Profilin promotes barbed-end actin filament assembly without lowering the critical concentration.
The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin.4.1. Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-beta4, both actin and Profilin. Actin (P.A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant ( approximately 10 x 10(6) M(-1) s(-1)) for actin monomer or Profilin. Actin. When measured in the absence of profilin, actin assembly curves over a 0.7-4 microM thymosin-beta4 concentration range fit a simple monomer sequestering model (1 microM K(D) for Thymosin-beta4. Actin). The corresponding constant for thymosin-beta4.pyrenyl-Actin, however, was significantly higher ( approximately 9-10 microM), suggesting that the fluorophore markedly weakens binding to thymosin-beta4. With solutions of actin (2 microM) and thymosin-beta4 (2 or 4 microM), the barbed-end assembly rate rose with increasing profilin concentration (0.7-2 microM). Actin assembly in presence of thymosin-beta4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M.-F. Carlier (1993) Cell 75, 1007-1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-beta4. Actin and ushers actin as a Profilin. Actin complex onto growing barbed filament ends.
Kang
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Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32610-0245, USA.
Purich
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Southwick
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D000199:Actins / Q000737:chemistry*; D000818:Animals; D003285:Contractile Proteins; D006801:Humans; D008027:Light; D008840:Microfilament Proteins / Q000494:pharmacology*; D011108:Polymers / Q000737:chemistry; D051304:Profilins; D011817:Rabbits; D012542:Scattering, Radiation; D013816:Thermodynamics; D013947:Thymosin / Q000494:pharmacology; D000099201:Thymosin beta(4)
D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D011108:Polymers; D051304:Profilins; D013947:Thymosin; D000099201:Thymosin beta(4); C495738:PFN1 protein, human