Modification of the mitochondrial F1-ATPase epsilon subunit, enhancement of the ATPase activity of the IF1-F1 complex and IF1-binding dependence of the conformation of the epsilon subunit.
Treatment of bovine heart submitochondrial particles with a low concentration of 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent for the Trp residue of the epsilon subunit [Baracca, Barogi, Lenaz and Solaini (1993) Int. J. Biochem. 25, 1269-1275], enhances the ATP hydrolytic activity of the particles exclusively when the natural inhibitor protein IF1 is present. Similarly, isolated F1 [the catalytic sector of the mitochondrial H+-ATPase complex (ATP synthase)] treated with the reagent has the ATPase activity enhanced exclusively if IF1 is bound to it. These experiments suggest that the modification of the epsilon subunit decreases the inhibitory activity of IF1, eliciting the search for a relationship between the epsilon subunit and the inhibitory protein. Certainly, a reverse relationship exists because HNB binds covalently to the isolated F1 exclusively when the inhibitory protein is present. This finding is consistent with the existence of the epsilon subunit in different conformational states depending on whether IF1 is bound to F1 or not. Support for this assertion is obtained by measurements of the intrinsic phosphorescence decay rate of F1, a probe of the Trp epsilon subunit conformation in situ [Solaini, Baracca, Parenti-Castelli and Strambini (1993) Eur. J. Biochem. 214, 729-734]. A significant difference in phosphorescence decay rate is detected when IF1 is added to preparations of F1 previously devoid of the inhibitory protein. These studies indicate that IF1 and the epsilon subunit of the mitochondrial F1-ATPase complex are related, suggesting a possible role of the epsilon subunit in the mechanism of regulation of the mitochondrial ATP synthase.
Solaini
G
G
Scuola Superiore di Studi Universitari e di Perfezionamento S. Anna, Via G. Carducci, 40, 56127 Pisa, Italy.
Baracca
A
A
Gabellieri
E
E
Lenaz
G
G
D015077:2-Hydroxy-5-nitrobenzyl Bromide / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007700:Kinetics; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology*
D046911:Macromolecular Substances; D015077:2-Hydroxy-5-nitrobenzyl Bromide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases