Abstract
The ability of adenyl-5'-yl imidodiphosphate (AMP-PNP), ADP, and PPi to dissociate the actin.myosin subfragment 1 (S-1) complex was studied using an analytical ultracentrifuge with UV optics, which enabled the direct determination of the dissociated S-1. At mu = 0.22 M, pH 7.0, 22 degrees C, with saturating nucleotide present, ADP weakens the binding of S-1 to actin about 40-fold (K congruent to 10(5) M-1), while both AMP-PNP and PPi weakens the binding about 400-fold (K congruent to 10(4) M-1). This 10-fold stronger dissociating effect of AMP-PNP and PPi compared to ADP correlates with our data showing that the binding of AMP-PNP and PPi to S-1 is about 10-fold stronger than the binding of ADP. In contrast, the binding constants of ADP, AMP-PNP, and PPi to acto.S-1 are nearly identical (K congruent to 5 x 10(3) M-1). At 4 degrees C, AMP-PNP has only a 3-fold stronger dissociating effect than ADP and, similarly, our data suggest that the binding of AMP-PNP and ADP to S-1 is quite similar at 4 degrees C. AMP-PNP and PPi are, therefore, somewhat better dissociating agents than ADP, but the difference among these three ligands is quite small. These data also show that actin and nucleotide bind to separate but interacting sites on S-1 and that the S-1 molecules bind independently along the F-actin filament with a binding constant of about 1 x 10(7) M-1 at 22 degrees C and physiological ionic strength.
Highlights
PNP),ADP,andPPi to dissociatethe actin.myosin both in vivo and in vitro studies there is considerable disasubfragment 1(S-1) complex was studied using an an- greement as towhether AMP-PNP dissociates actoalytical ultracentrifuge withW optics, which enabled myosin, i.e,whether it is a good analog of ATP in this respect
ADP weakens the bindingof S-1 to actin about 40-fold ( K = lo6 M”), while both AMP-PNPandPPi weakens the binding about 400-fold (K= lo4 M-‘).This 10-fold stronger dissociating effecot f AMP-PNP and PPi compared to ADP correlates with our data showing that the binding of AMP-PNP and PtPoi S-1is about 10-fold strongerthanthebinding of ADP
54 7 separate but interacting sites on S-1, as originally proposed he causedby an increase inthe preferred angleof the attached for ATP and actin by Eisenberg and Moos [26]
Summary
The binding of actin andnucleotide to S-1was investigated using three different ligands, two nucleotides(AMP-PNPand ADP) and onenucleotide analog(PPI).As originally suggested by Eisenberg and Moos [26] for ATP and actin, it is likely that nucleotide andactin bind to S-1 inaccordancewith Scheme 1: M. In order to solve Scheme 1 for each of these ligands, it is necessary to obtain values forXI,the binding constant of nucleotide (ornucleotide analog) to S-1 in the absenceof actin. The slope of 1.2 for the competition experiment between PP, and[“HIAMP-PNP(the circles)shows that AMP-PNP and PP, have quite similar binding constants to S-1 with PPi binding about 1.2 timesstrongerthanAMP-. Having determined KI in Scheme 1 for AMP-PNP, ADP, and PP,, we measured K3 and K, for each ligand. UV optics withvarying concentrations of ligand: AMP-PNP (Oj, ADP (O),and PP, (A).The dissociation by ADP was measured using. The amount of dissociation of the acto .S-1 complex was determined at varying ligand concentrations with AMP-PNP, ADP, and PPi. The data were plotted according to the following equation [13]:
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