Assessment of the number of nucleotide binding sites on chloroplast coupling factor 1 by the continuous variation method.
The method of continuous variation (Job plot analysis) and difference absorbance spectroscopy were used to investigate the binding of 2'(3')-(trinitrophenyl)-ADP and -ATP to chloroplast coupling factor 1 (CF1). Experiments performed at a low total concentration (30 microM) of nucleotide and enzyme binding sites (assuming three or four binding sites per CF1) could be interpreted in terms of approximately three nucleotide binding sites per CF1. At higher total concentrations (100 and 400 microM), the number of apparent binding sites increased to almost four. Computer-generated Job plots, using a protein-ligand complex formation scheme of n independent, nonequivalent binding sites, gave good fits to the experimental data at all concentrations when four binding sites were modeled. The dissociation constant of the fourth site was estimated to be approximately 20 microM. Additional nucleotide binding sites were not directly observed by this method and, if they exist, have very weak binding affinities (dissociation constants greater than approximately 1 mM).
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- Spectrophotometry
- Proton-Translocating ATPases
- Models, Chemical
- Computer Simulation
- Biochemistry & Molecular Biology
- Binding Sites
- Adenosine Diphosphate
- Adenine Nucleotides
- 1101 Medical Biochemistry and Metabolomics
- 0601 Biochemistry and Cell Biology
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Spectrophotometry
- Proton-Translocating ATPases
- Models, Chemical
- Computer Simulation
- Biochemistry & Molecular Biology
- Binding Sites
- Adenosine Diphosphate
- Adenine Nucleotides
- 1101 Medical Biochemistry and Metabolomics
- 0601 Biochemistry and Cell Biology