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) 0.021μm(site B), another slowly exchangeable high affinity site withKd1.6μm(site C) and several low affinity (Kd˜30μm) sites. TheKdvalues for sites B and C of the other nucleotides tested were 0.5μmand 16μm(GDP), 8μmand 34μm(CDP), 17μmand 20μm(UDP) and 1.4μmand 1.4μm(PP1).From a comparison of the observed UV spectral change and the amount of nucleotide bound to these sites, as calculated from the aboveKdvalues, 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−1ADPcm−1for ADP at 278 nm. Our conclusions were strengthened by the good agreement between the observed spectra and the calculated spectra (derived from theKdandΔεvalues 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 spe
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