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

Abstract

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.

On the Mechanism of Photosystem Ii Deterioration by Uv‐b Irradiation

“…7 show that UV-B irradiation does not significantly affect the polypeptide pattern. Despite the problems arising for quantitative analysis with Coomassie blue stained SDS-polyacrylamide gels (Camm et al, 1987) it can be concluded that the destruction of the water oxidizing enzyme system is not caused by a depletion of the extrinsic 33 kDa polypeptide. It seems more likely that UV-B irradiation causes structural changes in the apoprotein which leads to a distortion of the binding of part of the manganese.…”

Appearance of Type 1, 2, and 3 Light-Harvesting Complex II and Light-Harvesting Complex I Proteins during Light-Induced Greening of Barley (Hordeum vulgare) Etioplasts

“…The polyclonal anti-LHCII* antibodies that cross-react with a11 LHCII apoproteins (Dunahay and Staehelin, 1987) were used as a reference. In addition, we used polyclonal antibodies against the 33-, 23-, and 17-kD protein of the OEC (Camm et al, 1987;L.A. Staehelin and M. Seibert, unpublished results) and anti-peptide antibodies against the LHCI-specific sequence PLWFPGSTPPE (K. Sauer and L.A. Staehelin, unpublished results) of the Lhca 2*1 (LHCI-15) gene of petunia (Stayton et al, 1986).…”

Developing Seeds of Arabidopsis Store Different Minerals in Two Types of Vacuoles and in the Endoplasmic Reticulum

“…Proteins were resolved by SDS-PAGE on 12% (w/v) gels and electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA) using a semidry transfer technique. The polyclonal anti-MSD1 antibody, which recognizes Mn superoxide dismutase from Arabidopsis (Kliebenstein et al, 1998), was used at a concentration of 1:2000, and the anti-33-kD protein antibody (Camm et al, 1987) was used at a concentration of 1:3000. We used the monoclonal antibody anti-actin (Andersland et al, 1994) to ensure equal loading of cytoplasm protein.…”