Assay of N1-Acetylpolyamine Oxidase Activity with N1,N11-Didansylnorspermine as the Substrate by Ion-Pair Reversed Phase High Performance Liquid Chromatography

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概要

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• An assay method to measure N1-acetylpolyamine oxidase (PAO) activity with N1,N11-didansylnorspermine (DiDNS333) as the substrate by high performance liquid chromatography (HPLC) was developed. Dansylpolyamines were synthesized, and their activity as a substrate for partially purified PAO from rat liver was evaluated. Among the dansylpolyamines, DiDNS333 was a useful substrate for the development of the PAO assay method. DiDNS333 was degraded by PAO to 1-dansylamido-3-propanal (DNS3al) and N1-dansylnorspermidine (DNS33). As DNS3al was separated into two peaks by HPLC of the assay mixture containing aminoguanidine, determination of DNS33 was used for the development of the assay method. When the assay method was applied to inhibition studies, the DNS33 peak on the chromatogram was consistently produced, and weak interference was found in the incubation with higher concentrations of natural polyamines. This result suggested that contaminating polyamines in biological samples do not interfere with this method. When the assay method was applied to cell extract from Chinese hamster ovary cell samples, the PAO activity, even at the low level in the cells, could easily be detected with as little as 10 μg of protein, which corresponds to 1×105 cells. This HPLC method is a rapid, sensitive and useful assay for the measurement of PAO activity.

著者

• Shirahata Akira

  産業医科大学 医学部小児科学

• Shirahata A

  Fac. Of Pharmaceutical Sciences Josai Univ.

• Shirahata A

  聖マリアンナ医科大学 医学部医学統計学分野

• Takao Koichi

  Laboratory Of Cellular Physiology Department Of Clinical Dietetics & Human Nutrition Faculty Of

• Takao Koichi

  Department Of Analytical Chemistry Faculty Of Pharmaceutical Sciences Josai University

• Sugita Yoshiaki

  Laboratory of Cellular Physiology, Department of Clinical Dietetics & Human Nutrition, Faculty of Ph

• Shirahata Akira

  Laboratory of Cellular Physiology, Department of Clinical Dietetics & Human Nutrition, Faculty of Ph

• Sugita Yoshiaki

  Faculty Of Pharmaceutical Sciences Josai University

• Shirahata Akira

  Lab. Of Cellular Physiology Dep. Of Clinical Dietetics And Human Nutrition Fac. Of Pharmaceutical Sc

• Shirahata Akira

  Laboratory Of Bio-analytical Chemistry Department Of Pharmaceutical Sciences Faculty Of Pharmaceutical Sciences Josai University

• Shirahata Akira

  Laboratory of Cellular Physiology, Department of Clinical Dietetics & Human Nutrition, Faculty of Pharmaceutical Sciences, Josai University

• Sugita Yoshiaki

  Laboratory of Cellular Physiology, Department of Clinical Dietetics & Human Nutrition, Faculty of Pharmaceutical Sciences, Josai University

• Sugita Yoshiaki

  Laboratory of Bioorganic Chemistry, Department of Pharmaceutical Technochemistry, Josai University

関連論文

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