31P-NMR analysis of synaptic vesicles. Status of ATP and internal pH

The status of ATP in cholinergic synaptic vesicles from electric organ of Torpedo marmorata has been studied by 31P-NMR. In isolated vesicles, ATP was found to be the only major phosphate-containing constituent. The chemical shifts and the linewidths of the P gamma and P beta resonances are different from those of uncompartmented ATP added to the suspension. The spectrum of intact electric tissue contains two sets of ATP resonances; one of them belongs to vesicular ATP as identified by its linewidths and chemical shifts. Both spectral characteristics of vesicular ATP, linewidths and chemical shifts, could be explained by an exchange mechanism, which also occurs in model solutions containing acetylcholine, ATP and Mg2+ at a pH around 5.5. We therefore conclude that the cholinergic synaptic vesicles store ATP together with acetylcholine and Mg2+ essentially in free solution at an acidic pH. Since the chemical shift of the P gamma resonance of ATP is largely determined by factors other than pH, the concept of its use as an indicator for the internal pH has to be modified in the case of these organelles.