Interactions between the oligomycin sensitivity conferring protein (OSCP) and beef heart mitochondrial F1-ATPase. 1. Study of the binding parameters with a chemically radiolabeled OSCP.
Upon treatment of beef heart mitochondrial oligomycin sensitivity conferring protein (OSCP) with [14C]-N-ethylmaleimide ( [14C]NEM) or dithiobis(nitro[14C] benzoate), 1 mol of either SH reagent was incorporated per mol of OSCP. Radiolabeling occurred at the level of the only cysteine residue, Cys-118, present in the OSCP sequence reported by Ovchinnikov et al. [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I. V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22]; it did not alter the biological activity of OSCP tested in a reconstituted F0-F1 system that catalyzed oligomycin-sensitive ATPase activity or ATP-Pi exchange. The parameters of [14C]NEM-OSCP binding to isolated beef heart mitochondrial F1 were assessed by equilibrium dialysis. Addition of trace amounts of Tween 20 prevented unspecific adsorption of OSCP. The binding curves showed that each F1 possesses a high-affinity OSCP binding site (Kd = 0.08 microM) and two low-affinity OSCP binding sites (Kd = 6-8 microM). Binding of OSCP to the high-affinity site on F1 is probably responsible for the ability of OSCP to confer oligomycin sensitivity to F1 in the ATPase complex.
Dupuis
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Issartel
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JP
Lunardi
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J
Satre
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M
Vignais
P V
PV
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases; D009840:Oligomycins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014357:Trypsin
D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D008565:Membrane Proteins; D009840:Oligomycins; D010446:Peptide Fragments; D004228:Dithionitrobenzoic Acid; D014357:Trypsin; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein; D005033:Ethylmaleimide