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 glycosylceramidases. Endoglycoceramidases (EGCase) from bacteria preferred lactosides with an acylamide structure over simple n-alkyl lactosides. While ceramide glycanase (CGase) from leech did not show preference. N -Acylaminoethyl beta-lactosides and n -alkyl lactosides were substrates for both EGCase and CGase, but N-acylaminobutyl beta-lactosides, whose acylamide residue differs 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 EGCases and CGase and this chromogenic substrate may be an alternative substrate for endo-type glycosylceramidase activity. Km of the chromogenic lactoside for CGase and Rhodococcus EGCase were 28 microM and 2.9 mM, respectively.
Miura
Y
Y
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan.
Arai
T
T
Ohtake
A
A
Ito
M
M
Yamamoto
K
K
Yamagata
T
T
D002863:Chromogenic Compounds / Q000378:metabolism*; D005697:Galactosides / Q000378:metabolism; D010694:Lactase-Phlorizin Hydrolase / Q000145:classification* / Q000378:metabolism*; D013045:Species Specificity; D013379:Substrate Specificity
D002863:Chromogenic Compounds; D005697:Galactosides; D010694:Lactase-Phlorizin Hydrolase