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J. Biol. Chem., Vol. 279, Issue 32, 33519-33527, August 6, 2004

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Effects of Profilin and Thymosin ₄ on the Critical Concentration of Actin Demonstrated in Vitro and in Cell Extracts with a Novel Direct Assay^*

Elena G. Yarmola and Michael R. Bubb

From the Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32610 and the Research Service, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida 32608

The free actin concentration at steady state, A_c, is a variable that determines how actin regulatory proteins influence the extent of actin polymerization. We describe a novel method employing fluorescence anisotropy to directly measure A_c in any sample after the addition of a trace amount of labeled thymosin ₄ or thymosin ₄ peptide. Using this assay, we confirm earlier theoretical work on the helical polymerization of actin and confirm the effects of actin filament-stabilizing drugs and capping proteins on A_c, thereby validating the assay. We also confirm a controversial prior observation that profilin lowers the critical concentration of Mg²⁺-actin. A general mechanism is proposed to explain this effect, and the first quantitative dose-response curve for the effect of profilin on A_c facilitates its evaluation. This mechanism also predicts the effect of profilin on critical concentration in the presence of the limited amount of capping protein, which is the condition often found in cells, and the effect of profilin on critical concentration in cell extracts is demonstrated for the first time. Additionally, nonlinear effects of thymosin ₄ on the steady state amount of F-actin are explained by the observed changes in A_c. This assay has potential in vivo applications that complement those demonstrated in vitro.

Received for publication, April 21, 2004 , and in revised form, May 28, 2004.

^* This work was supported by the Medical Research Service of the Department of Veterans Affairs, National Science Foundation Grant NSF-0316015 (to M. R. B.), and NIAMS, National Institutes of Health Grant 5K25AR048918 (to E. G. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Box 100221, Dept. of Medicine, University of Florida, Gainesville, FL 32610. Tel.: 352-392-4681; Fax: 352-374-6170; E-mail: bubbmr{at}medicine.ufl.edu.

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