Quantitation by fast-atom bombardment/mass-analysed ion kinetic energy spectrometry: kinetic analysis of cyclic nucleotide phosphodiesterase activity.
Quantitation of cyclic nucleotide phosphodiesterase activity by means of fast-atom bombardment (FAB) mass spectrometry with mass-analysed ion kinetic energy (MIKE) spectrum scanning is described. Characteristic peaks of the substrate, cyclic AMP, and product, AMP, were identified in positive-ion FAB mass spectra and MIKE scans of the protonated molecules. By spiking enzyme incubates with known quantities of cyclic AMP and AMP and measuring peak heights in the MIKE spectra of both spiked and unspiked samples, the concentrations of cyclic AMP and AMP in solution at the end of a series of enzyme incubations have been estimated. From the data obtained the Km and Vmax of the enzymes were calculated as 181 microM and 28.6 nmol/min respectively, showing excellent agreement with values of the Michaelis constant, Km = 205 microM and the maximum velocity Vmax = 33.2 nmol/min obtained by radioactive assay.
Newton
R P
RP
Biochemistry Research Group, School of Biological Sciences, University College of Swansea, UK.
Walton
T J
TJ
Brenton
A G
AG
Kingston
E E
EE
Harris
F M
FM
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D066298:In Vitro Techniques; D013058:Mass Spectrometry / Q000379:methods; D009206:Myocardium / Q000201:enzymology*; D051381:Rats; D016339:Spectrometry, Mass, Fast Atom Bombardment / Q000379:methods
D015105:3',5'-Cyclic-AMP Phosphodiesterases