Extraction of chloroplasts

The use of Triton X-100 to disperse Triticum chloroplast membranes has been reported to increase the recoverable yield of GAg by 1000 as compared with methanol extraction. The authors suggest that the methanol causes irreversible binding of GAS to the plastid membrane (72). However, the enhanced recovery using Triton X-100 has been disputed (73) and was not beneficial in the extraction of chloroplasts of Pisum. Now that physico-chemical procedures are available for many of the PGS, more attention should be directed at improving the extraction procedures for PGS. 

One peripheral polypeptide with MW = 33 kDa is isolated with the PS II/OEC core (Table 1). Two other extrinsic peptides with molecular masses of 17 and 23 kDa have been implicated in the 02-evolving process, although they may be replaced in the core preparations by high concentrations of Ca " and CE salts with preservation of 02-evolution activity. The involvement of these three polypeptides in O2 evolution was first suggested by the Tris extraction experiments on so-called inside-out chloroplast vesicles by Akerlund et al. [71] and by cholate extraction of chloroplasts by Sayre and Cheniae [72]. A flurry of activity ensued and the situation with respect to these polypeptides is now reasonably clear. The biochemical properties of these polypeptides are discussed in detail in two recent reviews [7,8]. 

In an attempt to characterize the membrane-bound iron-sulfur proteins, Malkin, Aparicio and Amon isolated the iron-sulfur protein by acetone extraction of chloroplasts which had previously been freed of soluble ferredoxin. They estimated the molecular mass of the protein to be 8 kDa. They also found that its absorption spectrum displayed no characteristic features. The EPR spectrum of the isolated protein was, however, quite different from that of the protein in the native, membrane-bound state. 

Phenylalanine permease and y-aminobutyrate permease topology is also lipid dependent and secondary transporters for proline, melibiose, tryptophan and lysine are also defective in uphill transport in E. coli cells lacking PE (Bogdanov and Dowhan, unpublished) suggesting that function and possibly topology of a broad spectrum of transporters is dependent on membrane lipid composition. The orientation of OEP7, an outer envelope protein of spinach chloroplasts, was inverted with respect to its native orientation when reconstituted in liposomes made of a total lipid extract of chloroplasts containing... 

Fig. 8. The motion of a spin label incorporated into the polar lipids of chloroplast membranes from N. oleander as a function of temperature. The polar lipids were prepared from a total lipid extract of chloroplast membranes by chromatographic separation of the neutral lipids on a silicic acid column. The lipids were suspended in 0.01 M Tris-acetate buffer, pH 7.2, containing 5 mm EDTA, and vesicles formed by brief sonication. The vesicles (1 mg lipid/O.I ml) were labeled with 12NS, and the motion is expressed as roan empirical motion parameter which approximates the time for the N- 0 band of the probe to rotate through 90°. The motion of the spin label increases i.e., to decreases, as the temperature increases. The temperatures of 53°, 49°, and 43°C correspond to the temperature at which fluorescence intensity increased (see Fig. 7) for plants grown at 45°/30°C (circles), 20715°C (squares), and 24 h after plants acclimated to 45°/32°C were shifted to 20°/15°C (triangles). The straight lines were fitted by linear regression and the value for to at 53°, 49°, and 43°C are 8.6, 8.8, and 8.4 0.05 x I0 s, respectively.

Several experiments have involved measurement of photosynthetic parameters during modification of lamellar lipids. Unfortunately the methods are, at present, extremely crude, which makes the interpretation of results rather difficult. Thus, experiments with solvent extraction of chloroplasts may reveal no apparent changes in the morphology of the organelle (Magree et al., 1966). At the same time, heptane-extracted chloroplasts show a lipid... 

The phase structure of total polar lipid extracts of chloroplast thylakoid membranes when dispersed in physiological salt solutions is characterised by non-lamellar arrangements of lipid C4D. This contrasts markedly with the general belief that the dominant form of the lipids in the photosynthetic membrane is in a bilayer configuration. The conclusion that must be drawn from this is that the interation of the different polar lipids with the other membrane components imposes a bilayer structure and prevents phase separation of non-bilayer forming lipids into separate domains within the membrane. 

Quinn, P.J. and Williams, W.P. (1983). The structural role of lipids in photosynthetic membranes, Biochim. Biophys. Acta, 737, 223-266. Gounaris, K., Sen, A., Brain, A.P.R., Quinn, P.J. and Williams, W.P. (1983a), The formation of non-bilayer structures in total polar lipid extracts of chloroplast membranes, Biochim. Biophys. Acta, 728, 129-139. 

Synthesis in a Cell-free Extract of Spinach Chloroplasts. 277... 

Fig. 8.8 Macrophage cytotoxic assays of plant extracts. Supernatant samples were tested from Trgeneration of chloroplast transgenic line pLD-JWl (proteins were extracted in buffer containing no detergent and MTTwas added after 5 hours). pLD-JWl (extract stored for 2 days) pLD-JWl (extract stored for 7 days) -> PA 5 pg ml-1 —x— Control wild type (extract stored for 2 days) ...

Fig. 8.11 (A and B) Expression and purification of insulin-polymer fusion protein detected in copper (A) and Coomassie (B) stained gels. The same gel was first stained with copper, destained and restained with Coomassie R-250. Lane 1 Prestained marker Lane 2 Purified extract of polymer-insulin fusion protein from the chloroplast vector pSBL-OC-40Pris Lane 3 Reverse orientation of fusion protein from pSBL-OC-40Pris Lane 4 Purified extract of polymer-insulin fusion protein from the chloroplast vector pLD-OC-40Pris ...

