Requirement for a different hydrophobic moiety and reliable chromogenic substrate for endo-type glycosylceramidases

A series of synthetic lactosides with aglycones that differed in length and structure were used to determine the substrate specificity of endo-type gl ycosylceramidases, Endoglycoceramidases (EGCase) from bacteria preferred la ctosides with an acylamide structure over simple n-alkyl lactosides, While ceramide glycanase (CGase) from leech did not show preference. N-Acylaminoe thyl beta-lactosides and n-alkyl lactosides were substrates for both EGCase and CGase, but N-acylaminobutyl beta-lactosides, whose acylamide residue d iffers from that in ceramide, were not hydrolyzed by EGCases, Thus, EGCases , but not CGase, appear to require an N-acyl group at the same position as that of intact glycosphingolipid for substrate recognition. A p-nitrophenyl lactoside derivative possessing an N-acyl chain was degraded by both EGCas es and CGase and this chromogenic substrate may be an alternative substrate for endo-type glycosylceramidase activity. K-m of the chromogenic lactosid e for CGase and Rhodococcus EGCase were 28 mu M and 2.9 mM, respectively.