ESTIMATION OF CYTIDYLYL CYCLASE ACTIVITY AND MONITORING OF SIDE-PRODUCT FORMATION BY FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY

The enzyme cytidylyl cyclase catalyses the conversion of cytidine 5'-t riphosphate into cytidine 3',5'-cyclic monophosphate, a third naturall y occurring cyclic nucleotide currently under investigation to assign a biochemical function, Quantitation of the activity of this enzyme ha s been carried out by the positive-ion fast-atom bombardment mass spec trometric analysis of the enzyme incubation mixture after the reaction has been terminated, The data obtained are in good agreement with tho se obtained from the conventional radiometric and radioimmunoassays of the same enzyme preparations. The advantage of the mass spectrometer- based assay is the facility for multiple component monitoring. Thus, t he production of the cytidine diphosphates and monophosphates, and the production of four cytidine 3',5'-cyclic monophosphate analogues as s ide-products, were simultaneously estimated, The identities of two of the side-products, 2'-O-glutamyl- and 2'-O-aspartyl-cytidine-3',5'-cyc lic monophosphate, and of the cytidine 3',5'-cyclic monophosphate prod uct, were confirmed by mass-analysed ion kinetic energy spectra from t he collision-induced dissociation of the protonated molecules. (C) 199 7 by John Wiley & Sons, Ltd.