Spinach chloroplast isolation

Figure 9 Relationship between the thylakoid LpH, assayed by 9-aminoacridine fluorescence, and qE obtained by uncoupler titration of broken spinach chloroplasts isolated from dark adapted (closed symbols) or pre-illuminated leaves (open symbols).

During the 1960s, research on proteins containing iron—sulfur clusters was closely related to the field of photosynthesis. Whereas the first ferredoxin, a 2[4Fe-4S] protein, was obtained in 1962 from the nonphotosynthetic bacterium Clostridium pasteurianum (1), in the same year, a plant-type [2Fe-2S] ferredoxin was isolated from spinach chloroplasts (2). Despite the fact that members of this latter class of protein have been reported for eubacteria and even archaebacteria (for a review, see Ref. (3)), the name plant-type ferredoxin is often used to denote this family of iron—sulfur proteins. The two decades... 

Aman, R., A. Schieber, and R. Carle. 2005a. Effects of heating and illumination on trans-cis isomerization and degradation of (3-carotene and lutein in isolated spinach chloroplasts. J. Agric. Food Chem. 53 9512-9518. 

It will be noted that in isolated spinach chloroplasts, one hardly needs to worry about making an inhibitor too hydrophobic i.e. optimal log P = 5.2 for the N,N-dimethyl- and 5.4 for the methoxymethyl-ureas. In contrast to the isolated chloroplast studies, one sees from a list of commercially successful herbicides for which log P values have been measured or calculated, (Table IV) that getting the herbicide to the chloroplast in the living plant places much greater restrictions on its hydrophobic-hydrophilic balance. Indeed, the average log P of this set is only 2.54. 

Plant type ferredoxins. Tagawa and Arnon (16) described the isolation of a ferredoxin from spinach chloroplast. This ferredoxin is a protein of 12,000 molecular weight, and consists of 97 amino acids (17). Spinach ferredoxin has abosrbance maxima at 325, 420 and 465 nm (18). Ferredoxins of this type have been isolated from other sources of plants and algae, e.g., alfalfa (19), taro (20), Leuceana glauca (21) and Scenedesmus (22). The prot s of thes erredoxins are similar in their properties to ferredoxin from spinach. 

Electron rlVans for to a Hill Reagent Isolated spinach chloroplasts evolve 02 when illuminated in the presence of potassium ferricyanide (a Hill reagent), according to the equation... 

Rojas, I. S., Lotina-Hennsen, B., and Mata, R., 2000. Effect of lichen metabolites on thylakoid electron transport and photophosphorylation in isolated spinach chloroplasts. J. Nat. Prod 63, 1396-1399. 

Isolation of TPN-Reducing Factor" from spinach chloroplasts... 

In 1957, Arnon, Whatley, and Allen (10) reported isolation of a substance having the properties of a protein, which they obtained from spinach chloroplasts and designated the TPN-reducing factor . The TPN-reducing factor catalyzed photoreduction of TPN by chloroplast fragments. Their preparation catalyzed the photoreduction of TPN almost exclusively. 

Prior to 1961, all the factors that catalyzed the photoreduction of pyridine nucleotides had been isolated from either chloroplasts or leaves. The green plant was considered the only source of these proteins. However, in 1961 the association of these proteins with green plants ceased to be unique when Losada, Whatley, and Arnon (64) isolated a similar protein from a photosynthetic bacterium. This bacterial protein replaced the chloroplast protein in the photoreduction of TPN by illuminated spinach chloroplast fragments. 

Steup. M., Robenek, H., and Melkonian, M. 1983. In vitro degradation of starch granules isolated from spinach chloroplasts. Planta 158, 428-436. 

Mammalian pyruvate carboxylase has four identical subunits, and the isolated monomer will catalyze the complete reaction. By contrast, three distinct subunits can be isolated from acetyl CoA carboxylase of Escherichia coli and spinach chloroplasts a biotinyl carrier protein, biotin carboxylase, and carboxyl transferase. 

