Ca2+-dependent binding of [3H]calmodulin to the microsomal fraction of brain.

Abstract

The binding of calmodulin to a brain microsomal fraction rich in synaptic membranes and vesicles was studied using 3H-labeled calmodulin. The binding was Ca2+-dependent and highly specific to calmodulin since it was competitively displaced only by unlabeled calmodulin and not by 200-4,000-fold excess of other proteins that included troponin-C and S-100 protein. Within the physiological pH range, the specific binding, defined as the amount of bound [3H]calmodulin which is displacable by the addition of an excess of unlabeled calmodulin, agreed well with the Ca2+-dependent binding defined as the difference between the total binding in the presence of Ca2+ and the binding obtained with EGTA in place of Ca2+. Both binding activities appeared to be greatest at about pH 7.0. The binding, either specific or Ca2+-dependent, is a calmodulin concentration-dependent saturable process. The dose-dependent curve obtained for increasing concentrations of [3H]calmodulin agreed well with that obtained for mixtures of a fixed concentration of [3H]calmodulin and increasing concentrations of unlabeled calmodulin over the entire concentration range examined. The results serve as the basis for using [3H]calmodulin in binding studies. Scatchard plot analysis of the curve gave two different Kd values for calmodulin, 8.2 X 10(-8) and 5.3 X 10(-7) M. The corresponding maximum binding capacities were 1.0 X 10(14) and 1.6 X 10(14) calmodulin molecules per mg of microsomal protein, respectively. The binding ability of the microsomal fraction was completely abolished by prior treatment with proteolytic enzymes.