OXIDATIVE STRESS, THIOL REAGENTS, AND MEMBRANE-POTENTIAL MODULATE THEMITOCHONDRIAL PERMEABILITY TRANSITION BY AFFECTING NUCLEOTIDE-BINDINGTO THE ADENINE-NUCLEOTIDE TRANSLOCASE

Stimulation of the mitochondrial permeability transition (MPT) is de-e nergized mitochondria by phenylarsine oxide (PheArs) is greater than t hat by diamide and t-butylhydroperoxide (TBH), yet the increase in CyP binding to the inner mitochondrial membrane (Concern, C. P. and Hales trap, A. P. (1994) Biochem. J. 302, 321-324) is less. From a range of nucleotides tested only ADP, deoxy-ADP, and ATP inhibited the MPT. ADP inhibition involved two sites with K-i values of about 1 and 25 mu M which were independent of [Ca2+] and CyP binding. Carboxyatractyloside (CAT) abolished the high affinity site. Following pretreatment of mit ochondria with TBH or diamide, the K-i for ADP increased to 50-100 mu M, whereas pretreatment with PheArs or eosin maleimide increased the K -i to >500 mu M; only one inhibitory site was observed in both cases. Eosin maleimide is known to attack Cys(159) of the adenine nucleotide translocase (ANT) in a CAT-sensitive manner (Majima, E., Shinohara, Y. , Yamaguchi, N., Hong, Y. M., and Terada, H. (1994) Biochemistry 33, 9 530-9536), and here we demonstrate CAT-sensitive binding of the ANT to a PheArs affinity column. In adenine nucleotide-depleted mitochondria , no stimulation of the MPT by uncoupler was observed in the presence or absence of thiol reagents, suggesting that membrane potential may i nhibit the MPT by increasing adenine nucleotide binding through an eff ect on the ANT formation. We conclude that CsA and ADP inhibit pore op ening in distinct ways, CsA by displacing bound CyP and ADP by binding to the ANT. Both mechanisms act to decrease the Ca2+ sensitivity of t he pore. Thiol reagents and oxidative stress may modify two thiol grou ps on the ANT and thus stimulate pore opening by both means.