used to determine the binding constant of cation-free G-actin for nucleotide. The binding constant of cation-free actin for ATP and ADP was near 3 X 105 m-1, a value approximately Vioooth that of the binding constant for ATP in the presence of bound cation. Since, when cation is present, binding of
Ultraviolet absorbance changes occur when actin-bound nucleotide is changed from ADP to ATP or ITP. These absorbance changes may reflect a conformational change in actin when the nature of the bound nucleotide A XAdthough the validity of the sliding filament hypothesis as a model accounting for the events in muscle contraction appears to be well established (Huxley, 1960) the mechanism by which the energy of ATP is utilized to produce filament displacement is not understood. While it is generally believed that some shortening or change in shape of the myosin cross bridge consequent to ATP hydrolysis accounts for the movement of myosin past the actin filaments, the possibility remains that this movement is due to limited shortening of the actin filaments (Huxley, 1969). It has been suggested that the basis of flagellar contraction depends on an ATPinduced conformational shortening of flagellin, the flagellar analog of muscle actin (Silvester and Hoi will, 1965). Consequently, evidence that the actin molecule changes in conformation and, hence, possibly shortens when ATP is substituted for bound ADP would be of interest. That such a conformational change of actin leading to muscle contraction occurs has been suggested (Laki, 1969), but direct supporting evidence has been lacking. Higashi and Oosawa (1965) concluded from their spectral studies that there is no appreciable difference in the spectra of G-actin containing bound ATP, ADP, or ITP. No evidence of conformational differences between ATP and ADP-G-actin was detected in optical rotation studies (Nagy, 1966), although the ultraviolet absorbance changes which resulted from addition of EDTA to G-actin solutions differed depending on whether the actin
The binding of adenosine diphosphate (ADP) by G-actin was studied under various conditions. Binding constants were determined by measuring either the rates of denaturation of G-actin solutions in the presence of varying nucleotide concentrations, or the concentration of free nucleotide in G-actin solutions at equilibrium. The binding constant of G-actin for ADP at 0" was 2-5 X lo6 M -~; a t 25" it was 4 X l o 4 M -~, whereas at 0" ATP bound to G-actin with an affinity constant of 2.4 X IO7 M -~, Changing the I n view of the possible role of actin-bound nucleotide in energy-transfer processes leading to muscle contraction, studies of the properties of actin-bound nucleotide are important. Several recent publications have presented conflicting evidence on the extent to which ADP-G-actin dissociates in solution to form ADP and native G-actin. Higashi and Oosawa (1965) derived a binding constant of G-actin for ADP of 1.3 X lo4 M -~ from studies of changes in absorbance at 230 m p after addition of ADP to ADP-G-actin solutions. Thus, according to these data, at 1.5 mg/ml of ADP-G-actin in the absence of added ADP only 20% of the nucleotide was actually bound to the protein. Seidel et al. (1967) determined the amount of native actin containing no bound nucleotide and estimated that the binding constant of G-actin for ADP a t 4" was 4 X IO6 M -~. I n contrast, Hayashi and Rosenbluth (1964) inferred that less than 5 % of the total nucleotide present was free after studies of removal of free nucleotide by Dowex 1 from 1 mgjml of ADP-G-actin solutions at 0". West et al. (1967a) studied the kinetics of ADP hydrolysis by apyrase and found that only 7 of the nucleotide was dissociated in 1.5 mg/ml of ADP-G-actin solutions at 0". If the free nucleotide in these studies was equivalent to that found at equilibrium these results imply a binding constant greater than 7 X 106 M -~, a value 500-fold greater than that calculated by Higashi and Oosawa (1965) and 20-fold greater than that determined by Seidel et al. BBCB and a Veterans Administration Hospital Clinical Investigatorship. actin-bound cation from magnesium to calcium did not appear to alter ADP binding. Free magnesium at a concentration of 0.2 mM reduced the rate of G-actin denaturation by onethird but there was no change in the affinity of G-actin for ADP.It was concluded that the mechanism by which free magnesium stabilizes G-actin depends on a prolonged lifetime of nucleotide-free G-actin molecules without change in the properties of G-actin containing bound nucleotide. excess of magnesium at 0" retarded thermal denaturation of ADP-G-actin. It seemed possible that such a stabilizing effect of free MgCI2 could have been mediated by an increased affinity of G-actin for ADP.Our results show tighter ADP binding to G-actin at 0" than found in previous studies. A marked temperature dependence for ADP binding to G-actin was found. Nucleotide binding was not altered if calcium was substituted for actin-bound magnesium, nor did addition of free magnesium alter nucleotide b...
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