José J. García

Cross-Linking of the Endogenous Inhibitor Protein (IF1) with Rotor (γ,ε) and Stator (α) Subunits of the Mitochondrial ATP Synthase

The location of the endogenous inhibitor protein ( IF1) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F1I and intact F1F0I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of α–IF1, IF1–IF1, γ–IF1, and ε–IF1 cross-linkages in soluble F1I and in native F1F0I complexes. Cross-linking blocked the release of IF1 from its inhibitory site and therefore the activation of F1I and F1F0I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF1 is at a distance ≤12 Å,to γ and ε subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF1 inhibits conformational changes of the catalytic β subunits, the inhibitory mechanism of IF1 may involve the interference with rotation of the central stalk.

Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans

The F1F0 complex of Paracoccus denitrificans (PdF1F0) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF1F0-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur. J. Biochem.267, 993–1000) but its effect on the PdF1F0-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and 32Pi ⇔ ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal ΔpH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K0.5 for Mg2+-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC50 of Mg2+-ADP for inhibition of the PdF1F0-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF1F0 complex. These effects are not due to membrane or PdF1F0 uncoupling. Kinetic and structural modifications that could account for these results are discussed.

Inhibition by trifluoperazine of ATP synthesis and hydrolysis by particulate and soluble mitochondrial F1: competition with H2PO4-.

The effect of trifluoperazine (TFP) on the ATPase activity of soluble and paniculate F1ATPase and on ATP synthesis driven by succinate oxidation in submitochondrial particles from bovine heart was studied at pH 7.4 and 8.8. At the two pH. TFP inhibited ATP hydrolysis. Inorganic phosphate protected against the inhibiting action of TFP. The results on the effect of various concentrations of phosphate in the reversal of the action of TFP on hydrolysis at pH 7.4 and 8.8 showed that H2PO4− is the species that competes with TFP. The effect of TFP on oxidative phosphorylation was studied at concentrations that do not produce uncoupling or affect the aerobic oxidation of succinate (<15ΜM). TFP inhibited oxidative phosphorylation to a higher extent at pH 8.8 than at pH 7.4; this was through a diminution in theVmax, and an increase in theKm for phosphate. Data on phosphate uptake during oxidative phosphorylation at several pH showed that H2PO4− is the true substrate for oxidative phosphorylation. Thus, in both synthesis and hydrolysis of ATP, TFP and H2PO4− interact with a common site. However, there is a difference in the sensitivity to TFP of ATP synthesis and hydrolysis; this is more noticeable at pH 8.8, i.e. ATPase activity of soluble F1 remains at about 40% of the activity of the control in a concentration range of TFP of 40–100ΜM, whereas in oxidative phosphorylation 14ΜM TFP produces a 60% inhibition of phosphate uptake.

Hyposmolarity-sensitive release of taurine and free amino acids from human lymphocytes

This study has shown that human lymphocytes possess a hyposmolarity-sensitive release of FAA, particularly of the most abundant one, taurine. This release may contribute to the volume regulatory decrease in lymphocytes.

Unisite hydrolysis of [gamma 32 P]ATP by soluble mitochondrial F1-ATPase and its release by excess ADP and ATP. Effect of trifluoperazine.

Some of the characteristics of unisite hydrolysis of [γ32P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [γ32P]ATP bound at the catalytic sites of F1 under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [γ32P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [γ32P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [γ32P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30–40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [γ32P]ATP/32Pi bound at the catalytic site and a 50% diminution in the rate of 32Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F1 in the sense that in a fraction of F1 molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [γ32P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.

Unisite ATP hydrolysis by soluble Rhodospirillum rubrum F1–ATPase is accelerated by Ca2+

At saturating concentrations of ATP, soluble F1 from the Rhodospirillum rubrum (RF1) exhibits a higher rate of hydrolysis with Ca2+ than with Mg2+. The mechanisms involved in the expression of a higher catalytic activity with Ca2+ were explored by measuring the ATPase activity of RF1 at substiochiometric concentrations of ATP (unisite conditions). At a ratio of 0.25 [gamma-32P]ATP per RF1, the enzyme exhibited a 50 times higher hydrolytic rate with Ca2+ than with Mg2+. The rate of [gamma-32P]ATP binding to RF1 was in the same range with the two divalent metal ions. Centrifugation-filtration of RF1 exposed to substoichiometric [gamma-32P]ATP concentrations and Mg2+ through Sephadex columns yielded an enzyme that contained [gamma-32P]ATP and [32P]phosphate in a stoichiometry that was close to one. In the presence of Ca2+, the eluted enzyme did not contain [gamma-32P]ATP nor [32P]phosphate. This indicated that the rate of product release was faster with Ca2+ than with Mg2+. It was also observed that the ratio of multisite to unisite hydrolysis rates was of similar magnitude with both divalent cations. This suggests that they do not affect differently the cooperative mechanisms that may exist between catalytic sites. In consequence, the higher ATPase activity of RF1 in presence of Ca2+ strongly suggests that the retention time of products is decreased in the presence of this cation. Copyright 1998 Elsevier Science B.V.