Chromatophores isolation

NAD" photoreduction in chromatophores isolated from several purple non-sulfur bacteria [41,48-50] and from the purple sulfur bacterium Chromatium vinosum [51] but did not inhibit ATP-driven NAD reduction in the dark. 

FIGURE 32.1 The morphological response of chromatophores isolated from B. splendens, the Siamese fighting fish, upon application of a biologically active agent, (a) Control cells, (b) cells treated with a chemical agent. 

Kinase activity from the cytoplasmic fraction of R. rubrum is observed with chromatophores, isolated B875 complexes as well as histone V-S (from calf th3nnus) as substrates as demonstrated in fig. 4. 

Rhodobacter capsulatus cells lacking the B800/850 antenna complex (strain U43 with episomal expression of the reaction center and B875 antenna complex) and Rhodospirillwn rubrum cells (strain SI) were grown to late log phase anaerobically in the light at 30 C and harvested. Cells were either broken using a French press and chromatophores isolated as described by Woodbury et al. (8), or cells were ruptured by grinding in alumina and chromatophores isolated as describe by Jackson and Crofts (9). 

Fig 2. Sub-saturating photobleaching spectra of purified chromatophores isolated from A) Wild type cells, B) FM210 cells and C) LM210 cells. All three were grown semi-aerobically. The chromatophores were suspended in 50 mM MOPS, 100 mM KCl containing 9 iM valinomycin and 1 mM ascorbate. 

N,N,N, N -tetramethyl-1,4-phenyldiamine dihydrochloride (Hellingwerf et al., 1975). Membrane vesicles and chromatophores isolated from cells grown cin-aerobically in the light contain a functional cyclic electron transfer system (Michels, Konings, 1978). Upon illumination of these membrane preparations a proton motive force is formed and energy is supplied for solute transport and ATP-synthesis (see below). 

Hatefi et al. 220) have isolated the succinate dehydrogenase of Rhodospirillum ruhrum by extraction of chromatophores with NaC104. The enzyme has two subnits of molecular weights of approximately... 

Leu-amide) isolated from cockroach heads (16). MRCH/PBAN peptides do not resemble any of the crustacean chromatophorotropins, and the former peptides may not be active on crustacean chromatophores and eye pigment cells. It remains unknown whether MRCH influences rapid color changes brought about by intracellular pigment migration in insects. 

In chromatophores, the of Qa decreases by 59 mV/pH unit as the pH is raised, up to an apparent p A that is between 7.8 and 9.8, depending on the species [16,30,35,36]. The pA A probably reflects the binding of a proton to a group other than the quinone itself, because the absorption spectrum and EPR spectrum of Qa match those expected for an anionic semiquinone [31,37-40]. The EN-DQR spectrum of Qa suggests that the quinone is hydrogen-bonded to a histidine residue of the protein [41]. The E value of about -0.18 V measured above the p/ A may be the most relevant value when Qa is photoreduced, because Qa probably transfers an electron to Qg before proton uptake occurs. In isolated reaction centers of Rb. sphaeroides, the E j of Qg is about 0.07 V more positive than that of Qa [29,34,42- 4]. The difference between the two E values appears... 

Fig. 1. Kinetics and standard free energy changes of electron transfer steps in reaction centers isolated from Rb. sphaeroides. In the chromatophore membrane, a c-type cytochrome (Cyt c,) normally reduces before an electron moves from Qa to Qg. The cytochrome oxidation has a time constant of about 20 fis in Rb. sphaeroides. and 0.5 to 2 p in reaction centers of Rp. viridis and Ch. vinosum, which have bound cytochromes. When the reaction center is excited a second time, Ob" is reduced to...

The free energy gap between P and P C can be calculated from measurements of the fluorescence that occurs during the lifetime of the radical-pair in reaction centers that have electron transfer to blocked by the reduction or extraction of the quinone [65,78-81]. The fluorescence emitted by P at any given time is a measure of the amount of the excited singlet state that is in equilibrium with the radical-pair. By this measure, the earliest form of P I that can be resolved lies about 0.17 eV below P in free energy, both in chromatophores and in isolated reaction centers (Fig. 1). The amplitude of the fluorescence decays in several steps, possibly because of nuclear relaxations in the radical-pair. 

