Calmodulin regulation of the ATP-dependent calcium uptake by inverted vesicles prepared from rabbit synaptosomal plasma membranes

Calmodulin has been shown to activate the ATP-dependent Ca2+ uptake in inside-out vesicles which have been prepared from rabbit synaptosomal plasma membranes by the methodology of Gill et al. (Gill, D.L., Grollman, E.F. and Kohn, L.D. (1981) J. Biol. Chem. 256, 184-192). Following extensive washings of these membranes with EGTA/EDTA solutions, the Ca2+ uptake activity demonstrated an affinity for calmodulin of 30 nM and an affinity for Ca2+ of 2 microM. The activity was completely inhibited by the anticalmodulin compound R24571 (Ki congruent to 8 microM). The molecular weight of the ATPase molecule, revealed by a combination of the [125I]calmodulin overlay technique and [32P]phosphoenzyme electrophoresis, was 145 000. The overlay technique also revealed that the mechanism of activation is via a direct binding of calmodulin to the pump molecule.