Measurement of the reversibility of ATP binding to myosin in calcium-activated skinned fibers from rabbit skeletal muscle. Oxygen exchange between water and ATP released to the solution.
We have measured the rate constant for ATP release from myosin heads of Ca2+-activated, demembranated muscle fibers using the technique of phosphate-water oxygen exchange. Single rabbit psoas fibers were held in an activating solution in [18O]water ([MgATP] = 8 mM, ionic strength = 0.2 M, pH = 7.0, 24 degrees C). After about 20% hydrolysis of ATP, product Pi and remaining ATP were isolated, and the distribution of 18O in both molecules was analyzed using a mass spectrometer. The exchange in Pi was similar to that previously reported (Hibberd, M. G., Webb, M. R., Goldman, Y. E., and Trentham, D. R. (1985) J. Biol. Chem. 260, 3496-3501). The amount of 18O in ATP gave a rate constant of about 4 s-1 for ATP release, if it is assumed that each rate constant in the pathway of ATP hydrolysis has the same value for all myosin ATPase sites. However, the distribution of 18O in both released Pi and ATP is not well explained by a single pathway for ATP hydrolysis. We present a model that indicates how such distributions could arise from a range of values for the rate constants for Pi and ATP release from actomyosin, and this range is determined by differences in the amounts of strain in attached crossbridges. The kinetic information obtained from these isotope exchange experiments is compared to show that they give a compatible set of rate constants for actomyosin in fibers.
Bowater
R
R
Division of Physical Biochemistry, National Institute for Medical Research, London, United Kingdom.
Webb
M R
MR
Ferenczi
M A
MA
D000205:Actomyosin / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D009119:Muscle Contraction; D009218:Myosins / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D013816:Thermodynamics; D014867:Water / Q000378:metabolism
D010710:Phosphates; D014867:Water; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins; D010100:Oxygen; D002118:Calcium