Purification and partial characterization of vertebrate smooth muscle vinculin

Abstract

<p>Vinculin, isolated from turkey gizzard smooth muscle, was purified by chromatography on CM-cellulose after isolation from a DEAE-cellulose column. Vinculin sedimented as a single peak with a sedimentation coefficient;(DIAGRAM, TABLE OR GRAPHIC OMITTED...PLEASE SEE DAI);Circular dichroism spectra of vinculin indicated a considerable degree of secondary structure, with an alpha-helical content of approximately 50% as measured at 208 nm. The ultraviolet absorption spectrum of vinculin gave a measured;(DIAGRAM, TABLE OR GRAPHIC OMITTED...PLEASE SEE DAI);Digestion of vinculin with Ca('+2)-activated neutral protease yielded major fragments with molecular weights determined by SDS-polyacrylamide gel electrophoresis of 98,000, 85,000, and 26,000. The factor(s) in DEAE-cellulose-purified vinculin responsible for the marked decrease in the low shear viscosity of actin solutions can be isolated and removed by CM-cellulose chromatography. Under standard conditions (2 mM MgCl(,2), pH 7.5, 25(DEGREES)C), addition of vinculin (30 (mu)g/ml) causes a 30% increase in the viscosity of F-actin. Structural studies show this increase is paralleled by an increase in cross-linking of actin filaments. When the temperature is decreased to 15(DEGREES)C, vinculin still causes a slight, but consistent increase in the viscosity of F-actin due to an increase in cross-linking of the actin filaments. When the temperature was increased to 37(DEGREES)C, however, addition of vinculin had no measurable effect on actin viscosity or appearance. When the pH was increased to 8.0, a 60% increase in the low shear viscosity of F-actin resulted from addition of vinculin (30 (mu)g/ml) and this effect was paralleled by additional cross-linking of actin filaments. Surprisingly, when the pH was lowered to 7.0, vinculin (70 (mu)g/ml) caused a 40% decrease in F-actin viscosity. This was reflected by the formation of "irregular-appearing" actin filaments. Elevation of the ionic strength by addition of NaCl resulted in an increase in the viscosity of the F-actin controls, but addition of vinculin (70 (mu)g/ml) decreased the viscosity of these controls by approximately 65%. Electron microscope observations indicated that vinculin had disrupted the branched, paracrystalline-like bundles present in the F-actin formed at the higher salt concentrations. When tropomyosin was premixed with actin in a 1 to 7 molar ratio and low shear viscosity was measured at near physiological-like conditions (37(DEGREES)C, 150 mM NaCl), addition of vinculin (50 (mu)g/ml) caused a 90% increase in apparent viscosity.</p>