Characterization of the ATP-dependent calcium efflux in dialyzed squid giant axons.

Abstract

The magnitude of the activating effect of ATP on the Ca efflux was explored at different [Ca2+]i [inside concentration] in squid, Doryteuthis plei, axons previously exposed to cyanide seawater and internally dialyzed with a medium free of ATP and containing p-trifluoro methoxy carbonyl cyanide phenyl hydrazine. At the lowest [Ca2+]i used (0.06 .mu.M) more than 95% of the Ca efflux depends on ATP. At high [Ca2+]i (100 .mu.M, 50-60% of the Ca efflux still depends on ATP. The apparent affinity constant for ATP was not significantly affected in the range of [Ca2+]i from 0.06-1 .mu.M. Axons dialyzed to reduce their internal Mg failed to show the usual activation of the Ca efflux when the Tris or the sodium salt of ATP was used. Only in the presence of internal Mg is ATP able to stimulate Ca efflux. Nine naturally occurring high-energy phosphate compounds were ineffective in supporting Ca efflux. These compounds were UTP, GTP, CTP, UDP, CDP, ADP, AMP, cyclic AMP and acetyl phosphate. The compounds 2''deoxy-ATP and the hydrolyzable analog, .alpha.,.beta.-methylene ATP were able to activate the Ca efflux. The nonhydrolyzable analog .beta.,.gamma.-methylene ATP competed with ATP for the activating site, but was unable to activate the Ca efflux. The results are discussed in terms of the specificity of the nucleotide site responsible for the ATP-dependent Ca efflux.