An enzymatic assay for calmodulins based on plant NAD kinase activity.
NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5's ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5's for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.
D000818:Animals; D001487:Basidiomycota / Q000032:analysis; D001681:Biological Assay; D001921:Brain / Q000201:enzymology; D000252:Calcium-Transporting ATPases / Q000097:blood; D002147:Calmodulin / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D007887:Fabaceae / Q000032:analysis / Q000201:enzymology; D006801:Humans; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010946:Plants, Medicinal; D011863:Radioimmunoassay; D013045:Species Specificity; D013552:Swine
D002147:Calmodulin; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; D000252:Calcium-Transporting ATPases