4-methylumbelliferyl-glucoside and conduritol-epoxide

Glucocerebrosidase is present in considerable amounts in human urine. The enzyme is stable in concentrated urine for several days when stored at 0 degrees C. Like tissue glucocerebrosidase, the urinary enzyme is inhibited by conduritol B-epoxide and hydrolyses not only glucocerebroside but also the synthetic substrate 4-methyl-umbelliferyl-beta-D-glucoside. The enzyme is deficient in urine from patients with Gaucher disease (type 1). It is possible to discriminate completely between patients with type 1 Gaucher disease and control subjects by measuring the ratio glucocerebrosidase/beta-hexosaminidase in urine. The value of this ratio (mean +/- SE) with the synthetic substrates 4-methylumbelliferyl-beta-glucoside and p-nitrophenyl-beta-N-acetylglucosaminide, respectively, was 34.2 +/- 3.7 (n = 24) in the controls and 2.1 +/- 0.9 (n = 21) in the patients.

Topics: Adolescent; Adult; beta-N-Acetylhexosaminidases; Child; Female; Gaucher Disease; Glucosidases; Glucosides; Glucosylceramidase; Hexosaminidases; Humans; Hymecromone; Inositol; Male; Middle Aged; Substrate Specificity

We show that sialosylgangliotetraosylceramide (GM1) is a potent activator of delipidated (sodium cholate- and 1-butanol-extracted) lysosomal rat liver glucocerebroside:beta-glucosidase. Stimulation of 4-methylumbelliferyl-beta-D-glucopyranoside hydrolysis by the beta-glucosidase was markedly dependent upon the concentration of GM1 in the assay medium. Estimations of critical micellar concentration (CMC) performed fluorometrically using the dye N-phenylnaphthylamine revealed two CMC values of GM1 above 18 degrees C; the CMC of the primary micelles (3.32 microM) was temperature-independent whereas that of the secondary micelles decreased with decreasing temperature (17.2 and 10.8 microM at 37 and 20 degrees C, respectively). In the temperature range of 18-39 degrees C, beta-glucosidase activity increased sharply when the GM1 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 beta-glucosidase by GM1, it decreased the CMC only slightly (14.8 versus 17.2 microM at 37 degrees C). The heat-stable factor (8 micrograms/ml) changed the shape of the activation curve from sigmoidal to hyperbolic, suggesting that the heat-stable factor permits beta-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 beta-glucosidase by GM1 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 beta-glucosidase appears to consist of 50% protein and 50% ganglioside (56 molecules/178,500 g). Concentrations of GM1 below the CMC of secondary micelles increased the rate of inactivation of the enzyme by the irreversible inhibitor conduritol B epoxide at 37 degrees C, indicating that GM1 monomers or primary micelles do interact with the enzyme, even though they do not increase the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside by the enzyme.

Topics: Animals; beta-Glucosidase; Chemical Phenomena; Chemistry, Physical; Dose-Response Relationship, Drug; Enzyme Activation; G(M1) Ganglioside; Gangliosides; Gaucher Disease; Glucosidases; Glucosides; Hot Temperature; Humans; Hymecromone; Inositol; Kinetics; Liver; Lysosomes; Male; Micelles; Molecular Weight; Rats; Rats, Inbred Strains; Temperature