ADP Binding to TF1 and Its Subunits Induces Ultraviolet Spectral Changes1

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Abstract
Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF 1 ) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF 1 in the presence and in the absence of Mg 2+ , an UV absorbance change was induced (t1/2 ˜ 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated β subunits in the presence and in the absence of Mg 2+ , and with the isolated a subunits in the presence of Mg 2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF 1 in the presence of Mg 2+ , three high-affinity sites ( Kd = 61 nM) and three low-affinity sites ( Kd = 87μM). In the absence of Mg 2+ , TF 1 has one high-affinity site ( Kd < 10 nM) and five low-affinity sites ( Kd = 100 μM). Moreover, we found a single Mg 2+ -dependent ADP binding site on the isolated α subunit and a single Mg 2+ -independent ADP binding site on the isolated β subunit. From the above observations, we concluded that the three Mg 2+ -dependent high-affinity sites for ADP are located on the a subunit in TF 1 and that the single high-affinity site is located on one of the β subunits in TF 1 in the absence of Mg 2+ .

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