Leptinotarsin-D, a neurotoxic protein, evokes neurotransmitter release from, and calcium flux into, isolated electric organ nerve terminals.

Previous work has demonstrated that the neurotoxin leptinotarsin elicits release of neurotransmitter from mammalian nerve terminals, and it has been suggested that the toxin may act either as a direct agonist of voltage-sensitive calcium channels in these terminals (Crosland et al., 1984) or as a calcium ionophore (Madeddu et al., 1985a,b). Preliminary studies (Yeager et al., 1987) demonstrated that leptinotarsin also evokes transmitter release from isolated elasmobranch electric organ nerve terminals. We now report further investigations of the effects of leptinotarsin in this system. The action of the toxin is saturable, releasing about the same small fraction of total transmitter as that released by depolarization. An upper limit for the concentration for half maximal release is estimated to be 4 nM. Leptinotarsin-evoked transmitter release exhibits behavior very similar to depolarization-evoked release with respect to dependence on Ca2+, Ba2+, and Sr2+ and blockade by Co2+, Cd2+, and trifluoperazine. Leptinotarsin also promotes the uptake of calcium into synaptosomes to a degree similar to that caused by depolarization by K+. The binding of leptinotarsin to nerve terminals is probably Ca2+ dependent and receptor mediated. Taken together with the behavior of leptinotarsin-evoked release in other preparations, these results are consistent with the hypothesis that this toxin acts by opening a presynaptic calcium channel. However, the possibility that leptinotarsin is a calcium ionophore cannot be excluded.

Transmitter release from presynaptic terminals of electric organ: inhibition by the calcium channel antagonist omega Conus toxin.

Cholinergic synaptosomes from electroplax of the ray Ommata discopyge release both ATP and ACh when depolarized with high K+ concentration in the presence of Ca2+. Others have shown that the ATP and ACh are released in the molar ratio found in isolated synaptic vesicles. Thus, it is assumed that the release of ATP reflects exocytosis of synaptic vesicles, and that transmitter release can be indirectly monitored by assaying ATP release. We present further evidence for this assumption and examine the effects of presynaptic neurotoxins on this ATP release. As expected for transmitter release, we find that depolarization-evoked ATP release is supported by Sr2+ and Ba2+ and is inhibited by the Ca channel antagonists Co2+ and Mn2+. Likewise, the presynaptic toxins omega-CmTX and omega-CgTX, omega peptides from the venom of the marine snails Conus magus and Conus geographus, respectively, inhibit 80% of the depolarization-evoked ATP release. Half-maximal inhibition of ATP release occurs with approximately 0.5 microM of either toxin. The toxins' effects are reversible, and when toxin is washed away, the time dependence of recovery of release is approximately first order and half complete within 40 min with omega-CmTX and 15 min with omega-CgTX. The Ca2+ ionophore A23187 induces Ca2+-dependent ATP release from resting synaptosomes. As would be expected of a Ca channel antagonist, omega-CmTX does not affect this ionophore-induced release. Leptinotarsin-d (LPTd), a putative Ca channel agonist from the Colorado potato beetle, evokes Ca2+-dependent ATP release from resting synaptosomes. omega-CmTX does not block LPTd-evoked release of ATP, which suggests that omega-CmTX and LPTd act at different sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine inhibition of calmodulin-sensitive adenylate cyclase from bovine cerebral cortex.

Calmodulin (CaM)-sensitive adenylate cyclase has recently been purified extensively from bovine brain. In this study, the sensitivity of the CaM-sensitive adenylate cyclase to adenosine and adenosine analogs was examined. The highly purified enzyme preparation retained sensitivity to inhibition by adenosine and adenosine analogs with ribose ring modifications, but not to those with purine ring modifications. Adenosine inhibition of this enzyme was not dependent on GTP and was noncompetitive with respect to ATP. Enzyme that had been dissociated from functional guanine nucleotide binding protein interactions by gel filtration in the presence of the zwitterionic detergent 3-[3-(cholamidopropyl)-dimethylammonio]-propanesulfonate and Mn2+ retained sensitivity to adenosine inhibition. The Ki for adenosine inhibition of the CaM-sensitive adenylate cyclase was approximately 2.6 X 10(-4) M. 5'-Guanylylimidodiphosphate and CaM did not affect the Ki of 3'-deoxyadenosine for the enzyme, but the presence of Ca2+ in the millimolar range raised the Ki by a factor of 5. These results show that the CaM-sensitive form of adenylate cyclase from bovine brain is subject to adenosine inhibition, and strongly suggest that this inhibition is due to interaction of ligands with a purine-specific ("P") site located on the catalytic subunit of the enzyme.

Purification of the calmodulin-sensitive adenylate cyclase from bovine cerebral cortex.

A calmodulin-sensitive adenylate cyclase was purified 3000-fold from bovine cerebral cortex using DEAE-Sephacel, calmodulin-Sepharose, and two heptanediamine-Sepharose column steps. The purified enzyme activity was stimulated by calmodulin, forskolin, 5'-guanylyl imidodiphosphate, and NaF. The molecular weight of the protein component was estimated as 328 000 with a smaller form of Mr 153 000 obtained in the presence of Mn2+. The most highly purified preparations contained major polypeptides of 150 000, 47 000, and 35 000 daltons on sodium dodecyl sulfate (SDS) gels. Photoaffinity labeling of the preparation with azido[125I]iodocalmodulin gave one product of 170 000 daltons on SDS gels. It is proposed that the catalytic subunit of the calmodulin-sensitive enzyme is 150 000 +/- 10 000 daltons and that the enzyme exists as a complex of one catalytic subunit and the stimulatory guanyl nucleotide regulatory complex. These data are consistent with the previous report that the catalytic subunit of this enzyme has a molecular weight of 150 000 +/- 10 000 [Andreasen, T.J., Heideman, W., Rosenberg, G.B., & Storm, D.R. (1983) Biochemistry 22,2757].

Reconstitution of calmodulin-sensitive adenylate cyclase from bovine brain with phosphatidylcholine liposomes.

A partially purified calmodulin (CaM)-sensitive adenylate cyclase from bovine cerebral cortex was reconstituted with a series of phosphatidylcholine liposomes having variable fatty acid composition. The enzyme was successfully associated with dimyristoyl, dipalmitoyl, distearoyl, and dioleoylphosphatidylcholine liposomes. The specific activity of the enzyme in the various liposomes varied over a 4.6-fold range indicating some degree of specificity for fatty acid composition. The adenylate cyclase-liposome preparation retained sensitivity to both CaM and 5'-guanylylimidodiphosphate (GppNHp). Arrhenius plots of enzyme activity in the four different liposome preparations all exhibited a pronounced discontinuity at 30 degrees C +/- 2, even though the bulk-phase thermal transition points for the liposomes varied from -20 to 54 degrees C. Fluorescence anisotropy studies of reconstituted liposome systems illustrated that incorporation of protein did not alter the normal-phase transition point of these lipids. Since Arrhenius plots of the enzyme in Lubrol PX, prior to reconstitution with lipids, were strictly linear, it is concluded that the breaks at 30 degrees C may be the effect of a local enzyme-phospholipid environment. It appears that this adenylate cyclase is not particularly sensitive to phase transitions of the bulk lipid phase. The phospholipid reconstituted enzyme system appears suitable for examination of the influence of lipids on the CaM-sensitive adenylate cyclase.