Characteristics of a presynaptic plasma membrane Ca2(+)-ATPase activity from electric organ.
Ca2(+)-ATPase activity was measured in electric organ synaptosomal homogenates and their derived presynaptic plasma membranes using a low ionic strength medium, low in Ca2+ and Mg2+, and devoid of K+. The enzyme activity showed a high apparent affinity for Ca2+ (KCa:0.5 microM) and was: (1) 5-fold stimulated by 120 nM calmodulin, (2) highly sensitive to LaCl3 inhibition, and (3) not affected by 20 mM NaN3 or 0.1 mM ouabain. The addition of Mg2+ promoted the disappearance of Ca2(+)-ATPase activity. Incubation of synaptosomal homogenates in the above-mentioned assay medium with [gamma -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 NH2OH, indicating that the isotope incorporation required the presence of Ca2+ and the formation of an acyl-phosphate derivative. The results indicate that the Ca2(+)-ATPase activity from synaptosomal homogenates had characteristics corresponding to those of the enzyme that catalyzes an outward transport of Ca2+ in nerve terminals. Preincubation of synaptosomes in Ca2+ plus K+, a depolarizing procedure, induced a large and rapid decrease in the Ca2(+)-ATPase activity, possibly mediated via Ca2+ entry through voltage-gated Ca2+ channels. Furthermore, the muscarinic cholinergic agonist oxotremorine (at 15 microM concentration) did not significantly affect either the enzyme activity or the intensity of the Ca2(+)-dependent 32P incorporation into the 140 kDa band, suggesting that the enzyme is not coupled to muscarinic binding sites.
Donnet
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Instituto de Química y Fisicoquímica Biológicas (IQUIFIB, UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Medrano
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Otero
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Ochoa
E L
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Rossi
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JP
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004555:Electric Fish; D004557:Electric Organ / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D010095:Oxotremorine / Q000494:pharmacology*; D010761:Phosphorus Radioisotopes; D013570:Synaptic Membranes / Q000187:drug effects / Q000201:enzymology*; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology
D015220:Calcium Channels; D010761:Phosphorus Radioisotopes; D004533:Egtazic Acid; D010095:Oxotremorine; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium