Open AccessThe Ca 2+ ‐pumping ATPase and the major substrates of the cGMP‐dependent protein kinase in smooth muscle sarcolemma are distinct entities
Author(s) -
BALTENSPERGER Kurt,
CARAFOLI Ernesto,
CHIESI Michele
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb13848.x
Subject(s) - sarcolemma , protein kinase a , atpase , cgmp dependent protein kinase , microbiology and biotechnology , biophysics , chemistry , myocyte , kinase , biochemistry , biology , enzyme , mitogen activated protein kinase kinase
It has been proposed that the plasma membrane Ca 2+ 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 Ca 2+ 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 Ca 2+ pump were compared. Electrophoretic analysis of the phosphorylated intermediate of the sarcolemmal Ca 2+ ‐ATPase and the G1 phosphoprotein showed that these two proteins are not identical. The results were confirmed by using a 125 I‐calmodulin overlay technique and an antibody against human erythrocyte Ca 2+ ‐ATPase. Ca 2+ ‐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 Ca 2+ 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 Ca 2+ ‐uptake experiments at 37°C. However, the cGMP‐dependent thiophosphorylation failed to influence the Ca 2+ ‐uptake properties of sarcolemmal vesicles. The results show that the Ca 2+ 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.