1. Introduction Several laboratories have observed the activation by anions of Fr-ATPase from mitochondria [l-7] or chloroplasts [8 3. The mechanism of this activation is still a matter of discussion. On the basis of kinetic studies with mitochondrial ATPase of yeast, a strong influence of anions affecting the regulatory site for MgATP was reported and anions defined as allosteric ligands controlling the affinity of ATP to its hydrolyz- ing site [6]. In [7] the kinetics of the mitochondrial Fr-ATPase activity were studied at various tempera- tures with different ATP analogues. Anions were sug- gested not to affect the binding of nucleotides at regulatory sites but directly interfere with catalytic site(s) by promoting the release of the reaction prod- ucts [7]. The latter explanation was invoked in [5]. In [9], after preincubation of the mitochondrial F,-ATPase with ADP, ADP remained bound at regu- latory site(s) and induced a ‘hysteretic’ inhibition which progressively developed in the presence of MgATP. This process brought the enzyme into a stable conformation of low specific activity. This paper directly proves that anions do not modify the binding ofADP at regulatory site(s). More- over, it will be shown that, when the enzyme has reached its stable conformation of low specific activ- ity , the anions can no longer induce their characteristic activation. This suggests that, in this conformation, the anion binding site(s) is(are) no longer accessible to the external medium or that the activating effect of anions can no longer occur when the regulatory site(s) is(are) occupied by ADP. 2. Materials and methods Nucleotides, phosphoenolpyruvate, pyruvate kinase and lactate dehydrogenase were obtained from Boehringer Mannheim. Sodium bicarbonate, potas- sium chromate and 2,4dinitrophenol were procured from Prolabo. Maleic acid and malonic acid were obtained from Sigma. [‘H]ADP (22 Ci/mmol) was purchased from Amersham. Nucleotidedepleted Fr was prepared from pig heart mitochondria as in [lo]. It was stored frozen at -80°C in 100 mM Tris-HzS04-5 mM EDTA-50% glycerol (pH 8 .O) at 5 mg protein/ml. The ADP- induced hysteretic inhibition was measured as in [9,1 l] by preincubating the enzyme (1 mg protein/ ml) in 50 mM Tris-HzS04- 10% glycerol- 1.5 mM MgS04 (pH 8 .O) containing 200 E.~M ADP unless other- wise indicated. Magnesium at 1.5 mM was sufficient to entrap the ADP in its regulatory site(s) [93. After 20 min at 30°C a 0.5- 1 ~1 aliquot was removed and diluted in 0.62 ml of the ATPase assay mixture. The latter was made of 50 mM Tris-H.$04 buffer (pH 8.0) the activating anion as indicated below, 4 mM phosphoenolpyruvate, 0.3 mM NADH and 3.3 n&l ATP plus 3.3 mM MgSO& this Mg2+ level being neces- sary to form the ATP-Mg complex, the enzyme sub- strate, at saturating concentration. The pH was adjusted again to 8.0 with KOH and 50 pg pyruvate kinase and 10 Erg lactate dehydrogenase were added. The disappearance of NADH was recorded for several minutes at 340 nm except when chromate (3 15 nm) or 2,4dinitrophenolate (300 nm) was present. The ‘stable-inhibited rate’ will be referred to as the rate measured 1 min after the addition to the ATPase