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Title:Interactions of ATP, ADP and magnesium ion with rubisco activase and their effects on rubisco activation
Author(s):Wang, Zhen Yuan
Doctoral Committee Chair(s):Ogren, William L.
Department / Program:Crop Sciences
Discipline:Crop Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Biology, Plant Physiology
Abstract:Rubisco activase is a chloroplast protein that mediates a greatly enhanced activation of rubisco in the presence of ATP and Mg$\sp{2+}$. The fluorescent dye 1-anilinonaphthalene-8-sulfonate was used to study the binding of rubisco activase with ligands. The results indicated that rubisco activase bound ADP (k$\sb{\rm d}$ = 0.76 $\mu$M) more tightly than ATP (k$\sb{\rm d}$ = 41 $\mu$M). Alkaline medium (pH 8.0) and Mg$\sp{2+}$ (4 mM) favored binding of ATP, compared to ADP, to rubisco activase. Binding of ATP to the Mg$\sp{2+}$-protein complex also induced a transient increase in the tryptophan fluorescence of spinach rubisco activase. The kinetics of the fluorescence change was similar to that of ATP hydrolysis. ADP was a competitive inhibitor of the ATP induced fluorescence enhancement. This complex exhibited a higher molecular size ($>$600 kDa) than the enzyme-Mg-ADP complex (340 kDa) measured by gel permeation chromatography. Analysis of the initial catalytic rates of the enzyme indicated that aggregation of the rubisco activase-Mg-ATP complex was required for both ATP hydrolysis and rubisco activation.
The effect of rubisco activase mediated ATP hydrolysis on the dissociation of RuBP from inactive spinach rubisco was characterized with (1-$\sp3$H) RuBP. In the absence of rubisco activase, dissociation of rubisco bound (1-$\sp3$H) RuBP was quite slow (k$\sb{\rm Off}$ = 4.8 $\times$ 10$\sp{-4}$ s$\sp{-1})$ after 1 hour incubation. In the presence of rubisco activase and ATP (but not ATP-$\gamma$-S) and Mg$\sp{2+}$, the dissociation rate of bound RuBP was significantly enhanced, indicating that a conformational change at the binding site of rubisco was mediated by rubisco activase and required hydrolysis of ATP.
Purified rubisco activase from tobacco, petunia and barley all exhibited ATP-dependent activity of rubisco activation. Tobacco rubisco activase was a tetramer of 42 kDa peptide subunits and was more aggregated when bound to Mg$\sp{2+}$ and ATP-$\gamma$-S. Rubisco activase from spinach and barley could activate rubisco from spinach, barley, Arabidopsis, Chlamydomonas, soybean, corn, wheat and pea, but was less efficiently in activating rubisco from tobacco, petunia and tomato, members of the Solanaceae family. In contrast, rubisco activase from tobacco and petunia could activate rubisco from species of their own family but exhibited poor activation with other species. These results indicate that while the unicellular green alga and most higher plants appear to share common properties in the interaction between rubisco and rubisco activase, the Solanaceae have diverged and developed different characteristics.
Issue Date:1992
Rights Information:Copyright 1992 Wang, Zhen Yuan
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9215904
OCLC Identifier:(UMI)AAI9215904

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