A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral beta-glucocerebrosidases.
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 were much higher than those for 4-methylumbelliferyl-beta-d-glucoside (4MU-Glc), which is used prevalently for Gaucher disease diagnosis. As a result, AGC activity was detected quantitatively using fluorescent GlcCer, but not 4MU-Glc, using 5 microl of human serum or 1 x 10(4) cultured human fibroblasts. The current method clearly showed the decrease of AGC activities in fibroblasts and serum from the patient with Gaucher disease compared with normal individuals, suggesting that the method is applicable for the diagnosis of Gaucher disease. Furthermore, this method was found to be useful for measuring the activities of nonlysosomal NGC of various cells and tissues in the presence of CBE.
Hayashi
Yasuhiro
Y
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan.
Zama
Kouta
K
Abe
Eriko
E
Okino
Nozomu
N
Inoue
Takehiko
T
Ohno
Kousaku
K
Ito
Makoto
M
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D005962:Glucosylceramidase / Q000378:metabolism*; D006801:Humans; D051381:Rats; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D005962:Glucosylceramidase