Assay for spermidine synthase activity by micellar electrokinetic chromatography with laser-induced fluorescence detection.
An assay for spermidine synthase (SPDS) activity in rat liver has been developed using micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection to enable the discovery of SPDS inhibitors. The assay was established by estimating the amount of spermidine (SPD) produced from the putrescine (PUT) present by SPDS. The SPD in an enzyme reaction mixture of homogenized rat liver could directly react with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent. The NBD derivatives of SPD and PUT could be separated and detected by MEKC-LIF detection within 15 min. The IC(50) value measured for SPDS inhibitor, 4-methylcyclohexylamine, in rat liver by this assay was consistent with published data. Our SPDS assay using MEKC-LIF is simple and allows easy determination of SPDS activity in homogenized samples without troublesome procedures such as preparation of antibody or fluorescence-labeled substrate. The assay should be effective for discovering the SPDS inhibitors using biological samples.
Sano
Masatake
M
Shionogi Research Laboratories, Shionogi & Co. Ltd., 12-4, Sagisu-5-Chome, Osaka 553-0002, Japan. masatake.sano@shionogi.co.jp
Nishino
Ikuko
I
D000818:Animals; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D003514:Cyclohexylamines / Q000494:pharmacology; D004121:Dimethyl Sulfoxide; D005453:Fluorescence; D007834:Lasers; D008099:Liver / Q000201:enzymology; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012997:Solvents; D011436:Spermidine Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors
D003514:Cyclohexylamines; D012997:Solvents; C059116:4-methylcyclohexylamine; D011436:Spermidine Synthase; D004121:Dimethyl Sulfoxide