Ca2+ Uptake, Ca2+-ATPase Activity, Phosphoprotein Formation and Phosphate Turnover in a Microsomal Fraction of Smooth Muscle

Abstract

Vesicles capable of phosphate-stimulated Ca uptake were isolated from the microsomal fraction of the smooth muscle of the pig stomach according to a previously described procedure which consists in increasing the density of the vesicles by loading them with calcium phosphate and isolating them by centrifugation. These vesicles, which contain calcium phosphate deposits, are able to accumulate an additional amount of Ca. This Ca uptake is accompanied by Ca-stimulated ATPase activity and by the formation of an acid-stable phosphoprotein. The acid-denatured phosphoprotein is dephosphorylated by hydroxylamine, which indicates that an acylphosphate is formed. This phosphoprotein probably represents a phosphorylated transport intermediate similar to that seen with the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle. As with the Ca2+-ATPase of sarcoplasmic reticulum vesicles, this vesicular fraction catalyzes an exchange between inorganic phosphate and the .gamma.-phosphate of ATP (ATP-Pi exchange), which is dependent on the presence of intravesicular Ca and an exchange of phosphate between ATP and ADP (ATP-ADP exchange). The turnover rate of the Ca pump, calculated from the ratio of Ca-stimulated ATPase activity to the steady-state level of phosphoprotein, is evidently similar to that of the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle.