Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide.
The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D066298:In Vitro Techniques; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D009711:Nucleotides / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000502:physiology; D013816:Thermodynamics; D013997:Time Factors
D000199:Actins; D015879:Myosin Subfragments; D009711:Nucleotides; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D009218:Myosins