium. Estimations of critical micellar concentration (CMC) performed fluorometrically using the dye N-phenylnaphthylamine revealed two CMC values of G(M1) above 18°C; the CMC of the primary micelles (3.32 μM) was temperature-independent whereas that of the secondary micelles decreased with decreasing temperature (17.2 and 10.8 μM at 37 and 20°C, respectively). In the temperature range of 18-39°C, β-glucosidase activity increased sharply when the G(M1) concentration was above the CMC of the secondary micelles. Although a heat-stable factor, purified from the spleen of a patient with Gaucher's disease, had a profound effect on the activation of β-glucosidase by G(M1)), it decreased the CMC only slightly (14.8 versus 17.2 μM at 37°C). The heat-stable factor (8 μg/ml) changed the shape of the activation curve from sigmoidal to hyperbolic, suggesting that the heat-stable factor permits β-glucosidase to be activated by primary micelles or monomers. The results of gel filtration chromatography and sucrose gradient centrifugation in H₂O and D₂O revealed that the activation of β-glucosidase by G(M1) was associated with an increase in the size of the enzyme from 45,800 to 178,500 daltons and an increase in the partial specific volume from 0.697 to 0.740 ml/g. The active, reconstituted β-glucosidase appears to consist of 50% protein and 50% ganglioside (56 molecules/178,500 g). Concentrations of G(M1) below the CMC of secondary micelles increased the rate of inactivation of the enzyme by the irreversible inhibitor conduritol B epoxide at 37°C, indicating that G(M1) monomers or primary micelles do interact with the enzyme, even though they do not increase the rate of hydrolysis of 4-methylumbelliferyl-β-D-glucopyranoside by the enzyme.