The Ca2+-pumping ATPase and the major substrates of the cGMP-dependent protein kinase in smooth muscle sarcolemma are distinct entities.
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 degrees C. The difficulty was overcome by ATP[gamma S], which produced full and stable thiophosphorylation of G1 during the Ca2+-uptake experiments at 37 degrees 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.
Baltensperger
K
K
Laboratory of Biochemistry, Swiss Federal Institute of Technology, Zürich.
Carafoli
E
E
Chiesi
M
M
D000818:Animals; D001011:Aorta / Q000201:enzymology; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification*; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D012508:Sarcolemma / Q000201:enzymology*; D013552:Swine
D011494:Protein Kinases; D000252:Calcium-Transporting ATPases