Ca2+-dependent phosphorylation and Ca2+ uptake in membrane fractions of the mesenteric artery.
Ca2+-dependent phosphorylation of endogenous substrate proteins (mol. wt 30 800, 35 500, 38 600 and 53 200) is found in a membrane subcellular fraction from rabbit mesenteric arteries. Characteristics of 32P incorporation are suggestive of a phosphoester-type phosphorylation produced by a Ca2+-dependent protein kinase. Ca2+-dependent phosphorylation and Ca2+ uptake rate show comparable affinities for Ca2+ of 3.5 x 10(-7) M and 2.4 x 10(-7) M, respectively. The dependence of both phenomena on the MgATP concentration is also similar. Ca2+-dependent phosphorylation and Ca2+ uptake are inhibited by trifluoperazine with an IC50 of 3 x 10(-5) M and 5 x 10(-5) M, respectively. These results suggest that Ca2+ uptake might be modulated by a Ca2+-dependent protein kinase, which is possibly regulated by membrane-bound calmodulin. Endogenous Ca2+-dependent phosphorylation is stimulated up to 300% by the addition of boiled cytosol. This stimulation is due to phosphorylation of proteins of molecular weight 21 000 and 81 500 and is reversed by trifluoperazine. Since this stimulation cannot be mimicked by addition of calmodulin or phosphatidylserine, and since boiled cytosol does not stimulate Ca2+ uptake, it is proposed that an unknown cytosolic factor stimulates a second Ca2+ pump. Since cAMP-dependent protein kinase is shown to cause little phosphorylation and has no effect on Ca2+ uptake, it is concluded that a Ca2+-dependent rather than a cAMP-dependent protein kinase might modulate Ca2+ transport in vascular smooth muscle.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D008638:Mesenteric Arteries / Q000378:metabolism*; D010646:Phentolamine / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D013997:Time Factors
D002147:Calmodulin; D010718:Phosphatidylserines; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D002118:Calcium; D010646:Phentolamine