HYDROLYSIS OF LACTOSYLCERAMIDE BY HUMAN GALACTOSYLCERAMIDASE AND GM1-BETA-GALACTOSIDASE IN A DETERGENT-FREE SYSTEM AND ITS STIMULATION BY SPHINGOLIPID ACTIVATOR PROTEINS, SAP-B AND SAP-C - ACTIVATOR PROTEINS STIMULATE LACTOSYLCERAMIDE HYDROLYSIS

Two exo-beta-galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1-beta-galactosidase (EC 3.2.1.23) and galact osylceramidase (EC 3.2.1.46). Analyses were performed with both enzyme s, using lactosylceramides with varying acyl chain lengths as substrat es that were inserted into unilamellar liposomes and naturally occurri ng sphingolipid activator proteins sap-B and sap-C, rather than deterg ents, to stimulate the reaction. While sap-B was a better activator fo r the reaction catalyzed by GM1-beta-galactosidase, sap-C preferential ly stimulated lactosylceramide hydrolysis by galactosylceramidase. The enzymic hydrolysis of liposome-integrated lactosylceramides was signi ficantly dependent on the structure of the lipophilic aglycon moiety o f the lactosylceramide decreasing with increasing length of its fatty acyl chain (C-2>C-4>C-6>C-8>C-10>C-18). However, in the presence of de tergents the degradation rates were independent of the acyl chain leng th. Hydrolysis of liposomal lactosylceramide was compared with sap-B-s timulated hydrolysis of liposomal ganglioside GM1 by GM1-beta-galactos idase and sap-C-stimulated degradation of liposomal galactosylceramide by galactosylceramidase. Kinetic and dilution experiments indicated t hat sap-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-beta-galactosidase as opt imal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) B iochim. Biophys. Acta 481, 561-572]. GM1-beta-galactosidase was more a ctive on these complexes than on glycolipids (GM1 and lactosylceramide s) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lacto sylceramide by galactosylceramidase proceeds almost exclusively on lip osomal surfaces: both activators, sap-C and sap-B, stimulated the hydr olysis of lactosylceramide analogues with long acyl chains more than t he hydrolysis of lactosylceramides with short acyl chains.