Autoradiography enables visualization of a compound's distribution and can guide bioanalytical assay development by allowing convenient evaluation of factors, such as choice of paper, spotting volume, punch size, punch location, temperature and humidity.

Phosphorylation of the P-light chain of myosin might be involved in the regulation of cardiac contractility. Thus an enhanced phosphorylation level of the P-light chain catalyzed by Caz+-calmodulin-dependent myosin light chain kinase (MLCK) increased significantly the Ca*+ sensitivity of chemically skinned ventricular frbre bundles of the pig. This effect was reversible. Whereas force development at submaximal Ca*+ concentration (pCa 5.5) increased by N 50% in the presence of MLCK, maximum tension achieved at maximum Ca*+-concentration (pCa 4.3) was not affected.

Calmodulin contains several binding sites for hydrophobic compounds. The apparent specificity of various 'calmodulin antagonists' for these sites was investigated. The Ki values for the inhibition of calmodulin-activated cyclic-nucleotide phosphodiesterase and myosin light-chain kinase was determined. In addition, the Kd values of the same compounds for binding to calmodulin were measured. The compounds could be separated into four groups. Group I and I1 compounds inhibited competitively the activation of the phosphodiesterase and myosin light-chain kinase by calmodulin. Group I compounds inhibited the activation of the phosphodiesterase and myosin light-chain kinase at identical concentrations. In contrast, group I1 compounds inhibited the activation of the phosphodiesterase at 5 -10-fold lower concentrations than that of myosin light-chain kinase. Group I11 compounds inhibited the activation of these enzymes by an uncompetitive mechanism. Group IV compounds inhibited the activation of the phosphodiesterase with Ki values above 10 pM and did not affect the activation of myosin light-chain kinase.Binding of I3H]bepridil to calmodulin under equilibrium conditions yielded one high-affinity site (apparent Kd 0.4 pM) and four low affinity sites (apparent Kd 44 pM). Group I compounds interfered with the binding of bepridil to the high and low-affinity sites in a competitive manner. Group I1 compounds interfered in a noncompetitive manner with the high-affinity site and apparently competed only with one of the low-affinity sites. These data show that 'calmodulin antagonists' can be differentiated into at least three distinct groups. Kinetic and binding data suggest that the three groups bind to at least three different sites on calmodulin. Selective occupation of these sites may inhibit specifically the activation of distinct enzymes.Calmodulin is a highly conserved intracellular calciumbinding protein, which activates more than 30 different proteins in a calcium-dependent manner 11, 21. At present it is not clear how calmodulin regulates so many protein functions with any degree of selectivity. Calmodulin has four calciumbinding sites and a number of hydrophobic sites, which are exposed on its surface upon calcium binding [3 -51. The exposure of these hydrophobic sites is thought to be essential for the binding of calmodulin to and activation of different proteins. A large number of compounds, known as calmodulin antagonists [6], bind in the vincinity of these hydrophobic sites. The binding of an antagonist to calmodulin interfers with the activation of many calmodulin-dependent enzymes suggesting that the binding sites for calmodulin antagonists are close to those sites which interact with the enzymes. The exact localization of the hydrophobic sites, calmodulin-antagonist-binding sites and sites for interaction with enzymes is presently unknown, although the approximate localization of these sites has been deduced from primary and tertiary structure data in connection with binding and NMR studies.

The calmodulin antagonists W-7, trifluoperazine and R24571 in vitro inhibited calmodulin-dependent and independent myosin light chain kinase activity with IC50 values of about 300 pM, 140 pM and 18 pM in the presence of 8 mg/ml myosin light chains. These ICjo values decreased to 15 pM, 6 pM and 2.5 pM when the concentration of myosin light chains was decreased to 0.4 mginil in the presence of saturating concentrations of calmodulin. Endogeneous tyrosine fluorescence of myosin light chain measured at 334 nm was quenched concentration dependently by trifluoperazine and R24573. In addition, fluorescence of W-7 measured at 370 nm was quenched concentration dependently by myosin light chains. The quenching of fluorescence which was independent of calcium, suggested that all three compounds bound to myosin light chain. The values for trifluoperazine obtained from fluorescence quench curves at different concentrations of myosin light chain were almost identical with those obtained under similar conditions from inhibition curves of myosin light chain kinase. These results indicate that 'calmodulin antagonists' inhibit the activity of myosin light chain kinase independent of calmodulin by binding to myosin light chain. The implication of this finding for the interpretation of results obtained in uivo by the use of 'calmodulin antagonists' is discussed.

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