Characteristics of a presynaptic plasma membrane Ca 2+-ATPase activity from electric organ

Abstract

Ca 2+-ATPase activity was measured in electric organ synaptosomal homogenates and their derived presynaptic plasma membranes using a low ionic strength medium, low in Ca 2+ and Mg 2+, and devoid of K +. The enzyme activity showed a high apparent affinity for Ca 2+ ( K Ca:0.5 μM) and was: (1) 5-fold stimulated by 120 nM calmodulin, (2) highly sensitive to LaCl 3 inhibition, and (3) not affected by 20 mM NaN 3 or 0.1 mM ouabain. The addition of Mg 2+ promoted the disappearance of Ca 2+-ATPase activity. Incubation of synaptosomal homogenates in the above-mentioned assay medium with [γ −32P]ATP resulted in the appearance of a 140 kDa band as revealed by SDS-gel electrophoresis. Labeling of this band with 32P was inhibited by 1 mM EGTA or 10 mM NH 2OH, indicating that the isotope incorporation required the presence of Ca 2+ and the formation of an acyl-phosphate derivative. The results indicate that the Ca 2+-ATPase activity from synaptosomal homogenates had characteristics corresponding to those of the enzyme that catalyzes an outward transport of Ca 2+ in nerve terminals. Preincubation of synaptosomes in Ca 2+ plus K +, a depolarizing procedure, induced a large and rapid decrease in the Ca 2+-ATPase activity, possibly mediated via Ca 2+ entry through voltage-gated Ca 2+ channels. Furthermore, the muscarinic cholinergic agonist oxotremorine (at 15 μM concentration) did not significantly affect either the enzyme activity or the intensity of the Ca 2+-dependent 32P incorporation into the 140 kDa band, suggesting that the enzyme is not coupled to muscarinic binding sites.