Determination of enzyme kinetic parameters of cyclic CMP-specific phosphodiesterase by quantitative fast atom bombardment tandem mass spectrometry

✍ Scribed by Russell P. Newton; Jalal A. Khan; Dipankar Ghosh; James I. Langridge; A. Gareth Brenton; Frank M. Harris; Terence J. Walton

• Publisher: John Wiley and Sons
• Year: 1991
• Tongue: English
• Weight: 637 KB
• Volume: 26
• Category: Article
• ISSN: 1076-5174
• DOI: 10.1002/oms.1210260514

✦ Synopsis

The determination of cytidine 3',5'-cyclic monophosphate-specific phosphodiesterase activity by means of fast-atom bombardment (FAB) mass spectrometry with mass-analysed ion kinetic energy (MIKE) spectrum scanning is described. Initial efforts to determine the activity of the enzyme by this method were unsuccessful owing to the obfuscation of sample-related peaks by peaks emanating from the incubation buffer and cation adducts; dilution of buffer and a desalting procedure overcame these difficulties. In the resulting positive-ion FAB mass spectra, characteristic peaks of the enzyme substrate and product could be readily identified and the protonated molecular ions selected for MIKE scanning. By spiking enzyme incubates with known amounts of substrate and product, and measuring peak heights in the MIKE spectra of both spiked and unspiked samples, the substratelproduct ratio at the end of a series of phosphodiesterase incubations was determined. From the data obtained, the K,,, and V,,, of the phosphodiesterase were calculated as 6.08 mM and 11 pmol min-' mg-', respectively, showing good agreement with the analogous values of 8.06 mM and 5.8 pmol-' min-' mg-' obtained by radioactive assay.