Isolation and characterization of oxygen-evolving thylakoid membranes and Photosystem II particles from a marine diatom Chaetoceros gracilis |
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Authors: | Ryo Nagao Osamu Tada Naoshi Dohmae Akinori Okumura Takeshi Takahashi Isao Enami |
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Institution: | a Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan b Biomolecular Characterization Team, Discovery Research Institute, RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan c JST, CREST, Japan d Department of Integrated Sciences in Physics and Biology, College of Humanities and Sciences, Nihon University, Sakurajosui 3-25-40, Setagaya-ku, Tokyo 156-8850, Japan e Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki 2641, Noda, Chiba 278-8510, Japan f Department of Life Science, University of Hyogo, Harima Science Garden City, Hyogo 678-1297, Japan |
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Abstract: | Thylakoid membranes retaining high oxygen-evolving activity (about 250 μmol O2/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 μmol O2/mg Chl/h in the absence and presence of CaCl2, respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein. |
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Keywords: | CBB Coomassie brilliant blue Chl chlorophyll Cyt cytochrome DCBQ 2 6-dichloro-p-benzoquinone DCMU dichlorophenyldimethylurea DM d-maltoside" target="_blank">n-dodecyl-β-d-maltoside FCP fucoxanthin chlorophyll a/c-binding protein HPLC high-performance liquid chromatography HTG d-thioglucoside" target="_blank">n-heptyl-β-d-thioglucoside Mes 2-morpholinoethanesulfonic acid OG d-glucoside" target="_blank">n-octyl-β-d-glucoside PBQ phenyl-p-benzoquinone PMSF phenylmethyl sulfonyl fluoride PSI and PSII Photosystem I and Photosystem II PVDF polyvinylidene fluoride SM sucrose monolaurate TMBZ 3 3&prime 5 5&prime -tetramethylbenzidine |
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