Ca2+ Regulation in Vascular Smooth Muscle: II. Ca2+ Binding of Aorta Leiotonin1

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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 Ca2+ 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.