Characterization of an actin flow artifact that occurs during the presteady state dissociation of actosubfragment-1.
Models for the activation of the myosin subfragment-1 (S-1) ATPase activity by actin describe transitions that occur between kinetic intermediate states during steady state hydrolysis of ATP. These states consist of myosin-nucleotide complexes in rapid equilibrium binding with actin, but steady state measurements of actin binding during hydrolysis lead only to a weighted average of the individual binding constants involved. In the current work, in order to determine the individual binding constants involved in the activation process, we have investigated the presteady state kinetics of the dissociation of actomyosin by ATP. We find that an actin flow artifact appears to dominate the time course of dissociation, and characterization of this artifact reveals that its magnitude rises linearly (approximately) with the concentration of bound S-1. Attempts to subtract the actin flow artifact from the actoS-1 dissociation signal were not entirely successful due at least partially to the transient nature of the bound S-1 concentration in the first few milliseconds. However, further studies reveal that if the order of addition of actin, ATP, and S-1 are varied, the observed light scattering transients are essentially superimposable. One possible explanation of these data is that the binding constants for myosin-ATP and myosin-ADP-Pi to actin are equal. However, it is also possible that the flow artifact is so large that further analysis is precluded. In addition, we show that the actin flow artifact has little effect on the fluorescence measurements of the phosphate burst reported previously. Therefore, the prior interpretation of the fluorescence data remains unchanged.
Harwalkar
V A
VA
Department of Medicine, State University of New York, Stony Brook 11794.
Stein
L A
LA
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D005189:False Positive Reactions; D005453:Fluorescence; D007700:Kinetics; D008027:Light; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D012542:Scattering, Radiation; D013552:Swine; D014364:Tryptophan
D000199:Actins; D010446:Peptide Fragments; D010710:Phosphates; C033025:actin subfragments; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D009218:Myosins