Mg2+ and ATP or adenosine 5'-[gamma-thio]-triphosphate (ATP gamma S) enhances intrinsic fluorescence and induces aggregation which increases the activity of spinach Rubisco activase.
ATP and Mg2+ 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 Mg2+ 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'-[gamma-thio]-triphosphate (ATP gamma S). 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 ATP gamma S was sigmoidal, although at pH 8 the half-saturation requirements for both ATP gamma S (12 microM) and Mg2+ (1.5 mM) were not too dissimilar to the binding affinities (6 microM 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 Mg2+ and ATP or ATP gamma S was obtained by gel-filtration chromatography. However, the apparent molecular mass was heterogeneous 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 Mg2+ to increase aggregation eliminated the otherwise observed time delays in the Rubisco activation and ATP hydrolysis kinetics.
Wang
Z Y
ZY
Department of Agronomy, University of Illinois at Champaign-Urbana.
Ramage
R T
RT
Portis
A R
AR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008970:Molecular Weight; D010940:Plant Proteins; D010945:Plants, Edible / Q000201:enzymology*; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000378:metabolism*
D010940:Plant Proteins; C054765:rca protein, plant; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium