Quantitative separation of spinach thylakoids into Photosystem II-enriched inside-out vesicles and Photosystem I-enriched right-side-out vesicles |
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Authors: | Hans-Erik Åkerlund Bertil Andersson |
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Institution: | Department of Biochemistry, University of Lund, P.O. Box 740, S-220 07 Lund Sweden |
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Abstract: | Yeda press disruption of thylakoids in the presence of magnesium followed by aqueous polymer two-phase partitioning fractionated the total thylakoid membrane material into two distinctly different fractions. One fraction comprised approx. 60% of the material on a chlorophyll basis and contained inside-out vesicles while the other fraction (40%) contained right-side-out vesicles. The sidedness of the vesicles was determined from the direction of their light-induced proton translocation. The inside-out vesicles showed a pronounced Photosystem (PS) II enrichment as judged by their high PS II and low PS I activities. Moreover, they showed a high ratio between the PS II reaction centre chlorophyll-protein complex and the PS I reaction centre chlorophyll-protein complex (CP I). The chlorophyll ratio was as low as 2.3 compared to 3.2 for the starting material. In contrast, the right-side-out vesicles showed a pronounced PS I enrichment. Their chlorophyll ratio was 4.3–4.9. The tight stacking induced by Mg2+ allows a quantitative formation of inside-out vesicles from the appressed thylakoid regions while mainly non-appressed thylakoids turn right-side-out. The possibility of fractionating all of the thylakoid material into two sub-populations with markedly different composition with respect to PS I and PS II argues against a close physical association between the two photosystems and in favour of their spatial separation in the plane of the membrane. This fractionation procedure, which can be completed within 1 h and gives high yields of both PS II inside-out thylakoids and PS I right-side-out thylakoids, should be very useful for facilitating and improving studies on both the transverse and lateral organization of the thylakoid membrane. |
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Keywords: | Thylakoid subfractionation Thylakoid stacking Phase partition Membrane vesicle (Spinach chloroplast) Chl chlorophyll PS photosystem CP chlorophyll-protein complex DCIP 2 6-dichlorophenolindophenol |
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