Measurement of spermidine/spermine-N1-acetyltransferase activity by high-performance liquid chromatography with N1-dansylnorspermine as the substrate |
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Authors: | Takao Koichi Miyatake Shinji Fukazawa Kazuaki Wada Makiko Sugita Yoshiaki Shirahata Akira |
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Institution: | Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyaki-dai, Sakado, Saitama 350-0295, Japan |
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Abstract: | We developed a nonradioactive assay to measure spermidine/spermine N1-acetyltransferase (SSAT) activity by high-performance liquid chromatography (HPLC). N1-dansylnorspermine was prepared and evaluated as a substrate of acetylation with acetyl-CoA by SSAT in rat hepatoma (HTC) cells. Kinetic studies revealed that the Km values of N1-dansylnorspermine and acetyl-CoA were approximately 11 and 13 μM, respectively. When the assay method was applied to HTC cell samples, the SSAT activity, even at the control level, could easily be detected in as few as 20 μg protein of cell extract corresponding to 1 × 105 cells per determination, and 100 samples could be analyzed overnight. Thus, our HPLC method is a rapid and sensitive assay for the measurement of SSAT activity. |
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