title: Mitochondrial cytochrome c oxidase is inhibited by ATP only at very high ATP/ADP ratios.
abstract: In the past, divergent results have been reported based on different methods and conditions used for enzymatic activity measurements of cytochrome c oxidase (CytOx). Here, we analyze in detail and show comparable and reproducible polarographic activity measurements of ATP-dependent inhibition of CytOx kinetics in intact and non-intact rat heart mitochondria and mitoplasts. We found that this mechanism is always present in isolated rat heart mitochondria and mitoplasts; however, it is measurable only at high ATP/ADP ratios using optimal protein concentrations. In the kinetics assay, measurement of this mechanism is independent of presence or absence of Tween-20 and the composition of measuring buffer. Furthermore, the effect of atractyloside on intact rat heart mitochondria confirms that (i) ATP inhibition occurs under uncoupled conditions [in the presence of carbonly cyanide m-chlorophenyl hydrazone (CCCP)] when the classical respiratory control is absent and (ii) high ATP/ADP ratios in the matrix as well as in the cytosolic space are required for full ATP inhibition of CytOx. Additionally, ATP inhibition measured in intact mitochondria extends in the presence of oligomycin, thus indicating further that the problem to measure the inhibitory effect of ATP on CytOx is apparently due to the lack of very high ATP/ADP ratios in isolated mitochondria.
authors.affiliation: .
authors.forename: Rabia
authors.identifier: 0000-0001-7139-9387
authors.initials: R
authors.lastname: Ramzan
authors.affiliation: .
authors.forename: Andreas K
authors.initials: AK
authors.lastname: Schaper
authors.affiliation: .
authors.forename: Petra
authors.initials: P
authors.lastname: Weber
authors.affiliation: .
authors.forename: Annika
authors.initials: A
authors.lastname: Rhiel
authors.affiliation: .
authors.forename: Muhammad Saad
authors.initials: MS
authors.lastname: Siddiq
authors.affiliation: .
authors.forename: Sebastian
authors.initials: S
authors.lastname: Vogt
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000378:metabolism; D051381:Rats
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV
keywords: ADP; ATP; Tween-20; cytochrome c oxidase; kinetics; mitochondria