Mg 2+ and ATP or adenosine 5′-[γ-thio]-triphosphate (ATPγS) enhances intrinsic fluorescence and induces aggregation which increases the activity of spinach Rubisco activase

Abstract

ATP and Mg 2+ caused a transient increase in the intrisinc fluorescence of Rubisco activase which was inhibited by the presence of ADP. Only minor changes in fluorescence were observed with ATP or Mg 2+ alone. The fluorescence increase was stabilized by addition of an ATP regenerating system or by substitution of ATP with a non-hydrolyzable analog, adenosine 5′-[γ-thio]-triphosphate (ATPγS). The initial rate of increase in fluorescence also depended on the concentration of protein in a manner consistent with second-order kinetics. The concentration dependence for the effect of ATPγS was sigmoidal, although at pH 8 the half-saturation requirements for both ATPγS (12 μM) and Mg 2+ (1.5 mM) were not too dissimilar to the binding affinities (6 μM and 2 mM, respectively) determined indirectly with the fluorescent probe, 1-anilinonapthalene-8-sulfonate. However, the concentration dependence of ATP was about 5-fold higher than its binding affinity, also sigmoidal and quite similar to the concentration responses of ATP hydrolysis and activation of Rubisco by the protein. These characteristics of the intrinsic fluorescence indicate that it monitors a conformational change in the protein occurring after binding of the nucleotide and associated with increased aggregation. Direct evidence of increased aggregation in the presence of Mg 2+ and ATP or ATPγS was obtained by gel-filtration chromatography. However, the apparent molecular mass was heterogenous and also varied with temperature. The increased aggregation of the protein resulted in altered kinetic properties. The ATP hydrolysis activity of the protein increased and the half-maximal ATP concentration decreased as the protein concentration was increased in the assay. Also, a brief pretreatment of the protein with ATP and Mg 2+ to increase aggregation eliminated the otherwise observed time delays in the Rubisco activation and ATP hydrolysis kinetics.