Evidence for a calmodulin-dependent phospholipase A2 that inhibits Na-K-ATPase.
We tested whether calnaktin, a proposed Ca/calmodulin (CaM)-dependent protein that inhibits the Na-K-ATPase, was a kinase, a phosphatase, a phospholipase A2 (PLA2), or a Ca-dependent protease. Human red blood cell membranes were extracted to remove associated calmodulin but to retain the proposed endogenous calnaktin. Exclusively cytoplasmic proteins and cofactors were presumably absent. In these membranes, free Ca inhibited the Na-K-ATPase with an inhibition constant (K[i]) of > or = 9 microM at a Na concentration of 18 mM. Addition of 100 nM CaM decreased the Ki to < 2 microM and increased the percent inhibition at 2 microM free Ca from 18 +/- 1 to 68 +/- 2%. The inhibitory effect of Ca/CaM was reversible, indicating that calnaktin is not a protease. Neither staurosporine (500 nM), 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpipera zin e (5 microM), nor genistein (100 microM) diminished Ca/CaM inhibition of the Na-K-ATPase. Thus there is no evidence that this protein is a kinase. Likewise, the phosphatase inhibitors microcystin (1 microM) and okadaic acid (10 microM) had no effect. PLA2 inhibitors arachidonyl trifluoromethyl ketone (AACOCF3), parabromophenacyl bromide (pBPB), and quinacrine all abolished Ca/CaM inhibition of the Na-K-ATPase. Ca/CaM also increased PLA2 activity, as reflected by an increase in the slope of fluorescence signal of 10-pyrene phosphatidylcholine, a substrate for PLA2. This Ca/CaM-induced change in slope was inhibited by both pBPB and AACOCF3. These data suggest that human red cell membranes contain a form of PLA2 that is activated by Ca/CaM and that this enzyme may mediate Ca/CaM inhibition of the Na-K-ATPase.
Okafor
M C
MC
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Schiebinger
R J
RJ
Yingst
D R
DR
D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology / Q000502:physiology*; D004910:Erythrocyte Membrane / Q000201:enzymology; D006801:Humans; D010741:Phospholipases A / Q000378:metabolism / Q000502:physiology*; D054467:Phospholipases A2; D010744:Phosphoric Monoester Hydrolases / Q000502:physiology; D010770:Phosphotransferases / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism
D002147:Calmodulin; D010770:Phosphotransferases; D010741:Phospholipases A; D054467:Phospholipases A2; D010744:Phosphoric Monoester Hydrolases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium