FLUORESCENCE ASSAY OF GLUCOSYLCERAMIDE GLUCOSIDASE USING NBD-CEREBROSIDE

被引：3

作者：

ABE, A

SHAYMAN, JA

RADIN, NS

机构：

[1] UNIV MICHIGAN,MED CTR,DEPT OBSTET & GYNECOL,DIV NEPHROL,1150 W MED CTR DR,ANN ARBOR,MI 48109

[2] UNIV MICHIGAN,MENTAL HLTH RES INST,ANN ARBOR,MI 48109

来源：

LIPIDS | 1992年 / 27卷 / 12期

关键词：

D O I：

10.1007/BF02535587

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

A sensitive fluorometric assay for glucocerebroside beta-glucosidase [Dinur, T., Grabowski, G.A., Desnick, R.J., and Gatt, S. (1984) AnaL Biochem. 136,223-2341 has been reexamined. It was found that the lipids containing the NBD moiety (12-[N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] used for standardization of the assay are light-sensitive and that the yield of fluorescent light is very sensitive to the composition of the solvent used in the fluorometric measurement. Some protection against fading could be obtained by adding a free-radical trapping agent, SlowFade. The fading of the free NBD-acid, when used for standardization, could be prevented by adding ethanol to the solvent, but this reduced the fluorescence yield. It is recommended that some of the fluorescent substrate be enzymatically hydrolyzed completely to NBD-ceramide, which can be utilized as the standard without the need to add ethanol. A warning about enzyme reaction rate stability with time is given, with a suggestion for ensuring constancy of activity.

引用

页码：1052 / 1054

页数：3

共 12 条

[1] THE OCCURRENCE OF 2 IMMUNOLOGICALLY DISTINGUISHABLE BETA-GLUCOCEREBROSIDASES IN HUMAN SPLEEN [J].