Extraction of functionally active photosystem 2 pigment-protein complexes from pea thylakoids and their purification on Sepharose DEAE 6B.
A method for selective extraction of photosystem 2 (PS 2) pigment-protein complexes (PPC) from pea thylakoids and their purification from Sepharose DEAE 6B was developed. It was shown that extraction of thylakoids (3 mg chlorophyll/ml) with Triton X-100 (Triton X-100:chlorophyll = 15-20:1 w/w) in the presence of 1 M sucrose, 1 M NaCl, 50 mM MES in the solubilization medium (pH 5.0), tenfold dilution with 50 mM MES buffer (pH 5.0), and centrifugation at 16,000 g for 10 min provided selective extraction of PS 2 PPC. Spectral, electrophoretic and functional properties of the isolated PPC were determined. This method allowed us to isolate native pigment-protein oxygen-evolving complexes (core complexes) and D1-D2-cytochrome b559 complexes of the reaction centre (RC). The core complex immobilized on a Sepharose DEAE 6B column was treated with 2 mM hydroxylamine or 1 M CaCl2. In the former case, modification by hydroxylamine led to the removal of 4 atoms of Mn, but not of the 33-kDa protein; in the latter case, modification by CaCl2 led to the removal of the 33 kDa protein. The modified core complexes were unstable upon native electrophoresis in the presence of n-dodecyl-beta-d-maltoside and deriphat-160. We suggest that the structural organization of the Mn cluster and 33 kDa protein stabilized the core complex and increased its stability upon the action of the detergents.
Khristin
M S
MS
Institute of Soil Science and Photosynthesis, Russian Academy of Sciences, Pushchino, Moscow Region.
Nikitishena
O V
OV
Smolova
T N
TN
Zastrizhnaya
O M
OM
D002736:Chloroplasts / Q000737:chemistry* / Q000648:ultrastructure; D002852:Chromatography, Ion Exchange / Q000379:methods; D004983:Ethanolamines; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007425:Intracellular Membranes / Q000737:chemistry*; D045342:Light-Harvesting Protein Complexes; D018532:Pisum sativum / Q000737:chemistry*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012685:Sepharose; D012965:Sodium Chloride; D014783:Viscosity
D004983:Ethanolamines; D007202:Indicators and Reagents; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D012965:Sodium Chloride; D012685:Sepharose; C007369:2-diethylaminoethanol