Nucleotide exchange and control of ATPase activity in Rhodospirillum rubrum chromatophores |
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Authors: | Luit Slooten Adriaan Nuyten |
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Institution: | Vrije Universiteit Brussel, Faculty of Sciences, Laboratory of Biophysics, Pleinlaan 2, 1050, Brussels, Belgium |
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Abstract: | (1) Light-activated ‘dark’ ATPase in Rhodospirillum rubrum chromatophores is inhibited by preincubation with ADP or ATP (in the absence of Mg2+). I50 values were 0.5 and 6 μM, respectively, after 20 s of preincubation. (2) In the absence of MgATP, the rate constant for dissociation of ADP or ATP from the inhibitory site was less than 0.2 min?1 in deenergized membranes. Illumination in the absence of MgATP caused an increase of over 60-fold in both rate constants. (3) In some experiments hydrolysis was performed in the presence of 10 μM Mg2+ and 0.2 mM MgATP. Under these conditions, the ADP or ATP inhibition was reversed within about 20 or about 80 s, respectively, after the onset of hydrolysis. This suggests that recovery from ADP or ATP inhibition (i.e., release of tightly bound ADP or ATP) in the dark is induced by MgATP binding to a second nucleotide-binding site on the enzyme. (4) Results obtained with variable concentrations of uncoupler suggest that in the absence of bound Mg2+ (see below), MgATP-induced release of tightly bound ADP or ATP does not require a transmembrane . This, together with the inhibitor/substrate ratios prevalent during hydrolysis, suggests that these reactivation reactions involve MgATP binding to a high-affinity binding site (Kd < 2 μM). (5) At high concentrations of uncoupler, a time-dependent inhibition of hydrolysis occurred in the control chromatophores as well as in the nucleotide-pretreated chromatophores. This deactivation was dependent on Mg2+. In addition, MgATP-dependent reversal of ADP inhibition in the dark was inhibited by Mg2+ at concentrations above 20–30 μM. By contrast, MgATP-dependent reversal of ADP inhibition occurs within 3–4 s, despite the presence of high concentrations of Mg2+ if the chromatophores are illuminated during contact with the nucleotides. Uncoupler abolishes the effect of illumination. A reaction scheme incorporating these findings is proposed. (6) The implications of these findings for the mechanism of lightactivation of ATP hydrolysis (Slooten, L. and Nuyten, A., (1981) Biochim. Biophys. Acta 638, 305–312) are discussed. |
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Keywords: | ATPase activation Nucleotide binding Mg2+ effect Chromatophore Bacterial photosynthesis (R rubrum) |
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