Thylakoid vesicles, preparation

Several polypeptide components of PS II and OEC have been isolated from thy-lakoids and PS II preparations capable of O2 evolution, after the initial isolation by Kuwabara and Murata [31] of a 33-34 kDa polypeptide (see, for a review. Ref. 10). On the basis of several criteria, such as the extraction by different reagents and the accessibility to antibodies in thylakoids or in inside-out vesicles prepared from thylakoids, a tentative and certainly incomplete picture has been proposed... 

At variance with Hill s scheme [3], which has been discussed above in its recent developments, a three-light reaction scheme has been proposed by Arnon and coworkers [4,59]. According to this scheme, Fd and subsequently NADP would be reduced by PS II directly, and PS II would perform two different photoacts with two acceptors Fd and Q (Qa ) [4]. The role of PS I would be limited to the performance of cyclic photophosphorylation, catalysed by Fd as the electron carrier. Recent experiments showing that PS Il-enriched, inside-out thylakoid vesicles are capable of low rates of NADP reduction upon addition of Fd, FNR and plasto-cyanin [67] have been designed to investigate the view that only PS II is required to transfer electrons from water to NADP. However, the presence of PS I in the preparations, though in low proportions, was not ruled out, and the cause of the absolute requirement for PC, which is known to be oxidized by P-700 [29], was unexplained. 

Fig. 19. Chloroplast thylakoid-membrane structure revealed by freeze-fracture electron microscopy. The oxygen-evolving (BBY) PS-II particle its preparation (A) and electron micrographs (B). The inside-out and rightside-out vesicles preparation, structure, and properties (C) and electron micrographs (D). Figure source (A) and (B) Dunahay, Staehelin, Seibert, Ogilvie and Berg (1984) Structural, biochemical and biophysical characterization of four oxygen-evolving photosystem II preparations from spinach. Biochim Biophys Acta 764 190, 185 (C) and (D) from Andersson and Akerlund (1978) Inside-out membrane vesicles isolated from spinach thylakoids. Biochim Biophys Acta 503 465, 468. Figure (B) kindly furnished by Dr. Andrew Staehelin.

Preparation of Thylakoid Vesicles. Broken (class C) chloroplasts from peas (Pisum sativum) and tobacco (Nicotiana tobacum) were prepared according to the method of Avron (29). The chloroplasts were resuspended in a medium containing 0.4-M sucrose and 10-mM tris(hydroxymethyl)aminomethane (Tris pH 7.5), and stored at liquid nitrogen temperature in the same medium supplementated with 30% v/v of ethylene glycol (30). The chloroplasts (6-mg/mL chlorophyl) were heat inactivated for 3 min 51 °C and then diluted by 1 500 with a double-distilled water that was adjusted to pH 7.7 with Tris buffer. Large thylakoid vesicles were formed due to the swelling process under these hypotonic conditions. The size distribution of the thylakoid vesicles was determined as previously described (18). 

Envelope-free intact thylakoid vesicles were isolated from spinach. Membrane-free PSII -particles were prepared from spinach as in [3]-Fluorescence of a weak modulated measuring beam (< 1 pE m s" ) was measured with a PAM fluorimeter. Redox-potentials were measured in an 02 free chamber with a Pt-calomel-electrode (Ingold). 

The a- and -vesicle preparations were made by sonication of thylakoids followed by partitioning of the material in an... 

Cytochrome f determinations (Table 3.) show that the BS fraction has a substantial enrichment (about 302) compared to thylakoids. This support earlier results from measurements made in our laboratory where we find Cyt f somewhat enriched in the appressed region of the thylakoid membrane (5). For comparison an ordinary inside-out vesicle preparation, B3, was measured. As B3 shows an intermediate value for both Cyt f content (about 152 enrichment) and for PS II content (4) it is reasonable to conclude that PS II and the cytochrome b -f complexes are in close contact in the appressed membrane region. 

