Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin--biotin binding.
In order to study the affinity binding of c-type cytochromes to the photosynthetic reaction center (RC) by quantitative affinity chromatography (QAC), RC from Rhodobacter sphaeroides was reconstituted into liposomes composed of egg phosphatidylcholine (EPC) and 2 mol% of biotinyl phosphatidylethanolamine simultaneously as the liposomes were formed and immobilized in (strept)avidin-coupled gel beads by rotary detergent dialysis. The immobilized amount was up to 80 nmol of RC and 33 micromol of lipid/g of moist gel in streptavidin-coupled Sephacryl S-1000 gel. By QAC frontal runs, retardation of mitochondrial cyt c on immobilized RC liposome columns was demonstrated. The dissociation constant for the RC-cyt c interaction was determined to be 0.20-0.57 microM. QAC studies also allowed evaluation of the orientation of reconstituted RC in immobilized liposomes by comparison of the total amount of cyt c binding sites with the amount of available binding sites obtained by QAC. It seems that the RC proteoliposomes immobilized in Sephacryl S-1000 gel exposed the cyt c binding sites on the outer surface of the liposomes due to effects of the gel network pore size and the resulting liposomal size.
Yang
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National Institute for Advanced Interdisciplinary Research, Tsukuba, Ibaraki, Japan. miyake@nair.go.jp
Liu
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Hara
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Lundahl
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Miyake
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D000818:Animals; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D003574:Cytochrome c Group / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D003956:Dialysis; D006736:Horses; D008081:Liposomes; D008567:Membranes, Artificial; D008929:Mitochondria, Heart / Q000201:enzymology*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D011485:Protein Binding
D003574:Cytochrome c Group; D003902:Detergents; D008081:Liposomes; D008567:Membranes, Artificial; D045322:Photosynthetic Reaction Center Complex Proteins; D001360:Avidin; D001710:Biotin