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Toru Hisabori, Hidehiro Sakurai, Relation between Ultra-Violet Spectral Change and Nucleotide Binding of the Chloroplast Coupling Factor 1, Plant and Cell Physiology, Volume 25, Issue 3, April 1984, Pages 483–493, https://doi.org/10.1093/oxfordjournals.pcp.a076737
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Abstract
Properties of the nucleotide binding sites on chloroplast coupling factor 1 (CF1) were studied by equilibrium dialysis and UV spectroscopy. From our direct binding studies, we identified at least four kinds of ADP binding sites on CF1; a barely dissociable ADP binding site (site A), a slowly exchangeable high affinity site with dissociation constant (Kd) 0.021 μm (site B), another slowly exchangeable high affinity site with Kd 1.6 μm (site C) and several low affinity (Kd ˜30 μm) sites. The Kd values for sites B and C of the other nucleotides tested were 0.5 μm and 16 μm (GDP), 8 μm and 34 μm (CDP), 17 μm and 20 μm (UDP) and 1.4 μm and 1.4 μm (PP1).
From a comparison of the observed UV spectral change and the amount of nucleotide bound to these sites, as calculated from the above Kd values, we concluded that the nucleotide binding to site B or G induces UV spectral changes that are almost the same in shape and magnitude. The estimated difference molar absorption coefficient (Δε) was 3.4×103m−1ADP cm−1 for ADP at 278 nm. Our conclusions were strengthened by the good agreement between the observed spectra and the calculated spectra (derived from the Kd and Δε values of ADP and GDP) when ADP and GDP were added together to CF1.
The cause of the unusual behavior of GDP in the UV difference spectrum which was unexplained in our previous report was shown to be competition between the GDP added and previously bound ADP at sites B and C; this distorted the real spectrum induced by GDP.
