Assay of Adenylyl
Cyclase Activity by Ion-exchange High-performance Liquid Chromatography
LIAO Fei*, ZHANG Xiao-Ping1, ZHOU
Qi-Xin2, ZENG Zhao-Chun, CHEN Wen-Yuan
( Biochemistry Department,1Central Laboratory；2Pharmacology Department,Chongqing
University of Medical Science, Chongqing 400016, China )
Abstract Adenylyl
cyclase(AC)activity was determined by directly quantifying the product cAMP
with ion-exchange HPLC. When AC reaction was terminated, the reaction mixture
was kept at 0 ℃ for 2 h to remove residual ATP utilizing the action of
ATP-hydrolyzing enzymes existed in the crude AC preparation of membrane pellet.
Papaverine was removed by the dichloromethane extraction, and cAMP was
quantified by chromatography on a WAX-1 HPLC column with baseline-resolution.
It was found that cAMP was linearly formed within 10 min of the reaction, and
the amount of cAMP formed was proportional to the amount of enzyme. Mouse brain
AC activity determined by this method was 42 nmol/min per gram of protein,
similar to that obtained by the classical [3H] labeled ATP method.
This ion-exchange HPLC was more rapid and convenient.
Key words Adenylyl cyclase(AC)；Ion-exchange high-perfomance
liquid chromatography；3′, 5′-cAMP；ATP
*Corresponding author：Tel,86-23-68803798；Fax,86-23-68812985；e-mail,[email protected]