A fluorometric microassay procedure for monitoring the enzymatic activity of GM1-ganglioside β-galactosidase by use of high-performance liquid chromatography

Kazutoshi Kiuchi, Tatsuro Mutoh, Makoto Naoi

研究成果: Article査読

7 被引用数 (Scopus)

抄録

For the measurement of the enzymatic activity of GM1-ganglioside (II3 NeuAcGgOse4Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosyl-glucosylceramide) β-galactosidase in crude enzyme samples, a microassay using nonradioisotopic GM1-ganglioside was devised. To reduce the volume of the reaction mixture and eliminate the interferences due to the fluorescent contaminants in the reaction mixture, NADH, a product after the oxidation of the released galactose with NAD and β-galactose dehydrogenase, was fluorometrically estimated by use of high-performance liquid chromatography. By this method, as little as 10 pmol of galactose can be detected. Using rat brain homogenates as an enzyme sample, the several parameters were reexamined to define the optimal conditions for the assay. This assay method was also applied to human cultured skin fibroblast homogenates, and it was found that this method can be used for the diagnosis of GM1-gangliosidosis, instead of the usual method using the radioisotopelabeled natural substrate.

本文言語English
ページ(範囲)146-151
ページ数6
ジャーナルAnalytical Biochemistry
140
1
DOI
出版ステータスPublished - 07-1984
外部発表はい

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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