Thylakoid vesicles

The quantum yield of photosynthesis, the amount of product formed per equivalent of light input, has traditionally been expressed as the ratio of COg fixed or Og evolved per quantum absorbed. At each reaction center, one photon or quantum yields one electron. Interestingly, an overall stoichiometry of one translocated into the thylakoid vesicle for each photon has also been observed. Two photons per center would allow a pair of electrons to flow from HgO to NADP (Figure 22.12), resulting in the formation of 1 NADPH and Og. If one ATP were formed for every 3 H translocated during photosynthetic electron transport, 1 ATP would be synthesized. More appropriately, 4 hv per center (8 quanta total) would drive the evolution of 1 Og, the reduction of 2 NADP, and the phosphorylation of 2 ATP. 

Light-Induced Mg" Efflux from Thylakoid Vesicles... 

Membranes (high potential) thylakoid vesicles Cytosol... 

In most instances, either for mitochondrial suspensions or whole bacteria, ApH is less negative than -0.5 unit making a contribution of, at most, -30 mV to Ap. The exception is found in the thylakoid membranes of chloroplasts (Chapter 23) in which protons are pumped into the thylakoid vesicles and in which the internal pH falls dramatically upon illumination of the chloroplasts.185 The ApH reaches a value of -3.0 or more units and Ap is 180 mV, while Em remains 0. Reported values of Em for mitochondria and bacteria range from -100 to -168 mV and Ap from -140 to -230 mV.172 179 Wilson concluded that Em for actively respiring mitochondria, using malate or glutamate as substrates,... 

Chloroplasts in plant cells are surrounded by a double membrane and have an internal membrane system of thylakoid vesicles that are stacked up to form grana. The thylakoid vesicles contain chlorophyll and are the site of photosynthesis. Carbon dioxide (C02) fixation takes place in the stroma, the soluble matter around the thylakoid vesicles. 

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. 

At least two sites can be identified by chemiosmotic principles [12] that is, sites at which transthylakoid proton movement is coupled to electron transport. One is in the reduction of the non-heme iron protein by plastoquinone (i.e. between plastoquinone and Cyt f, in agreement with the former technique), and a second at the water oxidation reaction. Since water oxidation has been shown to occur on the inside of the thylakoid vesicles, each water molecule oxidized leaves two protons intravesicularly, resulting, by chemiosmotic principles, in the creation of a high-energy state. Two coupling sites should result in a maximal H /c2 of 4, in agreement with the above discussed conclusions from ATP/e2 measurements. 

Our present understanding of the phosphorylation reaction has been greatly aided by the ability to study several partial reactions of the overall process, and follow them during fractionation of the intact thylakoid vesicle. 

Thylakoid vesicles as normally isolated possess little or no ATPase activity, despite their ability to catalyse vigorous photophosphorylation. Several treatments elicit an ATPase activity which is catalysed by the membrane-bound ATP synthase. Such treatments involve both the imposition of a transmembrane proton electrochemical gradient (A/ah+) and the reduction of a disulfide group on the y subunit of the enzyme [29,30]. 

Fl-E "kerlund (1981) Partial reconstitution of photosynthetic water splitting in inside-out thylakoid vesicles. In G Akoyunoglou (ed) Proc Intern Congr on Photosynthesis, Vol. 2 465-468... 

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. ... 

The lateral mobility of proteins and lipids in natural and artificial lipid bilayer membranes was determined by different methods. For long-range mobility, fluorescence recovery after photobleaching (13-15) and electrophoresis of membrane components (16) were employed. We employed the electrophoresis method for determination of the eletrophoretic and diffusional mobilities of PSI in the plane of hypotonically inflated, spherical thylakoid vesicles. To monitor the redistribution of PSI particles, we made use of the spatial characteristics of the contribution of PSI particles to electrophotoluminescence (EPL) (17, 18). The contribution of PSII to EPL was eliminated by heat treatment of the chloroplasts (19). The EPL originates from the PSI particles at the hemisphere of the vesicles at which the induced electrical field destabilizes the photoinduced charge separation (18). The electrophoretic and diffusional mobilities were measured in vesicular suspensions to avoid immobilization for microscopic visualization (20). The photosynthetic membranes are devoid of cytoskeletal elements that might interfere with the lateral mobility. 

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). 

INTERACTION BETWEEN LHC-II ANTENNA AND PS 2 CORE IN THYLAKOID VESICLES. 

Figure 2 Levels of Dl-protein ( ) and 33 KDa ( ) extrinsic protein in illuminated inside-out thylakoid vesicles.

In another experiment, inside-out vesicles were first isolated and then exposed to strong light. The result revealed a concomitant release of the 33, 23 and 16 kDa proteins from the exposed inner surface of the everted thylakoid vesicles which correlated closely with the Dl-protein degradation (Fig.2). These results show that the extrinsic proteins are released into the thylakoid lumen following photoinhibition and that there is an association between the Dl-protein and the 33, 23 and 16 kDa proteins. Moreover, the observec Dl-protein disappearance in inside-out vesicles shows that the degradation system is present in the appressed thylakoid region. 

PSII exists in at least two subpopulations, of which only B-PSII takes part in the whole-chain electron transfer. Non-B-PSII cannot reduce plastoquinone but can donate electrons to certain quinone acceptors (1). In fact, PPBQ, a common quinone acceptor, is often used to measure the PSII activity of isolated stroma thylakoid vesicles which are known to contain only non-B-PSII. Non-B-PSII is insensitive to photoinhibition vitro (2), and it has been suggested that non-B-PSII is used to replace damaged PSII-centers (3). These properties of non-B-PSII can lead to severe under- or overestimations of photoinhibition vivo, if electron transfer to quinone acceptors is used as a criterion. The same is true for fluorescence due to their capability to primary photochemistry, the non-B-PSII s probably have high Fy/F j. 

Phosphorylation of spinach thylakoid proteins was performed at 0 C. Stroma thylakoid vesicles were isolated directly after the phosphorylation was terminated or after incubation of the sample at a higher temperature for a certain time period. 

Isolation of inside-out thylakoid vesicles (B3) from spinach and their subfractionation by sonication and phase partition was done essentially as previously described (2,3). The fractions named 180s, 540s and BS according to the nomenclature described in (2,3) were obtained. All these fractions derive from the B3 fraction, and hence from the grana partitions, and have a high PSII activity (2,3). Their chlorophyll a/b ratios are given in Table 1. 

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