The calcium-dependent association and functional coupling of calmodulin with myocardial phospholipase A2. Implications for cardiac cycle-dependent alterations in phospholipolysis.
Herein we demonstrate the calcium-dependent regulation of myocardial phospholipase A2 activity, which is mediated by a cytosolic protein constituent that can be chromatographically resolved from, and subsequently reconstituted with, purified myocardial phospholipase A2. Purification of this protein by sequential column chromatographies revealed an 18-kDa doublet, which was identified as calmodulin by Western blotting, calcium-dependent precipitation with W-7 agarose beads, and reconstitution of calcium-mediated phospholipase A2 inhibition with authentic homogeneous calmodulin. Calcium-induced calmodulin-mediated inhibition of myocardial phospholipase A2 was titrated by physiologic increments of calcium ion (Kd approximately 200 nM). Moreover, ternary complex affinity chromatography with calmodulin-Sepharose demonstrated that inhibition of myocardial phospholipase A2 activity by calmodulin resulted from the direct interaction of calmodulin with the myocardial phospholipase A2 catalytic complex. Exposure of cultured A-10 muscle cells to three structurally disparate calmodulin antagonists (W-7, trifluoperazine, and calmidazolium) resulted in the robust release of arachidonic acid, which was entirely ablated by pretreatment of cells with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2-H-tetrahydropyran-2-one. Collectively, this study identifies a novel mechanism whereby latent phospholipase A2 activity can be released from tonic inhibition by alterations in the interactions between the phospholipase A2 catalytic complex, calcium ion, and the intracellular calcium transducer, calmodulin.
Wolf
M J
MJ
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Gross
R W
RW
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000378:metabolism; D066298:In Vitro Techniques; D008066:Lipolysis; D009206:Myocardium / Q000201:enzymology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D011817:Rabbits; D013379:Substrate Specificity
D002147:Calmodulin; D004791:Enzyme Inhibitors; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium