Attachment of CuZn-Superoxide Dismutase to Thylakoid Membranes at the Site of Superoxide Generation (PSI) in Spinach Chloroplasts: Detection by Immuno-Gold Labeling After Rapid Freezing and Substitution Method |
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Authors: | Ogawa Ken'ichi; Kanematsu Sumio; Takabe Keiji; Asada Kozi |
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Institution: | 1Department of Botany, Faculty of Science, Kyoto University Kyoto, 606-01 Japan
2Department of Food Science and Technology, Minami-Kyushu University Takanabe, Miyazaki, 884 Japan
3Department of Wood Science and Technology, Faculty of Agriculture, Kyoto University Kyoto, 606-01 Japan
4The Research Institute for Food Science, Kyoto University Uji, Kyoto, 611 Japan |
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Abstract: | In chloroplasts O2 is photoproduced via the univalentreduction of O2 in PSI even under conditions that are favorablefor photosynthesis. The photogenerated O2 is disproportionatedto H2O2 and O2 in a reaction that is catalyzed by superoxidedismutase (SOD). The H2O2-scavenging ascorbate peroxidase isbound to the thylakoid membranes at or near the PSI reactioncenter Miyake and Asada (1992) Plant Cell Physiol. 33: 541],and the primary product of oxidation in the peroxidase-catalyzedreaction, the monodehydroascorbate radical, is photoreducedto ascorbate in PSI in a reaction mediated by ferredoxin Miyakeand Asada (1994) Plant Cell Physiol. 35: 539]. Therefore, SODshould be localized at or near the PSI complex. We report herethe microcompartmentalization of the chloroplastic CuZn-SODon the stromal-faces of thylakoid membranes where the PSI-complexis located. Spinach leaves were fixed and substituted by a rapidfreezing and substitution method that allows visualization ofintact chloroplasts. The embedded sections were immuno labeledwith the antibody against CuZn-SOD by the immunogold method.About 70% of the immunogold particles were found within 5 nmfrom the surface of the stromal-faces of thylakoid membranes.Of these particles, about 40% were found at the ends and marginsof the grana thylakoids and 60% were found on the stromal sideof the stromal thylakoids. From these results, the local concentrationof CuZn-SOD on the stroma-facing surfaces of the thylakoid membraneswas estimated to be about 1 mM. The effect of the microcompartmentalizationof CuZn-SOD on the scavenging of superoxide radicals is discussed. (Received November 25, 1994; Accepted February 23, 1995) |
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