Isolation and characterization of oxygen-evolving photosystem II particles and photosystem II core complex from the filamentous cyanobacterium Spirulina platensis

0422357 - MBÚ 2014 RIV CZ eng J - Journal Article
Šetlíková, Eva - Sofrová, D. - Kovář, V. - Budáč, Petr
Isolation and characterization of oxygen-evolving photosystem II particles and photosystem II core complex from the filamentous cyanobacterium Spirulina platensis.
Photosynthetica. Roč. 51, č. 4 (2013), s. 517-530. ISSN 0300-3604. E-ISSN 1573-9058
R&D Projects: GA ČR GA206/08/1683; GA MŠMT(CZ) ED2.1.00/03.0110
Institutional support: RVO:61388971
Keywords : antibodies * fluorescence spectra * IMAC chromatography
Subject RIV: EE - Microbiology, Virology
Impact factor: 1.007, year: 2013

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
Permanent Link: http://hdl.handle.net/11104/0228579