A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral beta-glucocerebrosidases

Analytical Biochemistry
Yasuhiro HayashiMakoto Ito

Abstract

The activity of lysosomal acid beta-glucocerebrosidase (AGC, EC 3.2.1.45), which hydrolyzes the O-glycosidic linkage between D-glucose and ceramide of glucosylceramide (GlcCer), is a marker for the diagnosis of Gaucher disease because the disease is caused by dysfunction of AGC due to mutations in the gene. The activity of AGC is potently inhibited by conduritol B epoxide (CBE), whereas CBE-insensitive nonlysosomal neutral beta-glucocerebrosidase (NGC) activities have been found in various vertebrates, including humans. We report here a new reliable method to determine AGC as well as NGC activities using normal-phase high-performance liquid chromatography (HPLC) and NBD (4-nitrobenzo-2-oxa-1,3-diazole)- or BODIPY (4,4-difluoro-4-boro-3a,4a-diaza-s-indacene)-labeled GlcCer as a substrate. The reaction products of the enzymes, C6-NBD-ceramide and C12-BODIPY-ceramide, were clearly separated from the corresponding substrates on a normal-phase column within 5 min using a different solvent system. Reaction products could be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol for C6-NBD-ceramide and from 10 fmol to 5 pmol for C12-BODIPY-ceramide. V(max)/K(m) values of human fibroblast AGC for fluorescent GlcCer w...Continue Reading

References

Jan 1, 1978·Annual Review of Biochemistry·R O Brady
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Sep 13, 2003·The Journal of Pediatrics·Ozlem Goker-AlpanEllen Sidransky
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Nov 3, 2006·The Journal of Clinical Investigation·Yildiz YildizDavid W Russell
Nov 16, 2006·The Journal of Biological Chemistry·Rolf G BootJohannes M F G Aerts
Jun 28, 2007·The Journal of Biological Chemistry·Yasuhiro HayashiMakoto Ito

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Citations

Oct 29, 2011·Analytical and Bioanalytical Chemistry·Omid MotabarWei Zheng
Jan 22, 2011·Journal of Medicinal Chemistry·Juan J MaruganChristopher P Austin
Jun 13, 2015·Clinical Biochemistry·Azusa YoneshigeJunko Matsuda

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