Measurement of spermidine/spermine-N1-acetyltransferase activity by high-performance liquid chromatography with N1-dansylnorspermine as the substrate

We developed a nonradioactive assay to measure spermidine/spermine N'-acetyltransferase (SSAT) activity by high-performance liquid chromatography (HPLC). N'-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 N'-dansylnorspermine and acetyl-CoA were approximately I I and 13 mu M, 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 mu g protein of cell extract corresponding to 1 x 10(5) 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. (C) 2008 Elsevier Inc. All rights reserved.