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

Yasuhiro Hayashi, Kouta Zama, Eriko Abe, Nozomu Okino, Takehiko Inoue, Kousaku Ohno, Makoto Ito

Research output: Contribution to journalArticle

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Abstract

The activity of lysosomal acid β-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 β-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-bora-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. Vmax/Km values of human fibroblast AGC for fluorescent GlcCer were much higher than those for 4-methylumbelliferyl-β-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 μl of human serum or 1 × 104 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.

Original languageEnglish
Pages (from-to)122-129
Number of pages8
JournalAnalytical Biochemistry
Volume383
Issue number1
DOIs
Publication statusPublished - Dec 1 2008

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Glucosylceramidase
Glucosylceramides
Gaucher Disease
High performance liquid chromatography
Ceramides
Assays
High Pressure Liquid Chromatography
Fibroblasts
Acids
Glucosides
Reaction products
Serum
Vertebrates
Substrates
Glucose
Mutation
Genes
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
Tissue
Enzymes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral β-glucocerebrosidases. / Hayashi, Yasuhiro; Zama, Kouta; Abe, Eriko; Okino, Nozomu; Inoue, Takehiko; Ohno, Kousaku; Ito, Makoto.

In: Analytical Biochemistry, Vol. 383, No. 1, 01.12.2008, p. 122-129.

Research output: Contribution to journalArticle

Hayashi, Yasuhiro ; Zama, Kouta ; Abe, Eriko ; Okino, Nozomu ; Inoue, Takehiko ; Ohno, Kousaku ; Ito, Makoto. / A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral β-glucocerebrosidases. In: Analytical Biochemistry. 2008 ; Vol. 383, No. 1. pp. 122-129.
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AU - Inoue, Takehiko

AU - Ohno, Kousaku

AU - Ito, Makoto

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N2 - The activity of lysosomal acid β-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 β-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-bora-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. Vmax/Km values of human fibroblast AGC for fluorescent GlcCer were much higher than those for 4-methylumbelliferyl-β-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 μl of human serum or 1 × 104 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.

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