Abstract
The rate of exchange of G-actin with subunits of F-actin and the rate of hydrolysis of ATP in solutions of F-actin at steady state have been measured simultaneously. Subunit exchange kinetics were analyzed by both a treadmill model and an exchange-diffusion model. The best fit to a treadmill model of the data obtained in 0.5 mM MgCl2 and 0.2 mM ATP at 30 degrees C gave a treadmill efficiency (net monomers incorporated per ATP hydrolyzed) of 0.26, in good agreement with the previously reported s-value of 0.25 (Wegner, A. (1976) J. Mol. Biol. 108, 139-150) for similar ionic conditions. However, in this and other conditions with excess free divalent cations (Ca2+ or Mg2+), the observed exchange kinetics were in better agreement with an exchange-diffusion model than with a treadmilling model over the entire time course of the experiment. In the absence of excess divalent cations (50 mM KCl), exchange was too slow to be analyzed adequately by either model. Using the measured filament length distribution and the observed fit of the exchange-diffusion model to the data in 0.5 mM MgCl2, an on-rate constant of 2.8 x 10(6) M-1 S-1 and an off-rate constant of 5.8 s-1 were calculated. These values, while in good agreement with previously measured pre-steady state polymerization rate constants under different ionic conditions (Pollard, T. D., and Mooseker, M. S. (1981) J. Cell Biol. 88, 654-659), are about 30-fold higher than the rate constants predicted from the rate of ATP hydrolysis at steady state. To rationalize these discrepancies, a model is proposed in which a segment of F-actin subunits at one or both ends of the filament contains bound ATP at steady state.
Highlights
The rate of exchange of G-actin with subunitsof F- The actin filament has two structurally and kinetically actin and the rate of hydrolysis of ATP in solutions of distinct ends called barbed (B) and pointed (P) from the
F-actin at steadystate have been measured simultane-arrowhead pattern seen when actin is decorated with muscle ously.Subunitexchangekineticswereanalyzed by heavy meromyosin. The both a treadmillmodelandanexchange-diffusion only reactions that occur during elongationand at steady state model.The best fit to a treadmill model of the data are the addition of G-ATP actin monomers to F-ADP actin obtained in0.5 m~ MgClz and0.2 m~ ATP at 30 “Cgave filaments, with the concomitant hydrolysis of ATP, and the a treadmill efficiency
Actin Polymerization and Associated ATP HydrolysisG-actin, 5%labeled with N-pyrenyliodoacetamide, was equilibrated in buffer G containing 0.2 m~ [y3'P]ATP, polymerization was initiated at 30 "C bythe addition of 0.5 m~ MgC12, and thepolymerizationkinetics were monitored by both light scattering and fluorescence (Fig. 1).In other experiments, it was found that these two assays gave indistinguishableresults over a range of concentrations of pyrenyl actin (0.2-6%of the total actin) and avariety of ionic conditions (50-100 m~ KC1, 0.5-2.0 m MgC12, 1-2 m~ CaC12).In addition, Fig. 2 shows that the critical concentrations determined by fluorescence, under several ionic conditions,were identical with those found by light scattering
Summary
The rate of exchange of G-actin with subunitsof F- The actin filament has two structurally and kinetically actin and the rate of hydrolysis of ATP in solutions of distinct ends called barbed (B) and pointed (P) from the. The observedfit of the exchange-diffusion modetol the Wegner [7] was the first to point out that when ATP is data in 0.5 m~ MgC12, an on-rate constant of 2.8 X 10‘ hydrolyzed in a solution containing F-actin and G-actin at. M-’ s-l and an off-rate constant of 5.8 s” were caIcu- steady state, the on- and off-reactions at each filament end lated. These values, while in good agreement with parree- not symmetrical, i.e.ATP is hydrolyzed inthe association viously measured pre-steady state polymerization ratreeaction but ATP is not synthesized in the dissociation reacconstants under different ionic conditions
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