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Isolation and characterization of oxygen-evolving photosystem II particles and photosystem II core complex from the filamentous cyanobacterium Spirulina platensis
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SYSNO ASEP 0422357 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Isolation and characterization of oxygen-evolving photosystem II particles and photosystem II core complex from the filamentous cyanobacterium Spirulina platensis Author(s) Šetlíková, Eva (MBU-M)
Sofrová, D. (CZ)
Kovář, V. (CZ)
Budáč, Petr (MBU-M)Source Title Photosynthetica. - : Ústav experimentální botaniky AV ČR, v. v. i. - ISSN 0300-3604
Roč. 51, č. 4 (2013), s. 517-530Number of pages 14 s. Language eng - English Country CZ - Czech Republic Keywords antibodies ; fluorescence spectra ; IMAC chromatography Subject RIV EE - Microbiology, Virology R&D Projects GA206/08/1683 GA ČR - Czech Science Foundation (CSF) ED2.1.00/03.0110 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Institutional support MBU-M - RVO:61388971 UT WOS 000327507500006 DOI 10.1007/s11099-013-0051-0 Annotation Photosystem (PS) II particles retaining a high rate of O-2 evolution were isolated from the mesophilic filamentous cyanobacterium, Spirulina platensis. To achieve high production of PSII complexes in the cells, irradiance from halogen incandescent lamps was used. Disruption of cells by vibration of glass beads proved to be the most suitable procedure for isolation of thylakoid membranes. The selectivity of detergents for PSII particle preparation rose in the order of Triton X-100 < decyl-beta-D-glucopyranoside < dodecyldimethyl-aminooxide < n-heptyl-beta-D-thioglucoside < N-dodecyl-N,N-dimethylammonio-3-propane sulphonate < n-octyl-beta-thioglycoside < octylglucoside < n-dodecyl-beta-D-maltoside. The last four detergents yielded extracts, from which pure PSII particles not contaminated by PSI complexes could be obtained by sucrose-gradient centrifugation (20-45%) at the 43% sucrose level. We assumed both the acceptor and donor sides of the isolated n-dodecyl-beta-D-maltoside (DM) particles to be intact due to high oxygen production by DM particles [1,500 meq(e(-)) mol(-1) (Chl) s(-1)] achieved in the presence of all artificial acceptors tested. The PSII particle fraction from the sucrose gradient was used with immobilized metal (Cu2+) affinity chromatography (IMAC) for the preparation of the PSII core complex. By washing the column with a MES buffer containing MgCl2 and CaCl2, the phycobiliproteins were stripped off. The PSII core complex was eluted in a buffer containing 1% DM, mannitol, MgCl2, NaCl, CaCl2, and E >-aminocaproic acid. SDS-PAGE of the core complex provided pure bands of D1 and D2 proteins and PsbO protein from thylakoid membrane, which were used to raise polyclonal antibodies in rabbits Workplace Institute of Microbiology Contact Eliška Spurná, eliska.spurna@biomed.cas.cz, Tel.: 241 062 231 Year of Publishing 2014
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