A/B phosphorylation by membrane vesicle preparations from maize chloropiasts is affected by the osmolarity of the medium (Fig, 6) just as had been reported for spinach preparations. Also, removal of CFi by NaCl-EDTA abolishes the A/B phosphorylating capacity but has little or no effect on osmotic responsiveness of maize thylakoid preparations (Fig. 6). Osmotic responsiveness is necessary, but not sufficient, for A/B phosphorylation. The other component of the system, as we have divided it, is CFi. 

Protein-lipid interaction in retinal-rod outer disc membranes in sonicated vesicles is suggested from comparison of the T1 data of these vesicles with those of extracted liposome preparations from the same source (Brown et al., 1976). Chloroplast thylakoids form micellar structures in chloroform and bilayer structures in water. It was shown by 13C relaxation (Johns et al., 1977) that T1 data are sensitive to this change in secondary structure. As in the... 

The evidence that water is split on the inner side of thylakoids is convincing early experiments by Fowler and Kok [33] and more recent ones [6] have shown that the protons generated by water splitting are detected inside the thylakoid lumen. Furthermore, it has been shown that the 24 and 18 kDa polypeptides are accessible to antibodies only in so-called inside-out preparations these polypeptides can be extracted in salt solutions from the inside-out vesicles, and subsequently rebound to them [34,35]. 

In recent years there have been many attempts to isolate PSII proteinaceous components containing Mn and possibly restoring the Oj evolving ability. It has been reported on proteins [162,163] partially able to restore O2 evolution in protein-depleted vesicles the Mn associated to these preparations was however insufficient to account for the Mn content correlated to O2 evolution in the native membranes. By washing inside-out thylakoids with 250 mM NaCl, Akerlund [164] has extracted a 23 kDa protein free of Mn but reconstituting O2 activity. Several proteinaceous components, not all associated with Mn, could be involved in the oxidizing side of PSII. 

The BS particles were prepared from inside out vesicles from spinage thylakoids that had been broken by a mechanical press treatment. These vesicles were separated further by partition with an aqueous polymer two-phase system (1). The vesicles thus obtained have a diameter of about 0. 2 pm and contain only trace amounts of PS 1 (P700). 

The bleb preparations for all three species consisted of single vesicles and did not contain grape-like structures as reported for other procedures (13/14). However/ a fraction of blebs displayed dark spots/ so-called patches/ which are considered as still stacked thylakoid membranes (13). ... 

To show the reliability of the procedure three independent preparations were made (data shown in table 1). The standard deviation was 0.01-0.07 for the chlorophyll a/b ratios and 0.4-1.4 for the yields. Note that the yield of BS is about six per cent of the thylakoids which is a two-fold increase compared to the earlier preparartion (4). The relatively low yield of inside-out vesicles, B3, is due to that it otherwise would be a very tedious preparation. Yield of B3 in the range of 30 to 40Z can easily be obtained but is time-consuming when large quantities are needed. The fact that the yield of BS is always about one third of B3 implies that the BS fraction represents a considerable large domain of the appressed part of the thylakoid membrane. 

The protein/chlorophyll ratio of these particles obtained from grana thylakoids is 4.6 +/- 0.4 and corresponds to that of vesicles from spinach [15]. With PSII-membrane preparations oiNicotiana tabacum, which have been obtained according to the method... 

The photosynthetic electron transport of cyanobacteria has a requirement for Ca2+ cations which is absent in eukaryotic plants. So far this requirement has been observed with cell-free thylakoid fragments only (Binder et al., 1976 Piccioni, Mauzerall, 1978 Brand, 1979 England, Evans, 1981). Such preparations consist, most likely, of mixed populations of sealed and unsealed vesicles, thus making difficult the decision on which side of the thylakoid membrane the Ca2+ cations really act. In the present work, we study the Ca2+ effects on thylakoids in situ, using ion permeable Anacystis nidulans cells prepared either by partial (permeaplasts) or by total (shperoplasts) hydrolysis of the cell wall peptidoglycan with lysozyme. 

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