Calcium binding of arterial tropomyosin: involvement in the thin filament regulation of smooth muscle

Abstract

Bovine aortic tropomyosin has been isolated by DEAE-Sepharose chromatography following isoelectric precipitation and ammonium sulfate fractionation. A single-polypeptide [Mr 36000 on a sodium dodecyl sulfate (SDS)-polyacrylamide gel] was obtained under different electrophoretic conditions. The amino acid composition of bovine tropomyosin was very similar to that of rabbit skeletal muscle; the amino-terminal residue is blocked. The molecular weight of the native tropomyosin (76000), which is twice the calculated from the SDS-polyacrylamide gel, suggests that the molecules is a dimer. The diffusion coefficient of 3.4 .times. 10-7 cm2 s-1 and the frictional coefficient of 1.7 indicate that the molecule is asymmetric. Comparative high-pressure liquid chromatography peptide mapping of rabbit skeletal and bovine aortic tropomyosins shows primary structure variation. Bovine aortic tropomyosin binds calcium under physiological conditions of pH and ionic strength (22 mol of Ca2+/mol of tropomyosin with a Kd of 1.4 mM). Such a property is not shared by skeletal tropomyosin. In low Mg2+ concentration, both skeletal and aortic actin activations of the skeletal myosin ATPase activity are calcium independent. Addition of aortic tropomyosin to a hybrid actomyosin (aortic actin, skeletal myosin) yields an enhancement of the actin activation of the myosin ATPase activity, but the addition of skeletal tropomyosin yields a decrease of this activity. However, both the enhancement and decrease are calcium dependent. Addition of skeletal or aortic tropomyosin to an actomyosin system, where both actin and myosin come from skeletal muscle, yields only an enhancement of the actin activation of the myosin ATPase activity. In this case, the calcium dependence of this enhancement was observed only with aortic tropomyosin. The calcium modulation obtained in the presence of aortic actin and skeletal tropomyosin suggests that the calcium site is formed by the actin-tropomyosin complex. These findings indicate that both aortic actin and tropomyosin are implicated in thin filament linked Ca2+ regulation of the ATPase activity of myosin in smooth muscle.