The Ca2+‐pumping ATPase and the major substrates of the cGMP‐dependent protein kinase in smooth muscle sarcolemma are distinct entities

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Abstract
It has been proposed that the plasma membrane Ca2+ pump of smooth muscle tissues may be regulated by cGMP‐dependent phosphorylation [Popescu, L. M., Panoiu, C., Hinescu, M. & Nutu, O. (1985) Eur. J. Pharmacol. 107, 393–394; Furukawa, K. & Nakamura, H. (1987) J. Biochem. (Tokyo) 101, 287–290]. This hypothesis has been tested on a smooth muscle sarcolemma preparation from pig thoracic aorta. The actomyosin‐extracted membranes showed ATP‐dependent Ca2+ uptake as well as cGMP‐dependent protein kinase (G‐kinase) activity. The molecular masses of the major protein substrates of the G‐kinase (G1) and that of the Ca2+ pump were compared. Electrophoretic analysis of the phosphorylated intermediate of the sarcolemmal Ca2+‐ATPase and the G1 phosphoprotein showed that these two proteins are not identical. The results were confirmed by using a 125I‐calmodulin overlay technique and an antibody against human erythrocyte Ca2+‐ATPase. Ca2+‐uptake experiments with prephosphorylated membrane vesicles were carried out to elucidate possible effects of cGMP‐dependent phosphorylation of membrane proteins on the activity of the Ca2+ pump. The cGMP‐dependent phosphorylation was found to be extremely sensitive to temperature leading to very low steady‐state phosphorylation levels at 37°C. The difficulty was overcome by ATP[γS], which produced full and stable thiophosphorylation of G1 during the Ca2+‐uptake experiments at 37°C. However, the cGMP‐dependent thiophosphorylation failed to influence the Ca2+‐uptake properties of sarcolemmal vesicles. The results show that the Ca2+ pump of smooth muscle plasma membrane is not a direct target of the cGMP‐dependent protein kinase and is not regulated by the cGMP‐dependent phosphorylation of membrane proteins.