Hatanaka, A., T. Kajiwara and J. Sekiya. Biosynthesis of trans-2-hex-enal in chloroplasts from Thea sinensis. Phytochemistry 1996 15 1125. Matsura, T., T. Tsunoda and M. Arai. Theanine manufacture with tissue cultures of tea. Patent-Japan Kokai Tokkyo Koho-03 187,388 1991 7 pp. Takeo, C., H. Kinugass, H. Oosu, T. Kawasaki, N. Takakuwa, M. Shimizu and H. Kondo. Extraction of hyper-glycemics from tea. Patent-Japan Kokai Tokkyo Koho-04 124,139 1992 8 pp. Stalcup, A. M., K. H. Ekborg, M. P. Gasper and D. W. Armstron. Enantiomeric separation of chiral components reported to be in coffee, tea or... 

In 1955, R. A. Morton and associates in Liverpool announced the isolation of a quinone which they named ubiquinone for its ubiquitous occurrence.484 485 It was characterized as a derivative of benzoquinone attached to an unsaturated polyprenyl (isoprenoid) side chain (Fig. 15-24). In fact, there is a family of ubiquinones that from bacteria typically contains six prenyl units in its side chain, while most ubiquinones from mammalian mitochondria contain ten. Ubiquinone was also isolated by F. L. Crane and associates using isooctane extraction of mitochondria. These workers proposed that the new quinone, which they called coenzyme Q, might participate in electron transport. As is described in Chapter 18, this function has been fully established. Both the name ubiquinone and the abbreviation Q are in general use. A subscript indicates the number of prenyl units, e.g., Q10. Ubiquinones can be reversibly reduced to the hydro-quinone forms (Fig. 15-24), providing a basis for their function in electron transport within mitochondria and chloroplasts.486 490... 

Chloroplasts will be isolated by careful extraction of spinach leaves, using tricine buffer containing sucrose. The crude extract contains both whole and fragmented chloroplasts, but both contain all the necessary photosyn-thetic components and are capable of photophosphorylation. The preparation described in this experiment retains almost all of the chlorophyll in the chloroplasts. The total chlorophyll content of the chloroplasts will be determined by extracting the pigment with aqueous acetone and measuring the absorption at A. = 652 nm. The chlorophyll concentration is calculated according to Equation E9.3 (Arnon, 1949),... 

Moore, T.C., Coolbaugh, R.C. "Conversion of geranyl-geranyl pyrophosphate to ent-kaurene in enzyme extracts of sonicated chloroplasts." Phytochemistry, 1976, 15, 12kl 12k7. 

If the target sequence is present on the filter in a low concentration, either because it is a low copy number sequence or because a particular group of taxa yield a low concentration of chloroplast DNA in a total DNA extract, a prehybridization/hybridization buffer that contains dextran sulfate can greatly enhance the rate of hybridization (up to 100-fold44 when nick-translated probes are used) and permit detection of the target se-... 

Previously described methods for plant DNA extraction (e.g., see Hillis et al.16 and references therein) have usually either required CsCl purification or resulted in a final DNA product that often contained other contaminating substances. For restriction site analysis of chloroplast or mitochondrial genomes, where usually less than 0.5 fig of DNA per individual is required, any procedure was appropriate and practical. However, for minisatellite restriction enzyme analyses, typically 5-10 jtg of DNA is required per individual per trial. For such analyses, obtaining enough DNA from an individual for several trials using CsCl is costly and time-consuming ... 

Even before the fluorescence-quencher hypothesis was proposed, however, it had been suggested that plastoquinone may be an important electron carrier in green-plant photosynthesis. Bishops reported in 1959 the significant finding that extraction of PQ from chloroplasts results in the loss ofthe ability to evolve oxygen but that upon reconstitution with plastoquinone, oxygen-evolution activity is restored. 

Fig. 6. (A) Effect of composition of a binary soivent mixture on the apparent Chi a Aotal chlorophyli moiar ratio in extracts of spinach leaf tissue (0) and chloroplasts ( ). (B) relationship between the Chi a/Chl a molar ratio and the Chi a/P700 molar ratio for a number of P700-enriched subchloroplast particles by chloroform extraction (o) and by acetone extraction ( ). The solid line ciosest to the open circles is for Chi aVP700=1 and that nearest the filled circles for Chi aVP700= 2. See text for details. Figure source (A) Watanabe, Kobayashi, Maeda, Oba, Yoshida, Van de Meent and Amesz (1992) Function of the C13 -epimer chlorophylls in type I photosystem reaction centers. In N Murata (ed) Research In Photosynthesis, Vol III 4. Kluwer Acad PubI (B) Maeda, Watanabe, Kobayashi and Ikegami (1992) Presence of two chlorophyll a molecules at the core of photosystem I. Biochim Biophys Acta 1099 78.

With the authentic red tetrapyrrolic RCC (11) available as a reference material from the synthetic work (76), an identical red compound was detected in senescent plant material and was identified as an elusive red chlorophyll catabolite, when Pheo a (5a) was incubated with aerated extracts of washed membranes of senescent Canola chloroplasts (see Scheme 8) (68, 69). In addition, incubation of chemically pre-... 

To ensure the extraction of DNA from foods, mainly from highly processed products and the presence or absence of PCR inhibitors, many assays for food allergens include a universal eukaryotic primer pair (Brezna et al., 2006a, b Brezna and Kuchta, 2008) or plant-specific primer pair used to amplify the noncoding region of chloroplast (Rossi et al., 2006 Watanabe et al., 2007 Yamakawa et al., 2007 Yano et al., 2007). 

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