The in vitro kinetics of mitochondrial and cytosolic creatine kinase determined by saturation transfer 31P-NMR.
Michaelis- and dissociation constants of sarcomeric mitochondrial creatine kinase (Mi(b)-CK) in solution were determined by enzyme assay and compared to those of cytosolic MM-CK under identical conditions at pH 7.4 and 25 degrees C. Saturation transfer 31P-NMR was used to determine the steady state fluxes mediated by Mi-CK and MM-CK in solution. The NMR detected fluxes of both Mi-CK and MM-CK exhibited, as expected, a linear dependence on Vmax (Vmax range 0-9 mM.s-1). Interestingly, the oligomeric state of Mi-CK, with the Mi-CK octamer/dimer ratio ranging from 2 to 9, did not have a significant effect on the flux/Vmax ratio. Furthermore, the flux/Vmax ratio of Mi-CK was twice as high as that of MM-CK under similar conditions (flux/Vmax for Mi-CK was 0.31 and for MM-CK was 0.15). This difference was primarily due to a 4-fold higher apparent affinity for MgADP of Mi-CK compared to MM-CK (K(m)(MgADP) = 22 +/- 9 microM and 80 +/- 17 microM, resp.). The NMR observed fluxes were in agreement with the fluxes as calculated from the rate equation, using the appropriate metabolite concentrations and the kinetic constants from the spectrophotometric assays. Thus we conclude, that Mi-CK and MM-CK, when in solution, catalyse an exchange-reaction, the flux of which is fully observable by saturation transfer 31P-NMR.
van Dorsten
F A
FA
Department of in vivo NMR, Bijvoet Center for Biomolecular Research, Utrecht University, Netherlands.
Furter
R
R
Bijkerk
M
M
Wallimann
T
T
Nicolay
K
K
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002645:Chickens; D003402:Creatine Kinase / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008928:Mitochondria / Q000201:enzymology*
D000244:Adenosine Diphosphate; D003402:Creatine Kinase