The release of a 10‐kDa polypeptide from everted photosystem II thylakoid membranes by alkaline tris

Ljungberg, Ulf; Åkerlund, Hans‐Erik; Andersson, Bertil

doi:10.1016/0014-5793(84)80746-2

Cited by 54 publications

(12 citation statements)

References 17 publications

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“…3). In view of these attributes, spot Va could be the 22 kDa intrinsic polypeptide reported by Ljungberg et al (1984a, b) and was observed in some PS2 preparations (Yamamoto et al 1984, Gounaris et al 1984. Silver-staining also revealed that associated with group V is an additional off-diagonal spot (designated sVc in Fig.…”

Section: Resultsmentioning

confidence: 89%

Investigation of the spatial relationships between photosystem 2 polypeptides by reversible crosslinking and diagonal electrophoresis

1987

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Nearest neighbour relationships within the LHC2-PS2 complex were investigated by using the reversible crosslinking agent dithiobis(succinimidyl propionate) (DSP). This was accomplished by treating PS2-enriched membranes, prepared from chloroplasts of Pisum sativum, with the crosslinker followed by diagonal electrophoresis of the solubilised polypeptides.Analysis of the off-diagonal spot patterns produced by crosslinker cleavage and second dimension electrophoresis was made on the basis of: staining with Coomassie blue or silver, labelling with [(35)S]-methionine, and sensitivity to 1 M NaCl washing. It was concluded that LHC2 polypeptides crosslinked with several components of the PS2 complex and that the extrinsic polypeptides associated with water oxidation, having approximate molecular weights of 16 and 23 kDa, crosslink to form homodimers. The latter finding suggests that there may be more than one copy of each of these polypeptides per PS2 complex.

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Section: Resultsmentioning

confidence: 89%

Investigation of the spatial relationships between photosystem 2 polypeptides by reversible crosslinking and diagonal electrophoresis

1987

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“…[9,19]). Other workers have similarly demonstrated less than complete removal (see figures in [24]). Therefore, the CaC12-wash method should be viewed only as a convenient method for significant depletion, but not necessarily for perfect removal of the 33 kDa EP.…”

Section: Discussionmentioning

confidence: 98%

Assocation of the 33 kDa extrinsic polypeptide (water-splitting) with PS II particles: immunochemical quantification of residual polypeptide after membrane extraction

et al. 1987

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Various washing procedures were tested on Triton-prepared PS II particles for their ability to remove the 33 kDa extrinsic polypeptide (33 kDa EP) associated with the water-splitting complex. Residual 33 kDa EP was evaluated by Coomassie blue staining of SDS gels of washed particles and by Western blotting with an antibody specific for the 33 kDa EP. A wash with 16 mM Tris buffer, pH 8.3, inhibited water-splitting activity but did not remove all the 33 kDa EP. Sequential washes with 30 mM octyl glucoside (pH 8.0 and 6.8), and a single wash with 0.8 M Tris were also ineffective in removing all the 33 kDa EP. Washing with 1 M CaCl2 was more effective in removing 33 kDa EP; while only a faint trace of protein was detectable by Coomassie-staining, immunoblotting revealed a considerable remainder. The treated particles retained some water-splitting activity. The two step procedure of Miyao and Murata (1984) involving 1 M NaCl and 2.3 M urea was most effective, removing all but a trace of antibody positive protein. Our finding suggests that (1) the degree of depletion of the 33 kDa EP cannot be judged on the basis of Coomassie stain alone, and (2) this extrinsic protein is very tightly associated with the membrane, perhaps via a hydrophilic portion of this otherwise hydrophilic protein. The results also suggest that the presence or absence of the 33 kDa protein per se is not the primary determinant of residual water splitting activity.

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“…Feltham First) by the method of Kalberer et al [23] except that sorbitol was replaced by sucrose. Thylakoid membranes enriched in photosystem II were prepared as described by Ford and Evans [ 16] and were washed at 0.5 mg ml-1chlorophyll at 4 °C for 30 min with 0.8 M Tris-HC1 pH 8.4 to remove the 33, 23 and 16 kDa polypeptides [30]. The 33 kDa polypeptide was purified from the supernatant by FPLC anion-exchange chromatography after centrifugation at 40 000 x g for 15 min and dialysis against 20 mM bis-Tris-HC1 pH 6.5.…”

Section: Purification Of the 33 Kda Polypeptidementioning

confidence: 99%

The extrinsic 33 kDa polypeptide of the oxygen-evolving complex of photosystem II is a putative calcium-binding protein and is encoded by a multi-gene family in pea

et al. 1989

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The extrinsic 33 kDa polypeptide of the water-oxidizing complex has been extracted from pea photosystem II particles by washing with alkaline-Tris and purified by ion-exchange chromatography. The N-terminal amino acid sequence has been determined, and specific antisera have been raised in rabbits and used to screen a pea leaf cDNA library in λgt11. Determination of the nucleotide sequence of positive clones revealed an essentially full-length cDNA for the 33 kDa polypeptide, the deduced amino acid sequence showing it to code for a mature protein of 248 amino acids with an N-terminal transit peptide of 81 amino acids. The protein showed a high degree of conservation with previously reported sequences for the 33 kDa protein from other species and the sequence contained a putative Ca(2+)-binding site with homology to mammalian intestinal calcium-binding proteins. Northern analysis of total pea RNA indicated a message of approximately 1.4 kb, in good agreement with the size of the cDNA obtained at 1.3 kbp. Southern blots of genomic DNA probed with the labelled cDNA give rise to several bands suggesting that the 33 kDa polypeptide is coded by a multi-gene family.

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The release of a 10‐kDa polypeptide from everted photosystem II thylakoid membranes by alkaline tris

Cited by 54 publications

References 17 publications

Investigation of the spatial relationships between photosystem 2 polypeptides by reversible crosslinking and diagonal electrophoresis

Investigation of the spatial relationships between photosystem 2 polypeptides by reversible crosslinking and diagonal electrophoresis

Assocation of the 33 kDa extrinsic polypeptide (water-splitting) with PS II particles: immunochemical quantification of residual polypeptide after membrane extraction

The extrinsic 33 kDa polypeptide of the oxygen-evolving complex of photosystem II is a putative calcium-binding protein and is encoded by a multi-gene family in pea

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