KINETIC-ANALYSIS AND MULTIPLE COMPONENT MONITORING OF EFFECTORS OF ADENYLYL-CYCLASE ACTIVITY BY QUANTITATIVE FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY

The enzyme adenylyl cyclase catalyses the conversion of adenosine 5'-t riphosphate (ATP) to adenosine-3',5'-cyclic monophosphate (cyclic AMP) , and is an important pharmaceutical target. Quantitation of this enzy me's activity has been carried out by positive-ion fast-atom bombardme nt mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The kinetic data obtained are in goo d agreement with those obtained by the conventional radiometric assay, and this mass spectrometry-based assay offers the facility to monitor the turnover of several components of the incubation simultaneously, This is utilized to study the relative efficiencies of two ATP-regener ating systems, three phosphodiesterase inhibitors and two modified sub strates, and to monitor the uptake and conversion of two competing sub strates, adenosine 5' triphosphate and 2'-deoxyadenosine-5-triphosphat e, to cyclic AMP and to cyclic deoxyAMP, respectively. (C) 1997 by Joh n Wiley & Sons, Ltd.