Evidence for the location of divalent cation binding sites on the chloroplast membrane |
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Authors: | Prochaska Lawrence J Gross Elizabeth L |
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Institution: | (1) Department of Biochemistry, Ohio State University, 43210 Columbus, Ohio;(2) Present address: Department of Biological Sciences, Purdue University, 47907 West Lafayette, Indiana |
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Abstract: | Summary We have used a combination of chemical labeling and detergent fractionation techniques to locate the divalent cation binding sites on the chloroplast membrane. We determined the Ca2+-binding properties of Triton X-100 subchloroplast particles. Photosystem II (TSFII) particles showed one binding site withn=8.4 moles-mg chl–1 andk
d
=20 m. Photosystem I (TSFI) particles contained two binding sites. The first had ann=1.5 moles-mg chl–1 andk
d
=4 m. The second had ann=9.6 moles-mg chl–1 andk
d
=160 m. We have previously shown (Prochaska & Gross,Biochim. Biophys. Acta
376:126, 1975) that the divalent cation binding sites could be blocked using a water-soluble carbodiimide plus a nucleophile. Chlorophylla fluorescence and lightscattering changes were affected at the same carbodiimide concentrations emphasizing the relationship between these processes. The carbodiimide-sensitive sites were found to be located on the Photosystem II particles. A direct correlation between the inhibition of calcium binding and the carbodiimide-mediated incorporation of a (14C)-nucleophile was observed upon varying such parameters as carbodiimide concentration, nucleophile concentration, pH, and time of reaction. The presence of CaCl2 during the carbodiimide plus nucleophile modification procedure decreased the incorporation of (14C)-nucleophile, emphasizing the competition of the CaCl2 and the modification reagents for some of the same sites. Sodium dodecylsulfate gel electrophoresis of chlorophyll protein aggregates suggested that the site of competition of the calcium chloride and the modification reagents was the light-harvesting chlorophylla/b protein. |
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