Properties of smooth muscle vinculin.
Vinculin, isolated from turkey gizzard smooth muscle, was purified by chromatography on CM-cellulose after isolation from a DEAE-cellulose column. Two-dimensional gel electrophoretic analysis of crude muscle fractions demonstrated that: 1) much of the approximately 130,000-dalton protein present in smooth muscle did not co-isoelectrically focus with the purified 130,000-dalton vinculin and 2) the purified vinculin consisted of three major, closely spaced isoelectric variants that were present only in small amounts in the original smooth muscle sample. Purified vinculin sedimented as a single peak with a sedimentation coefficient S0 20,w of 5.9. Circular dichroism spectra of purified 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 E1%(278) of 4.64. Digestion of vinculin, much of which is located at the cytoplasmic surface of the cell membrane, with Ca2+-activated neutral protease purified from skeletal muscle yielded major fragments with molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 98,000, 85,000, and 26,000. The factor(s) in DEAE-cellulose-purified vinculin responsible for decreasing the low shear viscosity of actin was removed and found in a crude fraction isolated by CM-cellulose chromatography. The purified vinculin had a small, but positive effect on the MgCl2-induced polymerization of actin as measured by low shear viscometry.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002154:Calpain; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005895:Gizzard, Avian; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification*; D009130:Muscle, Smooth; D011487:Protein Conformation; D014422:Turkeys; D016596:Vinculin; D014783:Viscosity
D000596:Amino Acids; D009124:Muscle Proteins; D016596:Vinculin; D010450:Endopeptidases; D002154:Calpain