Ca²⁺ Regulation in Vascular Smooth Muscle

II. Ca²⁺ Binding of Aorta Leiotonini

Abstract

The ability of leiotonin and actin of bovine aorta smooth muscle to form a tightly bound complex (Hirata et al. (1977) J. Biochem. 82, 1973-1976) was utilized to measure Ca²⁺ and Sr²⁺ binding to aortic leiotonin, and the physiological significance of this binding was investigated:
1) The leiotonin-actin complex showed strong affinity for Ca²⁺ whereas the actin filament without leiotonin showed weak affinity, binding Ca²⁺ only at relatively high Ca ²⁺ concentrations. When leiotonin C was removed from the leiotonin-actin complex, the Ca binding capacity of the complex fell to the level of the actin filament, and the original level was restored by the addition of chicken gizzard leiotonin C. Thus, leiotonin C was shown to be responsible for the Ca binding of the aortic thin filament at low Ca2+ concentrations.
2) The ability of the actin complex to bind Ca²⁺ mentioned above paralleled its ability to induce superprecipitation together with myosin.
3) The Ca²⁺ sensitivity of myosin light chain phosphorylation coincided with that of superprecipitation of myosin B, but the Sr²⁺ sensitivity of the phosphorylation was dissociated from that of superprecipitation, the phosphorylation being several times more sensitive to Sr²⁺ than superprecipitation. This supports the view that phosphorylation is not involved in the superprecipitation of aortic myosin B.
4) Modulator protein (calmodulin) could replace the function of leiotonin C in producing superprecipitation. However, the Sr2+ sensitivity of the superprecipitation of a reconstituted system containing modulator protein was in good accord with that of phosphorylation of this system, but was several times greater than that of the superprecipitation of a reconstituted system containing leiotonin C. It was, therefore, concluded that the Ca²⁺binding protein operating under physiological conditions is not modulator protein, but leiotonin.