Aggregation-linked kinetic heterogeneity in bovine cardiac myosin subfragment 1.

Studies of the cardiac myosin fragment 1 concentration dependence of the rate constants for adenosine 5'-triphosphate (ATP) binding and steady-state hydrolysis reveal that the observed rate constants are remarkably dependent on the protein concentration. The kinetics for ATP binding are biphasic, and both the fast- and slow-phase rate constants and the respective fractions of fast and slow material vary as a function of protein concentration. Two different types of kinetic experiments were conducted, one in which the ATP concentration was fixed but the subfragment 1 concentration was varied and another for which the ATP/subfragment 1 ratio was fixed but both concentrations were varied. The results of these two experiments on cardiac subfragment 1 are consistent with an ATP-dependent reversible aggregation. Light-scattering experiments confirm the presence of this aggregation and the ATP dependence. Similar studies on rabbit skeletal subfragment 1 give monophasic, protein-independent kinetics consistent with a monomeric species in solution. a simple monomer--dimer mechanism can account for the cardiac subfragment 1 kinetic results when changes in tryptophan fluorescence are used. However, the light-scattering results show that cardiac myosin subfragment 1 undergoes multiple reversible molecular weight changes in solution and may be tetrameric at high concentrations.