Structural,biochemical and biophysical characterization of four oxygen-evolving Photosystem II preparations from spinach |
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Authors: | Terri Goodman Dunahay L.Andrew Staehelin Michael Seibert Paula D. Ogilvie Steven P. Berg |
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Affiliation: | 1. Department of Molecular, Cellular and Developmental Biology, University of Colorado, Campus Box 347, Boulder, CO 80309 U.S.A.;3. Department of Biological Sciences, University of Denever, Denver, CO 80208 U.S.A. |
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Abstract: | Four procedures utilizing different detergent and salt conditions were used to isolate oxygen-evolving Photosystem II (PS II) preparations from spinach thylakoid membranes. These PS II preparations have been characterized by freeze-fracture electron microscopy, SDS-polyacrylamide gel electrophoresis, steady-state and pulsed oxygen evolution, 77 K fluorescence, and room-temperature electron paramagnetic resonance. All of the O2-evolving PS II samples were found to be highly purified grana membrane fractions composed of paired, appressed membrane fragments. The lumenal surfaces of the membranes and thus the O2-evolving enzyme complex, are directly exposed to the external environment. Biochemical and biophysical analyses indicated that all four preparations are enriched in the chlorophyll complex and Photosystem II, and depleted to varying degrees in the stroma-associated components, Photosystem I and the CF1-ATPase. The four PS II samples also varied in their cytochrome f content. All preparations showed enhanced stability of oxygen production and oxygen-rate electrode activity compared to control thylakoids, apparently promoted by low concentrations of residual detergent in the PS II preparations. A model is presented which summarizes the effects of the salt and detergent treatments on thylakoid structure and, consequently, on the configuration and composition of the oxygen-evolving PS II samples. |
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Keywords: | Photosystem II Oxygen evolution Freeze-fracture ESR Fluorescence (Spinach chloroplast) Chl chlorophyll DCIP 2,6-dichlorophenol indophenol EPR electron paramagnetic resonance Mes 4-morpholineethanesulfonic acid octyl glucoside P-430 a secondary PS I electron acceptor P-700 the PS I reaction center Pipes 1,4-piperazinediethanesulfonic acid PS I Photosystem I PS II Photosystem II SDS sodium dodecyl sulfate Tricine BBY membrane preparation described by Berthold, D.A. Babcock, G.T. and Yocum, C.F. (Ref. 17) K&M idem by Kuwabara, T. and Murata, N. (Ref. 19) YUSN idem by Yamamoto, Y., Ueda, T., Shinka, H. and Nishimura, M. (Ref. 27) LHC light-harvesting complex MDT magnesium-digitonin-Triton |
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