Chloroplasts thylakoids

Steponkus, P.L., Garber, M.P., Myers, S.P., Lineberger, R.D. (1977). Effects ofcold acclimation and freezing on structure and function of chloroplast thylakoids, Cryobiol. 14, 303-321. 

Cohen, Y., S. Yalovsky, and R. Nechushtai. 1995. Integration and assembly of photosynthetic protein complexes in chloroplast thylakoid membranes. Biochim. Biophys. Acta 1241 1-30. 

The reader may consult Sanchez-Ferrer et al. (58), Nunez-Delicado et al. (59), and Sajo et al. (29) for updates concerning the use of Triton surfactants to solubilize membrane-bound polyphenol oxidase. In addition, Kieselbach et al. (60) offers a contemporary view regarding the isolation and characterization of chloroplast thylakoids. Wilhelnova (61) compared the abilities of different detergents to fragment thylakoid membranes. 

G. F. W. Searle, J. Barber, and J. D. Mills, 9-Amino-acridine as a probe of the electrical double layer associated with the chloroplast thylakoid membranes, Biochim. Biophys. Acta 461, 413-425 (1977). 

F0Fi ATPase/ATP synthase of mitochondrial inner 19-58 membrane, chloroplast thylakoid, and bacterial plasma membrane... 

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... 

Our investigations into the structure and organisation of the chloroplast thylakoid membrane have involved the following approaches ... 

The association of cytochrome P450s with organelles other than the ER may have important implications for the biosynthesis of alkaloids. It may help to explain why biochemical localization studies have shown that certain reactions in a pathway occur within the chloroplast. In the case of Catharanthus alkaloids, the 3 rd to last step in vindoline biosynthesis involves a chloroplast thylakoid associated N-methyltransferase.22 The arguments for participation of chloroplasts in this reaction include the possibility that the previous step involving hydration of the 2,3 double bond might require a chloroplast based oxidation reaction (Fig 8.6). The conclusive identification of specific cytochrome P4S0 enzymes in chloroplasts suggests that this hypothesis should be tested. 

The electrical term in the chemical potential of H+ can also power ATP formation. For instance, when an EM of 0.16 V is artificially created across lamellar membranes, ATP formation can be induced in the dark. This is consistent with our prediction that an electrical potential difference of at least 0.13 V is necessary (Fig. 6-6). In chloroplast thylakoids, EM in the light is fairly low, e.g., near 0.02 V in the steady state (see Fig. 6-5). However, the electrical term can be the main contributor to A/xh for the first 1 or 2 seconds after chloroplasts are exposed to a high photosynthetic photon flux (PPF). The electrical component of the H+ chemical potential difference can be large for the chromatophores of certain photosynthetic bacteria such as Rhodopseudomonas spheroides, for which Em can be 0.20 V in the light in the steady state. 

Proton relaxation studies on chloroplast membranes have been presented. (791) Chloroplasts are the sites for the photosynthetic process, many details of which are still unknown. Manganese is known to be essential for oxygen evolution and thus it is important to be able to monitor manganese in its bound state. Water proton Ti values in chloroplast thylakoid membrane suspensions were used to monitor membrane-bound manganese. The results indicate that there may be a mixture of manganese oxidation states in the dark-adapted chloroplasts, with Mn(ii) and Mn(iii) being the most likely states. 

DSC has been used to study the individual protein components of biological membranes of relatively simply protein composition and the interaction of several of these components with lipids and with other proteins. The red blood cell membrane, which has been most intensively studied, exhibits five discrete protein transitions, each of which has been assigned to a specific membrane protein. The response of each of these thermal transitions to variations in temperature and pH as well as to treatment with proteases, phospholipases, specific labelling reagents, and modifiers and inhibitors of selected membrane activities, has provided much useful information on the interactions and functions of these components in the intact erythrocyte membrane (46-49). Similar approaches have been applied to the bovine rod outer segment membrane (50) and to the spinach chloroplast thylakoid membrane (51). 

In order to maintain a A/1h+ across a membrane, and to ensure that it is used for the synthesis of ATP and not dissipated by leakage, the membrane must be closed and not leaky to protons. From the rate at which a pH gradient across the membrane decayed, it was shown that the effective proton conductance of the mitochondrial inner membrane [8], bacterial plasma membrane [9], and chloroplast thylakoid membrane [10] have a value of only some 0.5 jttS2/cm, or a million-fold less than the aqueous phases on either side. 

The respiratory and photosynthetic electron-transfer pathways are proton pumps operating with the same polarity as does the A TP synthase when hydrolyzing A TP Since it is difficult to detect protons circulating in the steady-state, Mitchell and Moyle [19] studied the transient extrusion of protons when a small amount of oxygen is injected into an anaerobic incubation of mitochondria in the presence of substrate. Prior to this, Neumann and Jagendorf [20] had observed a light-dependent proton uptake into chloroplast thylakoid membranes. 

Generation of Superoxide Radicals and Hydrogen Peroxide in Chloroplasts. Isolated, illuminated chloroplast thylakoids slowly take up oxygen in the absence of added electron acceptors. This phenomenon was first observed by Mehler (7) and is often known as the "Mehler reaction. The reaction appears to result from the reduction of O2 to the... 

