Conditions limiting the use of ionophore A23187 as a probe of divalent cation involvement in biological reactions. Evidence from the slow fluorescence quenching of type A spinach chloroplasts.

The conditions under which ionophore A23187 can be used as a probe of Mg2+ involvement in the reactions of intact (Type A) spinach chloroplasts have been investigated by monitoring ionophore-induced reversal of slow fluorescence quenching. The following observations were made: (1) A23187-dependent reversal of quenching is a strong function of pH. This is consistent with competition between protons and divalent cations for the carboxylic acid moiety of the ionophore. (2) In the presence of exogenous Mg2+, quenching reversal by A23187 is significantly slowed. It is suggested that formation of the dimeric A23187 . Mg2+ complex delays action of the ionophore at the thylakoid membrane by slowing equilibration of the ionophore among chloroplast membrane phases. (3) In the absence of Mg2+, significant interaction of A23187 with certain monovalent cations--Li+ and Na+, but not K+--is observed. Evaluations of the interaction of ionophore A23187 with specific biological systems and inferences of divalent cation involvement, or lack thereof, must take these limitations into account.