Reconstitution of glucosylceramidase on binding to acidic phospholipid-containing vesicles.
Studies were conducted to investigate the mechanism by which acidic phospholipid-containing vesicles stimulate purified placental glucosylceramidase activity towards the water-soluble substrate 4-methylumbelliferyl-beta-D-glucopyranoside (MUGlc). Vesicles composed of pure phosphatidic acid (PA) or pure phosphatidylserine (PS) stimulated the activity of the enzyme about 20-fold. The inclusion of cholesterol and phosphatidylcholine (PC), beside PA, into the vesicles slightly improved their stimulatory effect. Further addition of oleic acid (OA) markedly increased the stimulation (50-fold). By ultracentrifugation and gel permeation procedures it was shown that, under optimal conditions for stimulation of the MUGlc hydrolysis by acidic phospholipid-containing vesicles, purified glucosylceramidase spontaneously binds to their surface. Interestingly, the molar fraction of the acidic phospholipid into the mixed vesicles, rather than its concentration in the assay, is the crucial parameter for activation and binding of the enzyme. The importance of glucosylceramidase association with appropriate vesicles for enzyme activation was indicated by observing that the presence of 0.2 M citrate-phosphate buffer (pH 5.5), that prevented the binding to PA-containing surfaces, also inhibited the enzyme activity. Our results indicate that the reconstitution of glucosylceramidase activity occurs through the spontaneous tight association of the enzymatic protein with preformed acidic phospholipid-containing vesicles.
Vaccaro
A M
AM
Department of Metabolism and Pathological Biochemistry, Istituto Superiore Sanità, Roma, Italy.
Tatti
M
M
Ciaffoni
F
F
Salvioli
R
R
Roncaioli
P
P
D002499:Centrifugation, Density Gradient; D002845:Chromatography; D005960:Glucosides / Q000378:metabolism*; D005962:Glucosylceramidase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*
D005960:Glucosides; D008081:Liposomes; D010743:Phospholipids; C014110:4-methylumbelliferyl glucoside; D006923:Hymecromone; D005962:Glucosylceramidase