Identification of the 29,000-dalton protein and its relevance to oligomycin-sensitive 32Pi-ATP exchange in bovine heart electron transport particles.
There have been several reports on the involvement of a 29,000-dalton protein in the regulation of ATP synthesis and 32Pi-ATP exchange (Zimmer, G., Mainka, L., and Heil, B. M. (1982) FEBS Lett. 150, 207-210). The present communication demonstrates that incubation of electron transport particles with 50 microM copper-o-phenanthroline results in reversible loss of 32Pi-ATP exchange but not of oligomycin-sensitive ATPase. Dependence of the inhibition on oxygen, its prevention by EDTA, ATP, or 2-mercaptoethanol, and subsequent restoration of the activity by 2-mercaptoethanol point to a thiol-disulfide interchange as the cause of inhibition. Analysis of copper-o-phenanthroline-treated samples by polyacrylamide gel electrophoresis conducted under nonreducing conditions shows four major changes. There is a decrease in the staining intensity of two bands with molecular weights of 34,000 and 29,000 with concomitant appearance of two new bands with molecular weights of 28,000 and 58,000-60,000. The 34,000-dalton band is tentatively identified as the phosphate transport protein. The 28,000-dalton component is formed by intramolecular and the 58,000-60,000-dalton component by intermolecular cross-linking of the 29,000-dalton protein. Pretreatment of electron transport particles with 2 mM N-ethylmaleimide does not affect 32Pi-ATP exchange or its inhibition by copper-o-phenanthroline but prevents cross-linking of the 34,000- and 29,000-dalton proteins. Evidence is presented to demonstrate that the purified H+-ATPase preparation has a single 29,000-dalton protein, identical to the adenine nucleotide translocase, and that it is not essential for 32Pi-ATP exchange or oligomycin-sensitive ATPase.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004579:Electron Transport; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D013367:Submitochondrial Particles / Q000378:metabolism*
D002352:Carrier Proteins; D008565:Membrane Proteins; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein; D005033:Ethylmaleimide