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Estimation of Cytidylyl Cyclase Activity and Monitoring of Side‐product Formation by Fast‐atom Bombardment Mass Spectrometry
Author(s) -
Newton Russell P.,
Groot Nancy,
van Geyschem Jan,
Diffley Penny E.,
Walton Terence J.,
Bayliss Mark A.,
Harris Frank M.,
Games David E.,
Brenton A. Gareth
Publication year - 1997
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(19970131)11:2<189::aid-rcm741>3.0.co;2-h
Subject(s) - chemistry , cytidine , mass spectrometry , fast atom bombardment , enzyme , protonation , nucleotide , cyclase , stereochemistry , chromatography , biochemistry , ion , organic chemistry , gene
The enzyme cytidylyl cyclase catalyses the conversion of cytidine 5′‐triphosphate into cytidine 3′,5′‐cyclic monophosphate, a third naturally occurring cyclic nucleotide currently under investigation to assign a biochemical function. Quantitation of the activity of this enzyme has been carried out by the positive‐ion fast‐atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data obtained are in good agreement with those 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, the production of the cytidine diphosphates and monophosphates, and the production of four cytidine 3′,5′‐cyclic monophosphate analogues as side‐products, were simultaneously estimated. The identities of two of the side‐products, 2′‐ O ‐glutamyl‐ and 2′ O ‐aspartyl‐cytidine‐3′,5′‐cyclic monophosphate, and of the cytidine 3′,5′‐cyclic monophosphate product, were confirmed by mass‐analysed ion kinetic energy spectra from the collision‐induced dissociation of the protonated molecules. © 1997 John Wiley & Sons, Ltd.