Pigment content of D1-D2-cytochrome b559 reaction center preparations after removal of CP47 contamination: an immunological study.
Isolated D1-D2-cytochrome b559 photosystem II reaction center preparations with pigment stoichiometry higher than 4 chlorophylls per 2 pheophytins can be contaminated with CP47 proximal antenna complex. Reaction center prepared by a modification of the Nanba-Satoh procedure and containing about 6 chlorophylls per 2 pheophytins showed immuno-cross-reactivity when probed with a monoclonal antibody raised against the CP47 polypeptide. Furthermore, they could be fractionated successfully by Superose-12 sieve chromatography into two different populations. The first few fractions off the column contained a more definitive 435 nm shoulder corresponding to increased chlorophyll content, and showed strong immuno-cross-reactivity with the CP47 antibody. The peak fractions off the column displayed a less prominent 435 nm shoulder, and did not cross-react with the antibody. Moreover, when a 6-chlorophyll preparation was mixed with Sepharose beads coupled to CP47 antibody, the eluted material corresponded to a preparation of about 4 chlorophylls per 2 pheophytins and did not show any cross-reaction with the antibody against CP47. The amount of CP47 protein in the 6-chlorophyll preparation as quantitated using Coomassie Blue staining or from gel blots was sufficient to account for most of the extra 2 chlorophylls. We conclude that D1-D2-cytochrome b559 preparations containing more than 4 chlorophylls per 2 pheophytins can be contaminated with small amounts of CP47-D1-D2-Cyt b559 complex and that native photosystem II reaction centers contain 4 core chlorophylls per 2 pheophytins.
Pueyo
J J
JJ
Department of Plant Nutrition, Estación Experimental de Aula Dei (CSIC), Zaragoza, Spain.
Moliner
E
E
Seibert
M
M
Picorel
R
R
D002734:Chlorophyll / Q000032:analysis*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000032:analysis*; D015151:Immunoblotting; D045342:Light-Harvesting Protein Complexes; D010674:Pheophytins / Q000032:analysis*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000032:analysis* / Q000302:isolation & purification*; D045332:Photosystem II Protein Complex; D013053:Spectrophotometry; D018724:Spinacia oleracea / Q000737:chemistry
D003573:Cytochrome b Group; D045342:Light-Harvesting Protein Complexes; D010674:Pheophytins; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; C070970:photosystem II, chlorophyll-binding protein, CP-47; D002734:Chlorophyll; C019313:cytochrome b559