Abstract
The interaction of Acanthamoeba actin and Acanthamoeba profilin was evaluated as a function of ionic conditions. In the presence of 2 mM MgCl2 or 1 mM MgCl2 and 50 mM KCl, profilin decreased the concentration of F-actin at steady state, and inhibited the rates of filament elongation and spontaneous nucleation and polymerization. All of the experimental data were quantitatively accounted for on the basis of a 1:1 complex between profilin and monomeric actin with a Kr between 4 and 9 microM, the same value obtained previously in the absence of MgCl2. Therefore, the Mg2+ concentration did not affect the KD of the profilin-actin complex in these experiments. On the other hand, profilin did greatly amplify the decrease in concentration of F-actin at steady state caused by lowering the Mg2+ concentration. This results from the effect of Mg2+ on the critical concentration of the actin monomer with which the profilin-actin complex is in equilibrium. When the Mg2+ concentration is lowered, the critical concentration of actin monomer increases so that more profilin-actin complex is formed. Thus, appreciably more F-actin depolymerizes than in the absence of profilin. In this way, profilin could function intracellularly to convert small changes in critical concentration into large changes in the concentration of F-actin.
Highlights
Of anyagentthatperturbsthecriticalconcentration.For profilin greatly amplified the decrease in the extent of actin example, in addition topossible local changes in ionic condipolymerization at steady statecaused by lowering the concen- tions, the critical concentration might be regulated intraceltration of Mg'+
In the presence of profilin, any increase in the critical concen
The F-actin concentration we have restricted our experiments to changeisn was reduced by 95%
Summary
Protein Preparations-Profilin was purified by the procedure of Reichstein and Korn (6). + 2 mM MgCI, or 1mM MgCI, 50 mMKC1 in buffer containing 5 mM imidazole, pH 7.3, 0.1 mM ATP, 0.1 mM dithiothreitol, and 0.01% NaN3.The actin was diluted into multiple 0.5-ml samples at a series of actin concentrations both in the presence and absence of profilin. The two methods gave identical results for the critical concentration of actin At these wavelengths, profilin does not change the fluorescence of pyrenyl G-actin. The critical concentration of actin in 2.0 mMMgC1, was determined independently by measuring the fluorescence intensity as a function of actin concentration (data not shown). Actin (37.2 p ~ w)as polymerized in 1.17 mMMgC12, sonicated 5 s, and diluted to 12.4 p M actin in solutions containing 27.5 p M profilin, 0.1 mM ATP, 5 mM imidazole,pH 7.3,O.l mM dithiothreitol, 0.01%NaN3 and 0.4-2.0 mMMgC1,. + incubation at 25 "C for 5 min, polymerization was induced by adding (final concentrations) 2 mM MgCl, or 1mMMgC1, 50 mMKC1
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