EFFECT OF SAPOSIN-A AND SAPOSIN-C ON THE ENZYMATIC-HYDROLYSIS OF LIPOSOMAL GLUCOSYLCERAMIDE

The degradation of glucosylceramide in lysosomes is accomplished by gl ucosylceramidase with the assistance of, at least, another protein, sa posin C (Sap C), which is generated from a large precursor together wi th three other similar proteins, saponins A, B, and D. In the present study, we have examined the effects of saposins on the enzymatic hydro lysis of glucosylceramide inserted in large and small phospholid lipos omes. The glucosylceramide contained in large unilamellar vesicles (LU V) was degraded by glucosylceramidase at a rate 7-8-fold lower than gl ucosylceramide inserted in small unilamellar vesicles (SUV). The separ ate addition of either Sap A of Sap C to the LUV system partially stim ulated the sphingolipid degradation while saposins B and D had no effe ct. In the presence of both Sap A and Sap C, the rate of sphingolipid degradation was higher than the sum of the rates with the two saposins individually, indicating synergism in their actions. The stimulatory effect of the two saposins depended on the incorporation of an acidic phospholipid such as phosphatidylserine (PS) into LUV. The characteris tics of glucosylceramidase activation by Sap C were different from tho se of Sap A. Sap C increased the rate of hydrolysis of both the artifi cial water soluble substate, 4-methylumbelliferyl-beta-D-glucopyranosi de, and the lipid substrate, glucosylceramide, while Sap A only stimul ated degradation of the sphingolipid. Also the binding properties of S aps A and C were markedly different. At acidic pH values, Sap C bound to PS-containing LUV and promoted the association of glucosylceramidas e with the membrane. In contrast, Sap A had poor affinity for the memb rane even in the presence of glucosylceramide; moreover, Sap A did not potentiate the capacity of Sap C to mediate glucosylceramidase bindin g. In conclusion, our results show that both Sap A and Sap C are requi red for maximal hydrolysis of glucosylceramide inserted in PS-containi ng LUV, that their effects are synergistic, and that their mode of act ion is different. Sap C is responsible for the membrane binding of glu cosylceramide, while Sap A stimulation is possibly related to its effe ct on the conformation of the enzyme. It can be envisaged that Sap A i n conjunction with Sap C might have a physiological role in glucosylce ramide degradation.