Contribution to the structural characterization of eucaryotic PSI reaction centre ? II. Characterization of a highly purified photoactive SDS-CP1 complex

Abstract

Under precise conditions, SDS PAGE† allows purification of a photoactive P700-chla-protein complex from eucaryotic cells. The yield of P700 recovery is close to 100%. A total protein content equivalent to about 140 kD for one mole of P700 has been estimated by chemical analysis, and electrophoresis revealed the presence of two peptidic chains with MWs close to 65 kD. Photochemical and structural properties of this complex are given and compared with those of other complexes previously isolated.

A single step separation of PS 1, PS 2 and chlorophyll-antenna particles from spinach chloroplasts

“…Protein content of fraction 1 was low as determined by the UV absorption spectrum and also by the amino acid composition analysis; we found 3.5 mg protein for 1 mg chlorophyll. In fraction 1 there was the same protein content per chlorophyll as in the CPI preparation [20], which migrated in a single band on SDS polyacrylamide gels. Fraction 1 contained more chlorophylls per P700 than CPI and, as the two preparations contained the same amount of protein per chlorophyll, fraction 1 contained more Wavelength ~, (nm) Figure 4.…”

“…Pigments and proteins were quantified as previously reported [20]: carotenoids after saponification of the lipid extract, chlorophylls by the method of Mackinney [10] and proteins by amino acid analysis.…”

“…P700 concentration was measured as previously reported [20]. P680 concentration was measured as in [24].…”

“…We have previously described a simple SDS-electrophoretic method to obtain a photoactive PS 1 enriched fraction [20]. Unfortunately, the use of SDS for membrane solubilization destroys PS 2 activities as measured by Hill activity, + P68o formation or Q-formation.…”

“…Each new preparation allows us to increase our knowledge of membrane structure, electron transport or the primary reactions of photosynthesis. There now exist some very good photosystem 1 (PS 1) preparations characterized either by enrichment of the primary donor (P700) [21,11,9,3] or by protein purity [5,6,13,20]. Progress in photosystem 2 (PS 2) preparation has been delayed by difficulties in protecting photochemical activities during the isolation procedure, as well as in characterizing a given enrichment.…”