On the mechanism of the reconstitution of F1-depleted ATPase complex with purified F1: Possible conformational effects |
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| Authors: | Shengguang Li Ying Zhang Zhihuan Lin |
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| Affiliation: | (1) Institute of Biophysics, Academia Sinica, Beijing, China |
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| Abstract: | The membrane sector (F0) of H+-ATPase was prepared by trypsin and urea treatment of F1-F0 and reconstituted with purified F1. The oligomycin sensitivity of the reconstituted F1-F0 complex obtained by treating F1 or F0 with Mg2+ before binding is much higher than that obtained without Mg2+ treatment. The greater change in the intrinsic fluorescence of the reconstituted F1-F0 complex obtained by Mg2+ treatment suggests that conformational changes may occur during the reconstitution. We deduce that Mg2+ binds to membrane lipids, thus decreasing membrane fluidity and changing the physical state of the lipids to provide a suitable microenvironment for conformational changes in F0. The data also suggest that the conformational change in the F0 portion of the F1-F0 complex can be transmitted to the F1 portion, the conformation of which is in turn altered, resulting in the formation of an F1-F0 complex with high oligomycin sensitivity. On the other hand, Mg2+ may act on F1 directly to induce a suitable conformational change which is then trnsmitted to F0, resulting in the formation of an H+-ATPase with greater sensitivity to oligomycin.Abbreviations STED 0.25 M sucrose, 10 mM Tris-SO4, 0.2 mM EDTA, and 1 mM dithiothreitol, pH 8.0 - NADH nicotinamide adenine dinucleotide, reduced form - olig. oligomycin - OSCP oligomycin sensitivity conferring protein - F6 coupling factor 6 - F1 coupling factor one (or F1-ATPase) - F1+Mg2+ and F1–Mg2+ the F1 treated and untreated with 1 mM Mg2+ respectively - F0 the membrane sector proteins of the H+-ATPase - TUF0 trypsin-urea – F0 - EUF0 EDTA-urea – F0 - F0+Mg2+ and F0–Mg2+ the F0 treated and untreated with 1 mM Mg2+ respectively - (F1 · F0)+Mg2+ and (F1 · F0)–Mg2+ the reconstituted F1 · F0 complex containing Mg2+-treated F1 and F0 and untreated F1 and F0 respectively - F1 · F0+Mg2+ and F1 · F0–Mg2+ the reconstituted H+-ATPase complex derived from the binding of purified F1 to the F0 treated and untreated with Mg2+ respectively - F1+Mg2+ · F0 and F1–Mg2+ · F0 the reconstituted H+-ATPase derived from the binding of F0 to the purified F1 treated and untreated with Mg2+ respectively |
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| Keywords: | Conformational interaction Mg2+ H+-ATPase reconstitution soluble F1-ATPase mitochondria |
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