Aminoacid-tRNA synthetases were isolated from spinach chloroplasts by methods developed at Jealott s Hill. Cytoplasmic aaTRS s from carrot were isolated by standard procedures. Compounds were assayed as inhibitors of the chloroplastic aaTRS s at a single rate, 10 pM. The reaction was followed by phosphate generation and the percentage inhibition determined using literature methods. The IC50 value for the spinach chloroplastic enzymes and inhibition data for carrot cytoplasmic aaTRS s were determined for compounds of interest. All compounds were tested for post-emergence herbicidal activity, at 0.125, or at 0.5, or at 2.0 k a in standard screens." ... 

Much effort has been put into the isolation, purification and reconstitution of both photosystems. Work on PSII has been concerned especially with the nature of the dioxygen-evolving site, which is thought to be a manganese protein. ESR studies on spinach chloroplasts have led to the postulate of the involvement in oxygen evolution of a pair (or possibly a tetramer) of antiferromagnetically... 

B Andersson and JM Anderson (1980) Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim Biophys Acta 593 427-440 P-" Albertsson (1985) Partition of Cell Particles and Macromolecules (3rd edition) John Wiley H-E "kerlund, B Andersson and P-" Albertsson (1976) Isolation of photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition. Biochim Biophys Acta 449 525-535 P Grber, A Zickler and H-E "kerlund (1978) Electric evidence for the isolation of inside-out vesicles from spinach chloroplasts. FEES Lett 96 233-237... 

NK Boardman and JM Anderson (1964) Isolation from spinach chloroplasts of particles containing different proportions of chlorophyll a and chlorophyll b and their possible role In the light reactions of photosynthesis. Nature 203 166-167... 

Fig. 9. Room-temperature absorption spectrum of spinach Cytbe in the presence of dithionite (A) reduced-minus-oxidized difference spectra as indicated for spinach Cyt bg compiex measured at room temperature in (A) inset and at low temperature (77 K) in (B) (C) 77 K difference spectra of the isolated (spinach) Cyt bef complex titrated to the potentials indicated. See text for details. Figure source (A) and (B) Hurt and Hauska (1981) A cytochrome flb complex of five polypeptides with plastoquinol-plastocyanin-oxidoreductase activity from spinach chloroplasts. Eur J Biochem 117 594 (C) Hurt and Hauska (1983) Cytochrome /) from isolated cytochrome complexes. Evidence for two spectral forms with different midpoint potentials. FEBS Lett 153 415.

Oxidant-induced reduction of cytochrome b The oxidant-induced reduction of Cyt b means the reduction ofCyt-Z>6 linked to the oxidation ofquinol is mediated by the Rieske iron-sulfur protein. This reaction has long been documented for the mitochondrial and photosynthetic-bacterial Cyt-icj complex. The same reaction is expected to occur in the Cyt-f)6/complex of higher plants and cyanobacteria. Oxidant-induced reduction of Cyt b was illustrated in the early reaction steps in Fig. 11 (C) above. We describe here, with the help of Fig. 12, the work of Hurt and Hauska detailing the spectrophotometric evidence for the reaction steps in the Cyt-b(f complex isolated from spinach chloroplasts. 

The electron-microscopic technique was subsequently used by Boekema, Berden and van Heel " in 1988 to study theCF, complex of ATP synthase isolated from spinach chloroplasts. 

H Baltscheffsky (1963) Inhibition of photophosphorylation in isolated spinach chloroplasts by lower aliphatic straight-chain alcohols. Acta Chem Scand 17 (supp.1) 308-312... 

Aguilar, M. L Romero, M. G. Chavez, M. L King-Diaz, B. Lotina-Hermsen, B., Biflavonoids isolated from Selaginella lepidophylla inhibit photosynthesis in spinach chloroplasts, J. Agric. Food Chem., 2008, 56, 6994-7000. 

---