Ulf Ljungberg

Immunological studies on the organization of proteins in photosynthetic oxygen evolution

Abstract Studies on inside-out thylakoid vesicles and several Photosystem-II particles have suggested the involvement of three proteins of 33, 23 and 16 kDa in photosynthetic oxygen evolution. In this study, monospecific antibodies were raised against the purified 33, 23 and 16 kDa proteins. The antibodies were used to investigate the organization and function of these proteins in the oxygen-evolving complex. Quantification of the 33, 23 and 16 kDa proteins by rocket immunoelectrophoresis revealed one copy of each polypeptide per some 200 chlorophylls in unfractionated thylakoids. Isolated inside-out thylakoids, derived from the grana partitions, showed 6–8 times more of the 33, 23 and 16 kDa proteins, on a chlorophyll basis, compared to the stroma lamellae vesicles. Agglutination studies revealed that the proteins are exposed on the lumenal side of the thylakoid membrane. An obligatory role for the 23 kDa protein in the photosynthetic water oxidation is suggested from the close correlation obtained between the inhibition of oxygen evolution and the release of this protein as caused by washing the inside-out thylakoids with increasing concentrations of NaCl. For the 16 kDa protein no such correlation was obtained. Rebinding experiments, using both salt-washed and alkaline Tris-washed inside-out thylakoids revealed that the 33 kDa protein was required for the binding of the 23 kDa protein, which in turn enhanced the binding of the 16 kDa protein. It is concluded that the three proteins are closely organized as a complex at the inner thylakoid surface in association with membrane spanning proteins of Photosystem II.

Studies on the polypeptide composition of the cyanobacterial oxygen-evolving complex

Abstract Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum . Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria ( Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis ). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl 2 or MgCl 2 inhibited O 2 evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O 2 evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O 2 evolution in parallel.

Identification of polypeptides associated with the 23 and 33 kDa proteins of photosynthetic oxygen evolution

Abstract An immunological approach was used for nearest-neighbor analyses for the 23 and 33 kDA proteins of the oxygen-evolving complex. Functional Photosystem II particles with a simple polypeptide composition were partly solubilized with detergent and incubated with monospecific antibodies against either the 23 or the 33 kDa protein. SDS-polyacrylamide gel electrophoresis revealed that the immunoprecipitates, apart from the antigenic proteins, also contained polypeptides at 24, 22 and 10 kDa. In contrast, polypeptides of the light-harvesting and Photosystem II core complexes showed very poor coprecipitation with the 23 and 33 kDa proteins. The 24, 22 and 10 kDa polypeptides were not precipitated by the antibodies if the 23 and 33 kDa proteins had been removed from the particles prior to solubilization. These observations demonstrate a close association between the 24, 22 and 10 kDa polypeptides and the 23 and 33 kDa proteins of the oxygen-evolving complex. None of these precipitated polypeptides contained any manganese. It is suggested that the 24, 22 and 10 kDa polypeptides are subunits of the oxygen-evolving complex and involved in the binding of the extrinsic 23 and 33 kDa proteins to the inner thylakoid surface.

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

The release of polypeptides from inside‐out thylakoid vesicles and photosystem II by alkaline Tris treatment was reinvestigated, using SDS‐polyacrylanude gel electrophoresis (PAGE) in the presence of urea, with highly increased resolution in the low molecular mass region. In addition to the 33‐, 23‐, and 16‐kDa proteins of the oxygen‐evolving complex, a 10‐kDa polypeptide was released. This 10‐kDa polypeptide is an entrinsic polypeptide located at the inner grana thylakoid surface and with a likely role in photosynthetic oxygen evolution.

The presence of low-molecular-weight polypeptides in spinach Photosystem II core preparations. Isolation of a 5 kDa hydrophilic polypeptide

Abstract Spinach Photosystem II core preparations have been analyzed by sodium dodecyl sulphate urea-polyacrylamide gel electrophoresis, with very high resolution in the low-molecular-weight region. In preparations active in oxygen evolution five low-molecular-weight polypeptides of 7, 6.5, 5.5, 5 and 4 kDa were resolved, in addition to the previously identified subunits of the Photosystem II core. These low-molecular-weight polypeptides were present even if the preparation was performed in the presence of several protease inhibitors. Four of these polypeptides showed a high degree of copurification with the larger Photosystem II core polypeptides during the isolation. At least one of the low-molecular-weight polypeptides is hydrophilic, and has been isolated in a pure form. It has an apparent molecular weight of 5 kDa and contains no prosthetic group or metal. A Photosystem II core preparation, unable to perform oxygen evolution but able to perform electron transport with diphenylcarbazide as donor, lacked two of the low-molecular-weight polypeptides (6.5 and 7 kDa), in addition to the hydrophilic 33 kDa protein. These 6.5 and 7 kDa polypeptides may therefore be necessary for the oxygen-evolving reaction, while the other low-molecular-weight polypeptides may be associated with the primary reactions of Photosystem II.