Spontaneous dismutation is thus most rapid at the acidic pH values needed to protonate 02% but the rate at neutral or alkaline pH values is greatly accelerated by the presence in chloroplasts of a superoxide dis-mutase enzyme, which catalyzes Reaction 4. Superoxide dismutase in the form of a copper-zinc enzyme is found both free in the stroma and bound to the outside of the chloroplast thylakoids (24-29). 

Photophosphorylation due to electron flow (blue) and proton flow (red) in chloroplast thylakoid membranes... 

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.

W Ortiz, E Lam, S Chollar, D Munt and R Malkin (1985) Topography of the protein complexes of the chloroplast thylakoid membrane. Plant Physiol 77 389-397... 

The most recent measurement was carried out by Setif and Mathis , who looked particularly for the probable cause for the variance in the redox-potential values reported in the literature. These authors used chloroplast thylakoid lamellae and simpler complexes fractionated by treatment with digitonin, Triton and other detergents or combinations thereof. A CPI complex i.e., one in which all iron-sulfur clusters were removed) prepared from tobacco by SDS fractionation was also used in their study. 

Fig. 2. (A) A model for b/in chloroplast thylakoid membrane (B) Topological arrangement of the four subunits of the purified Cyt b/complex (C) A densitometric scan of an SDS-PAGE gel for b/ (B) from Hauska, Schiitz and Biittner (1996) The cytochrome b/ complex - composition, structure and function. In DR Ort and CF Yocum (eds) Oxygenic Photosynthesis - The Light Reactions, p 384. Kluwer (C) from Black, Widger and Cramer (1987) Large-scale purification of active cytochrome b f complex from spinach chloroplasts. Arch Biochem Biophys 252 657.

The chloroplast thylakoids were first incubated in apH=5 medium and then rapidly changed to a basic medium with pH fixed at 8.2. The acid incubation period was restricted to 30 sec to minimize inactivation of the ATP synthase activity. AT values were maintained at 5,44 or 60 mV. At ApH=3.2 and each given AT, a linear increase in the ATP yield was observed up to a reaction time of 200 ms. The authors thus concluded that within this time span both ApH and AT remained practically constant. The slopes of the three curves, representing the rates of ATP synthesis at ApH=3.2, are 120,260 and 380 mmol ATP/mol Chi for AT equal to 5,44 and 60 mV, respectively. The rate of ATP synthesis gradually decreased after the initial linear portion and eventually approached zero, as ApH and AT decayed to approach threshold values completely. The three separate plots clearly show that the eventual ATP yield as well as the initial ATP rate increases with increasing amplitude of the diffusion potential AT. 

Fig. 15. Conceptual development of a membane vesicle subjected to voltage pulses to create a potential difference across the membrane. (A) A1 pm-dlameter sphere of water is Imagined placed between two platinum electrodes 1 mm apart (B) The water sphere is replaced by a sphere of lipid (C) The Interior of the lipid sphere is replaced by a sphere of water, resulting in a lipid shell surrounding an aqueous medium to form the equivalent of a membrane vesicle. See text for details. (D) A schematic representation of a chloroplast thylakoid membrane containing ATP synthase to be subjected to voltage pulses and then the amount of ATP formed determined. Plots of actually measured ATP formation by voltage pulses (E) or light pulses (F) as a function of the number of pulses. (A), (B), (C), (E) and (F) from Witt (1987) Examples for the cooperation of photons, excitons, electrons, electric fields and protons in the photosynthesis membrane. Nouveau Journal deChimie 11 97 (D) adapted from Bauermeister, Schlodderand Graber(1988) Electric field-driven ATP synthesis catalyzed by the membrane-bound ATP-synthase from chloroplasts. Ber Bunsenges Phys Chem 92 1037.

In Fig. 15 (D), the imaginary membrane vesicle is replaced either by an actual chloroplast thylakoid-membrane vesicle containing ATP synthase, as shown in the example of Bauermeister, Schloddder and Graber" or by a liposome reconstituted with isolated CFo Fi-ATP synthase. In the actual experiment, two platinum electrodes each 5 cm in area were spaced 2 mm apart and filled with a chloroplast suspension. The cuvette containing the chloroplast suspension and the electrodes was then thermostated at 4 °C and kept from light. 

In chloroplast thylakoid, the FIAC at 515 nm has been shown by Schliephake, Junge and Witt to be comprised of approximately equal contributions from photosytems 1 and 11. When electron carriers and inhibitors were used to isolate the two photosystems, it could be shown that the amplitude of the FIAC produced by either ofthe two photosystems was approximately half ofthe total amplitude of that produced by both photosystems together, as shown in Fig. 20 (B). Fig. 20 (C) shows the linear relationship between the number of ATP molecules synthesized per flash per electron-transport (e t) chain and the amplitude of the FIAC change at 515 nm (bottom scale), or the corresponding change in electric potential (top scale). 

A EXPERIMENTAL FIGURE 8-23 Synthesis of ATP by FqFi depends on a pH gradient across the membrane. Isolated chloroplast thylakoid vesicles containing FqFi particles were equilibrated in the dark with a buffered solution at pH 4.0. When the pH in the thylakoid lumen became 4.0, the vesicles were rapidly mixed with a solution at pH 8.0 containing ADP and P. ... 

Unlike mitochondria, chloroplasts contain a third membrane—the thylakoid membrane—on which photosynthesis occurs. The chloroplast thylakoid membrane is believed to constitute a single sheet that forms numerous small, interconnected flattened vesicles, the thylakoids, which commonly are arranged in stacks termed grana (see Figure... 

---