Studies on glucosylceramidase binding to phosphatidylserine liposomes: the role of bilayer curvature.
The influence of phosphatidylserine (PS) liposome size on their capacity to activate and bind purified glucosylceramidase was investigated. Gel filtration and flotation experiments showed that large unilamellar vesicles (LUV) of either pure PS or PS in admixture with phosphatidylcholine (PC) are unable to tightly bind purified glucosylceramidase, and thus, to fully stimulate its activity. By contrast, small unilamellar vesicles (SUV) of PS adsorb glucosylceramidase can either be favoured or inhibited by factors affecting the bilayer curvature of PS liposomes. An increase of PS vesicle size induced by a fusogenic agent such as poly(ethylene glycol) (PEG), decreased enzyme binding and activity. On the contrary, the reduction of PS LUV size by sonication increased their stimulating ability. Enzyme association with PS SUV is reversible. In fact, glucosylceramidase bound to PS SUV was released from the lipid surface when the SUV were transformed into larger vesicles by PEG; dissociation from the vesicles resulted in a dramatic decrease of enzyme activity. Although PS LUV are unable to reconstitute glucosylceramidase, their association with oleic acid (OA) promotes the interaction with glucosylceramidase. This phenomenon is best explained in terms of OA-induced surface defects of PS LUV, with consequent exposure of the more hydrophobic part of the membrane and hence the improved binding of hydrophobic region/s of glucosylceramidase. Our data indicate that the physical organization of the PS-containing liposomes is of critical importance of glucosylceramidase reconstitution. The observation that physical changes of the lipid surface can markedly affect the enzyme activity offers a new approach to the study of glucosylceramidase regulation.
Vaccaro
A M
AM
Department of Metabolism and Pathological Biochemistry, Istituto Superiore di Sanità, Roma, Italy.
Tatti
M
M
Ciaffoni
F
F
Salvioli
R
R
Barca
A
A
Roncaioli
P
P
D004789:Enzyme Activation; D005962:Glucosylceramidase / Q000378:metabolism*; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010316:Particle Size; D010718:Phosphatidylserines / Q000378:metabolism*
D008051:Lipid Bilayers; D008081:Liposomes; D009829:Oleic Acids; D010718:Phosphatidylserines; D019301:Oleic Acid; D005962:Glucosylceramidase