Glycerol stabilizes oxygen evolution and maintains binding of a 9 kDa polypeptide in photosystem II particles from the cyanobacterium, Phormidium laminosum

High concentrations of glycerol (⩾20% ) stabilize oxygen evolution in photosystem (PS) II particles from the thermophilic cyanobacterium, Phormidium laminosum. Treating PS II particles with lower glycerol concentrations inhibits activity and, in parallel, detaches a 9 kDa polypeptide from PS II. These results suggest that the 9 kDa polypeptide is essential for PS II activity, and that it is bound to PS II primarily by hydrophobic interactions that are strengthened in vitro by glycerol. The 9 kDa polypeptide is distinct from cytochrome b‐559 and does not appear to be associated with manganese or with the calcium or chloride requirements for oxygen evolution.

Reconstitution of oxygen evolution in high salt washed photosystem II particles

Photosystem II thylakoid particles possessing high rates of oxygen evolution, were shown to have a very simple polypeptide composition. Upon washing of these particles with 250 mM NaCl the oxygen evolution was inhibited up to 80% concomitant with a release of two polypeptides of 23 and 16 kDa. Readdition of the pure 23 kDa protein to the depleted thylakoids under low ionic strength reconstituted more than half of the lost activity. No stimulation was obtained with the 16 kDa protein alone or in combination with glycerol. The results give further strong evidence that the 23 kDa protein is an essential component in the oxygen evolving complex. The possible involvement of other proteins in this complex is discussed in light of the demonstrated simple polypeptide pattern of the photosystem II particles.

Low Molecular Weight Polypeptides in Photosystem II and Protein Dependent Acceptor Requirement for Photosystem II

Recently, several preparations of Photosystem II (PS II) core particles, with oxygen evolving capacity, have been reported (1–4). They all show a quite similar and simple polypeptide pattern. These PS II core preparations are of great interest for the elucidation of what polypeptides are required for the catalytic PS II functions. It is, therefore, important that all polypeptides in these preparations are identified and characterized. Recently, we found, by improved gel electrophoresis, that the PS II core particles isolated according to Ikeuchi et al. (2) contained a number of low molecular weight polypeptides (5) that were not reported in the original work. This study has now been extended to include comparison with two additional PS II core preparations, those described by Ghanotakis et al. (3) and Franren (4). Our results suggest that three out of eight identified low molecular weight polypeptides (4, 5 and 5.5 kDa) are obligatory for catalytic PS II function.

Discrimination by immunological analysis between two 33–34 kDa polypeptides involved in photosynthetic oxygen evolution

Abstract A 34 kDa protein, which is modified in a mutant of Scenedesmus obliquus with impaired oxygen evolution and decreased levels of managese, has been equated to a hydrophilic spinach 33 kDa protein. In the present study, we show immunological evidence that these two proteins are not identical. Scenedesmus wild type, mutant and revertant were analyzed by western blotting using antibodies against the spinach 33 kDa protein. The antibodies did not react with the 34 kDa protein, but with a 30 kDa polypeptide unaltered in the mutant. Thus, in Scenedesmus, and probably also in higher plants, there are two distinct proteins with similar molecular weights, both of which are essential for oxygen evolution and possibly also for managese binding.

The 5 kDa, 10 kDa and 22 kDa Polypeptides of Higher Plant Photosystem II

Higher plant photosystem (PS) II is a supramolecular complex with both integral and peripheral protein subunits, mainly located Ln the grana region region of the thylakoid membrane. in recent years, knowledge about this protein complex has accumulated, including the identification and isolation of a few polypeptide components /reviewed in 1/. However, a full and detailed picture of the structure and function of PS II requires that each protein component is identified and characterized. Earlier studies have suggested that two polypeptides of 10 kDa and 22 kDa are components of photosystem II /2,3/. in addition, a 5 kDa polypeptide has been found to be a component of the photosystem II core /4/. The present work reports the methods for isolation and the characterization of the three PS II components of 5 kDa, 10 kDa and 22 kDa.