Chloroplasts Subject

It is generally accepted that photosynthetic water oxidation involves five steps, termed the S states, each of these holds one of the oxidizing equivalents found in the OEC. This notion stems from the observation [127] that dark-adapted spinach chloroplasts subjected to short flashes of light exhibited a consistent pat-... 

In the total leaf homogenate of the Vigna leaves subjected to a gradual heat shock treatment, in addition to three high molecular mass HSPs (96,80 and 70 kDa) detectable in the rapid heat-shocked leaves, a 60-kDa HSP was detected as a distinct band in the fluorographic profile (Fig.4). Furthermore, two new HSPs in the size of 85 and 70 kDa were also detectable as a faint band. These three HSPs (85,70 and 60 kDa) detectable in the total leaf homogenate of the leaves heat shocked gradually are comparable to the HSPs detectable in the chloroplasts subjected to heat shock in vitro. 

The effect of iodosobenzoate is reversible (Vallejos, Andreo, 1976). Apparently the situation is similar in light-chloroplasts where two accessible sulfhydryl groups may be blocked by M-ethylmaleimide (Vallejos et al , 1983). However, the proton ATPase is active in this Ccise but not in broken chloroplasts subject to illumination. Thus, activation in vivo of the proton ATPase may involve reduction of the second disulfide bond of the y subunit or may required other changes in the complex. 

A decade after the discovery of the Rieske protein in mitochondria (90), a similar FeS protein was identified in spinach chloroplasts (91) on the basis of its unique EPR spectrum and its unusually high reduction potential. In 1981, the Rieske protein was shown to be present in purified cytochrome Sg/complex from spinach (92) and cyanobacteria (93). In addition to the discovery in oxygenic photosynthesis, Rieske centers have been detected in both single-RC photosynthetic systems [2] (e.g., R. sphaeroides (94), Chloroflexus (95)) and [1] (Chlo-robium limicola (96, 97), H. chlorum (98)). They form the subject of a review in this volume. 

For studies of membrane composition, the first task is to isolate a selected membrane. When eukaryotic cells are subjected to mechanical shear, their plasma membranes are torn and fragmented, releasing cytoplasmic components and membrane-bounded organelles such as mitochondria, chloroplasts, lysosomes, and nuclei. Plasma membrane fragments and intact organelles can be isolated by centrifugal techniques described in Chapter 1 (see Fig. 1-8). 

Metribuzin is a member of the substituted as-triazinone group of herbicides. Activity is due to interference with photosystem II electron transport in plant chloroplasts (Dodge, 1983). The metabolism of metribuzin in plants has been the subject of many short-term and long-term studies dating back to the early 1970s. 

The plastocyanins are blue copper proteins found in the chloroplasts of higher plants and algae where they mediate electron transport between cytochrome f and P-700 (Barber, 1983 Haehnel, 1984, 1986 Cramer etal., 1985 Sykes, 1985 Andersen et al., 1987). Plastocyanins each contain one copper bound by a single polypeptide chain of molecular weight around 10500 (Sykes, 1985). The spectroscopic properties of the copper are those of a typical blue site. The properties of the plastocyanins have been the subject of detailed reviews (Sykes, 1985 Haehnel, 1986 Chapman, 1991). 

Leaf discs have commonly been used for bioassays to determine if herbicides inhibit photosynthesis (Table 16.2). The simplest leaf-disc bioassay uses small discs cut from fully expanded cucumber or pumpkin cotyledons, floated in the light on a phosphate buffered medium containing suspected photosynthesis inhibitors.115 Qualitatively, if photosynthesis is inhibited, the leaf disc sinks. There are several variations of this method that can provide quantitative data. Evolution of O2 in the test solution can be measured with an oxygen electrode, CO2 induced pH changes colorimetrically determined with bromothymol-blue, or electrolyte leakage monitored with a conductivity meter. Leaf strips, algae, isolated chloroplasts, and duckweed (Lemna minor) have been used as test subjects. Although the bioassays presented in Table 16.2 are fairly easy to perform, few allelochemicals have been tested as possible inhibitors of photosynthesis. Many... 

Based on current knowledge, it seems likely that whatever form the final hypothesis may take, it will center around what happens when the formation of ATP or NADPH, or both, is inhibited after interference with the photochemical reactions of the chloroplasts. Hopefully, the postulates will serve as models that can be subjected to rigorous and sophisticated experimentation, and will be modified as our knowledge of biochemical control systems in higher plants increases. 

The normal one-electron reduction of occurs with a midpoint potential lower than 0 mV but the actual value is still a subject of some controversy (see Section 3.3.3 below). The value often cited by those not wishing to get bogged down in that controversy is that obtained by measuring the redox potential dependence of Cyt b-559 photooxidation at 77 K in chloroplasts [106]. A value was obtained which was pH-dependent at pH values below pH 8.6. The value at and above pH 8.6 (the p of Qa ) was -130 mV. This value is usually considered the operative E, since Qa is not protonated on a functional time scale. This assumption was also made earlier for the E of Qa/Qa in purple bacteria [107]. Arguments for and against the use of the E -pK are discussed in detail in a recent review [108]. 

Another mechanism of light-dependent enzyme activation has been proposed in which a membrane-bound dithiol-containing factor (light-effect mediator or LEM) reduced by the photosynthetic electron transport system reductively activates regulated enzymes in the chloroplast [28]. Certain facets of this mechanism may be identical to the ferredoxin/thioredoxin system while other aspects are still the subject of debate [18,33],... 

In addition to an external cell membrane (called the plasma membrane), eukaryotic cells also contain internal membranes that form the boundaries of organelles such as mitochondria, chloroplasts, peroxisomes, and lysosomes. Functional specialization in the course of evolution has been closely linked to the formation of such compartments. Specific systems have evolved to allow targeting of selected proteins into or through particular internal membranes and, hence, into specific organelles. External and internal membranes have essential features in common, and these essential features are the subject of this chapter. 

Regardless of how mitochondria and chloroplasts evolved, most of their oligomeric membrane protein complexes are now synthesized partly by cytoplasmic and partly by organelle ribosomes. This complicates the biogenesis of the organelles, but also makes it an exciting subject to study [13-15]. 

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.

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