In purple photosynthetic bacteria, and specifically in Rps. sphaeroides and Rps. capsulata, three cytochromes of b type have been identified by means of redox titration, in the dark, of isolated chromatophores [116]. They are characterized by midpoint potentials at pH = 7.0 equal to 0.155, 0.050 and -0.090 V (in Rps. sphaeroides)-, the of the 0.050 V species is pH dependent ( — 60 mV per pH unit) [116,117]. The presence of a cytochrome cc in these organisms, interfering spectrally with cytochrome b, makes the situation unclear as far as the existence of cyt. b E j = 0.155 V) is concerned [118]. The two other cytochromes E = 0.050 and — 0.090 V) have also been resolved kinetically in studies on the photosynthetic electron transport and on the basis of their spectral characteristics (band at 561 nm and a spht bands at 558 and 556 nm, respectively these two cytochromes will be referred to as 6-561 and 6-566 in the following) [119]. 

The g values of the ESR spectral lines (g = 2.02, 1.89 and 1.81 in mitochondria) have been ascribed to an FejSj (S Cys)4 center, i.e., containing two tetracoordinated iron atoms bridged by two sulphurs [126]. Similar spectra can be observed in chromatophores or in the isolated complex. The high-potential Fe-S protein appears to be the carrier involved in the binding of specific inhibitors of the oxidoreductase, such as UHDBT and DBMIB, which are structurally analogues of ubiquinone [127]. 

As stated on several occasions in the previous sections, electrons are delivered to bacterial and PSI-RC by electron carriers which can be isolated as water soluble homogeneous proteins, cytochromes of c type or plastocyanine. These carriers represent also the physiological electron acceptors for the 6/Cj complexes. It has been conceived, therefore, that these proteins can act as diffusable redox mediators between the different complexes, which in turn are thought to be laterally and independently mobile in the membrane lipid bilayer [219]. The location of these carriers would be the interface on one side of the asymmetrically arranged coupling membrane, namely towards the periplasmic space in bacteria (corresponding to the internal volume of chromatophores) or the inner lumen of thylakoids. 

The latter process was shown to require ATP, but the source of this ATP was unclear and a matter of considerable dispute. The breakthrough came in 1954 when Arnon and his colleagues demonstrated light-induced ATP synthesis in isolated chloroplasts. The same year Frenkel described photophosphorylation in cell-free preparations from bacteria. Photophosphorylation in both chloroplasts and bacteria was found to be associated with membranes, in the former case with the thylakoid membrane and in the latter with structures derived from the plasma membrane, called chromatophores. In the following years work in a number of laboratories, including those of Arnon, Avron, Chance, Duysens, Hill, Jagendorf, Kamen, Kok, San Pietro, Trebst, Witt and others, resulted in the identification and characterization of various catalytic components of photosynthetic electron transport. Chloroplasts and bacteria were also shown to contain ATPases similar to the F,-ATPase of mitochondria. 

Since the bacteriochlorophyll present in the light-harvesting complex accounts for the majority of all the bacterial pigments, its absorption bands can readily be identified even in the spectrum of the unfractionated membrane. On the other hand, the pigments belonging to the reaction center amount to only "1% of the total BChl and its absorption is often masked by the bulk pigments. The BChl a present in the reaction center may be identified however in a purified reaction-center preparation isolated from the chromatophore membrane. This may be illustrated with Chromatium vinosum following fractionation and isolation of the reaction-center complex and the three antenna complexes from the chromatophore membrane. Fig. 2 (B) shows the absorption spectrum of the unfractionated Chromatium chro-... 

Based on the nature of the cytochromes, there are two kinds of photosynthetic bacterial reaction centers. The first kind, represented by that of Rhodobacter sphaeroides, has no tightly bound cytochromes. For these reaction centers, as shown schematically in Fig. 2, left, the soluble cytochrome C2 serves as the secondary electron donor to the reaction center the RC also accepts electrons from the cytochrome bc complex by way ofCytc2- The rate of electron transfer from cytochrome to the reaction center is sensitive to the ionic strength of the medium. Functionally, cytochrome C2 is positioned in a cyclic electron-transport loop. In Rb. sphaeroides, Rs. rubrum and Rp. capsulata cells, the two molecules of cytochromes C2 per RC are located in the periplasmic space between the cell wall and the cell membrane. When chromatophores are isolated from the cell the otherwise soluble cytochrome C2 become trapped and held by electrostatic forces to the membrane surface at the interface with the inner aqueous phase. These cytochromes electrostatically bound to the membrane can donate electrons to the photooxidized P870 in tens of microseconds at ambient temperatures, but are unable to transfer electrons to P870 at low temperatures. 

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