title: Factors affecting the binding of glucosylceramidase to its natural substrate dispersion.
abstract: This paper reports the results of ultracentrifugation experiments devised for investigating the interactions occurring in the conditions of the enzymatic assay between glucosylceramidase and the components of the substrate dispersion. This dispersion contains, besides glucosylceramide, taurocholate and oleic acid. It has been found that glucosylceramide aggregates with oleic acid, while taurocholate is unable to associate with the sphingolipid, but improves the stability of the dispersion. When a crude glucosylceramidase placental preparation is incubated with the assay mixture the enzyme is almost totally bound to the glucosylceramide-oleic acid particles. The binding between glucosylceramidase and the substrate-containing particles is dramatically depressed by changes of experimental conditions which negatively influence also the enzyme activity such as: (1) a decrease in the molarity of the citrate/phosphate buffer; (2) an increase of the buffer pH, and (3) an increase of the taurocholate concentration. An excess of oleic acid neither inhibits the binding nor the activity. These results strongly suggest that glucosylceramidase activity is directly correlated with the binding of the enzyme to the lipid interface of the substrate-containing particles. We conclude that the enzymatic mechanism of glucosylceramide hydrolysis involves at least two steps: first the physical localization of the enzyme at the lipid-water interface, second the hydrolysis of the substrate glucosidic bond.
authors.affiliation: Department of Metabolism and Pathological Biochemistry, Istituto Superiore Sanità, Rome, Italy.
authors.forename: A M
authors.initials: AM
authors.lastname: Vaccaro
authors.forename: M
authors.initials: M
authors.lastname: Tatti
authors.forename: F
authors.initials: F
authors.lastname: Ciaffoni
authors.forename: R
authors.initials: R
authors.lastname: Salvioli
mesh_terms: D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D005776:Gaucher Disease / Q000201:enzymology; D005959:Glucosidases / Q000378:metabolism*; D005962:Glucosylceramidase / Q000302:isolation & purification / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D011485:Protein Binding; D013154:Spleen / Q000201:enzymology
chemical_list: D005959:Glucosidases; D006026:Glycoside Hydrolases; D005962:Glucosylceramidase