ABSTRACT
According to functional studies, the higher IF(1) content reported in mitochondria of cancerous cells is supposed to induce a higher association with the F(1)F(0) complex than in normal cells and therefore a better inhibition of its ATPase activity. The first structural evidence supporting this prediction is here presented. Densitometric analyses of Western blotting experiments indicated a 2-fold increase in IF(1) content of AS-30D submitochondrial particles compared to normal rat liver controls. The ratio of IF(1)/F(1) alpha subunit increased similarly as judged by Westernblot analyses. This IF(1) overexpression correlated with a slower rate of IF(1) release (F(1)F(0)-ATPase activation) from the F(1)F(0) complex in AS-30D than in normal rat liver submitochondrial particles. The IF(1)-IF(1), gamma-IF(1), and alpha-IF(1) cross-linkages previously formed with dithiobis(succinimidylpropionate) in bovine F(1)F(0)I and IF(1) complexes were reproduced in the F(1)F(0)I-ATP synthase of hepatoma AS-30D cells. However, a much lower yield of IF(1) cross-linkages was found in normal rat liver particles which made them almost undetectable in SMP as well as in the immunoprecipitated F(1)F(0)I complex. Modeling in vivo IF(1) overexpression of cancerous cells by in vitro reconstitution of excess recombinant IF(1) with rat liver submitochondrial particles devoid of IF(1) reproduced the same IF(1) cross-linkages observed in AS-30D particles.
Subject(s)
Cross-Linking Reagents/metabolism , Enzyme Inhibitors/metabolism , Liver Neoplasms, Experimental/metabolism , Mitochondria/metabolism , Mitochondrial Proton-Translocating ATPases/metabolism , Animals , Blotting, Western , Densitometry , Electrophoresis, Polyacrylamide Gel , Oligonucleotides , Plasmids/genetics , RatsABSTRACT
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 delta pH 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.
Subject(s)
Paracoccus denitrificans/enzymology , Proton-Translocating ATPases/drug effects , Sulfites/pharmacology , Adenosine Triphosphate/biosynthesis , Adenosine Triphosphate/metabolism , Cell Membrane , Kinetics , Phosphates/metabolism , Phosphorus Radioisotopes , Proton-Translocating ATPases/metabolismABSTRACT
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 alpha-IF1, IF1-IF1, gamma-IF1, and epsilon-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 < or = 12 angstroms to gamma and epsilon 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 beta subunits, the inhibitory mechanism of IF1 may involve the interference with rotation of the central stalk.
Subject(s)
Enzyme Inhibitors/chemistry , Mitochondrial Proton-Translocating ATPases/antagonists & inhibitors , Mitochondrial Proton-Translocating ATPases/chemistry , Animals , Binding Sites , Cattle , Cross-Linking Reagents , Electrophoresis, Gel, Two-Dimensional , In Vitro Techniques , Mitochondria, Heart/enzymology , Protein Subunits , Submitochondrial Particles/enzymology , SuccinimidesABSTRACT
Este estudio muestra que el analgésico no esteroideo, Ketorolac, actúa como ionóforo para calcio, ya que es capaz de transportar este metal a través de fases hidrofóbicas. Esta propiedad no afecta la capacidad respiratoria de las mitocondrias, lo que indica que el efecto ionoforético no es debido a una acción desacoplante del Ketorolac. El efecto de este compuesto se analizó en experimentos de reperfusión de corazones isquémicos. Se observó que el Ketorolac a una dosis de 1 mg/Kg de peso, administrado por vía intravenosa 30 minutos antes de inducir el período de anoxia, es capaz de prevenir el efecto arritmogénico de la reperfusión. Al analizar los niveles plasmáticos de las enzimas creatina cinasa y lactato deshidrogenasa, se observó un nivel más bajo de estas enzimas en las ratas tratadas con Ketorolac, en comparación con las ratas no tratadas. Estos resultados indican que el Ketorolac previene del daño por reperfusión, posiblemente evitando la sobrecarga de calcio en las células cardiacas.
