A high-throughput colorimetric assay to characterize the enzyme kinetic and cellular activity of spermidine/spermine N1-acetyltransferase 1.
Spermidine/spermine N(1)-acetyltransferase 1 (SSAT1) is a key enzyme that catalyzes the catabolism of polyamines. SSAT1 is a very important enzyme because it not only maintains the homeostasis of polyamines but also is involved in many physiological and pathological events. As such, a rapid assay of SSAT1 activity is valuable in drug screening and clinical diagnostics. Here, we report a novel colorimetric assay for monitoring SSAT1 activity in zebrafish (zSSAT1). In comparison with the available SSAT1 assays, this new method is cost-effective and simple. The optimal zSSAT1 activity was obtained below 55°C in a mild alkaline environment. The K(m) values of zSSAT1 for spermidine and spermine are 55 and 182 μM, respectively, whereas putrescine is not a good substrate for zSSAT1. In addition to enzyme kinetic studies, the colorimetric assay was also used to detect the cellular activity of SSAT1. Thus, the current method is a reliable assay for determining SSAT1 activity with many potential applications in medical biology.
Lin
Han-Jia
HJ
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan. hanjia@ntou.edu.tw
Lien
Yi-Chin
YC
Hsu
Chun-Hua
CH
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D003124:Colorimetry / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007700:Kinetics; D011073:Polyamines / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D011073:Polyamines; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; D000099228:Diamine N-Acetyltransferase