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Photosynthetica

, Volume 51, Issue 4, pp 517–530 | Cite as

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

  • E. Šetlíková
  • D. Sofrová
  • V. Kovář
  • P. Budáč
Original Papers

Abstract

Photosystem (PS) II particles retaining a high rate of O2 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-β-D-glucopyranoside < dodecyldimethyl-aminooxide < n-heptyl-β-D-thioglucoside < N-dodecyl-N,N-dimethylammonio-3-propane sulphonate < n-octyl-β-thioglycoside < octylglucoside < n-dodecyl-β-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-β-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 ɛ-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. These antibodies recognized D1 and D2 not only as monomers of 31 and 32 kDa proteins, but also as heterodimers of D1, D2 corresponding to the band of 66 kDa on SDS-PAGE. This was in contrast to antibodies of synthetic determinants, which reacted only with the monomers of D1 and D2 proteins. These negative reactions against heterodimers of D1, D2 supported the hypothesis that dimeric forms of PSII reaction centre proteins have a C-terminal sequence sterically protected against a reaction with specific antibodies.

Additional key words

antibodies fluorescence spectra IMAC chromatography selectivity of detergents 

Abbreviations

APC

allophycocyanine

BQ

p-benzoquinone

Chl

chlorophyll

CP43, CP 47

chlorophyll-proteins with Rm 43 and 47 kDa

Cyt

cytochrom

D1, D2

proteins of PSII reaction centre

DCBQ

2,6-dichloro-p-benzoquinone

DGP

decyl-β-Dglucopyranoside

DM

n-dodecyl-β-D-maltoside

DM-PSII

PSII particles isolated upon extraction of TM with DM

DMBQ

2,6-dimethyl-p-benzoquinone

ETR

electron transport rate

FeCy

ferricyanide

HR

Hill reaction

HTG

n-heptyl-β-D-thioglucoside

IMAC

immobilized metal affinity chromatography

LDAO

dodecyldimethyl-aminooxide

LT-FER

low temperature (77 K) fluorescence ratio estimated according to the areas under the major bands assumed to originates in the complexes of PSII and PSI

MES

2-morpholinoethanesulfonic acid

Mr

relative molecular mass

MR

Mehler reaction

OEC

oxygen-evolving complex

OG

octylglucoside

OTG

n-octyl-β-thioglycoside

PC

phycocyanine

PS

photosystem

PS-ETR

electron transport through PSII or PSI

PSQII/I ETR

(PSII ETR:PSI ETR)

PSQII/I (LT-FER)

ratio of areas under the bands of 77 K fluorescence emission spectra originated in the components of PSII and PSI

PSQQ

[PSQII/I (ETR):PSQ II/I (LT-FER)]

QA

primary quinone acceptor of PSII

QB

secondary quinone acceptor of PSII

RC

reaction centre

SB12

N-dodecyl-N,N-dimethylammonio-3-propane sulphonate

ROΔA

redox difference spectroscopy

SDS-PAGE

sodium dodecyl sulphate polyacrylamide gel electrophoresis

TM

thylakoid membranes

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • E. Šetlíková
    • 1
  • D. Sofrová
    • 2
  • V. Kovář
    • 1
  • P. Budáč
    • 1
  1. 1.Department of Autotrophic Microorganisms, Institute of MicrobiologyAcademy of Sciences of the Czech Republic, Opatovický mlýnTřeboňCzech Republic
  2. 2.Department of Biochemistry, Faculty of ScienceCharles UniversityPragueCzech Republic

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