Abstract
A23187 is a carboxylic acid antibiotic which simultaneously uncouples oxidative phosphorylation and inhibits ATPase of rat liver mitochondria incubated in a magnesium-free medium. The antibiotic acts as a freely mobile carrier to transport calcium and magnesium, but not potassium, from an aqueous medium buffered at pH 7.4 into a bulk organic phase. A23187 causes a progressive release of endogenous magnesium and inorganic phosphate from mitochondria incubated with substrate, but little change in calcium or potassium content. If EDTA is added, A23187 severely depletes intramitochondrial calcium and magnesium in Release of State 4 succinate oxidation by A23187 is inhibited by lanthanum chloride, ruthenium red, ((ethylene bis)oxyethylene nitrilo)tetraacetic acid (EGTA), EDTA, MgCl2, and ATP. Inhibition by EGTA (or EDTA) of A23187′s uncoupling is reversed readily by calcium but not magnesium. In the presence of A23187 plus EGTA, mitochondria retain only 2 to 3 nmoles of Mg2+ per mg of protein but remain coupled and phosphorylate ADP at 80 to 85% of control rates with β-hydroxybutyrate or succinate as substrate. Under these conditions, coupled phosphorylation supported by glutamate, and ATP hydrolysis are severely depressed, but increasing concentrations of magnesium in the incubating medium prevent these inhibitions. It is proposed that a cyclic, energy-dissipating flux of mitochondrial calcium accounts for uncoupling by A23187, whereas inhibition of ATPase and glutamate oxidation and increased potassium permeability of mitochondria result from antibiotic-mediated magnesium efflux.
Highlights
MethodsMitochondria were prepared as described by Johnson and Lardy [16] and suspended in 250 rnbr mannitol plus 70 mM sucrose
Release of State 4 succinate oxidation by A23187 is inhibited by lanthanum chloride, ruthenium red, ((ethylene bis)oxyethylene nitrilo)tetraacetic acid (EGTA), EDTA, MgCL, and ATP
Transport of Metal Cations by A,%3187 and Efects of Antibiotic on Mitochondrial Ions-A23187 transferred magnesium and calcium from an aqueous phase buffered at pH 7.4 or 9.8 into a bulk organic phase (Table I)
Summary
Mitochondria were prepared as described by Johnson and Lardy [16] and suspended in 250 rnbr mannitol plus 70 mM sucrose. Some sucrose solutions contained appreciable amounts of calcium and these solutions were passed over a Dowex 50 H+ cation exchange resin and brought to pH 7.4 with triethanolamine before use. All incubations of mitochondria were at 30”. Substrates were the free acids (except sodium nn-/?-hydroxybutyrate and potassium pyruvate) and nucleotides were the Tris salts; both were brought to pH 7.4 with triethanolamine base. A23187 was the generous gift of Dr Robert Hamill, Eli Lilly and Co., Indianapolis, Indiana, and the free acid was used in all experiments. Oxidative phosphorylation [17], ATPase activity [18], inorganic phosphate [19], and protein [20] were assayed by the methods referred to. The dissolved oxygen was taken to be 0.445 pg atom per ml of medium at 30”
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