Nucleotide sequence of the Acinetobacter calcoaceticus trpGDC gene cluster

A plasmid library of Acinetobacter calcoaceticus HindIII fragments was constructed, and clones that complemented an Escherichia coli pabA mutant were selected. Plasmids containing a 3.9-kb fragment of A. calcoaceticus DNA that also complemented E. coli trpD and trpC-(trpF+) mutants were obtained. We infer that complementation of E. coli pabA mutants was the result of the expression of the amphibolic anthranilate- synthase/p-aminobenzoate-synthase glutamine-amidotransferase gene and that the plasmid insert carried the entire trpGDC gene cluster. In E. coli minicells, the plasmid insert directed the synthesis of polypeptides of 44,000, 33,000, and 20,000 daltons, molecular masses that are consistent with the reported molecular masses of phosphoribosylanthranilate transferase, indoleglycerol-phosphate synthase, and anthranilate-synthase component II, respectively. A 3,105- bp nucleotide sequence was determined. Comparison of the A. calcoaceticus trpGDC sequences with other known trp gene sequences has allowed insight into (1) the evolution of the amphibolic trpG gene, (2) varied strategies for coordinate expression of trp genes, and (3) mechanisms of gene fusions in the trp operon.      

Differential extraction and structural specificity of specialized ubiquinone molecules in secondary electron transfer in chromatophores from Rhodopseudomonas sphaeroides, Ga

In chromatophores from the facultative photosynthetic bacterium, Rhodopseudomonas sphaeroides, Ga, the function of ubiquinone-10 (UQ-10) at two specialized binding sites (Q_B and Q_Z) has been determined by kinetic criteria. These were the rate of rereduction of flash-oxidized [BChl]₂⁺ through the back reaction, or the binary pattern of cytochrome b₅₆₁ (for the Q_b site), and the rapid rate of rereduction of flash-oxidized cytochrome c, or the relative amplitude of the antimycin-sensitive Phase III ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{~ 1.5}\text{ms}$) of the carotenoid spectral shift induced by a single turnover flash at E_h ~ 100 mV (for the Q_Z site). The phenomenon associated with the two binding sites behaved differently on extraction of UQ from lyophilized chromatophores using isooctane. By this selective extraction procedure it has been possible to show that UQ-10 molecules are required at different concentrations in the membrane for specific redox events in secondary electron transfer. The reduction of cytochrome b occurs in particles which no longer show the phenomena associated with Q_Z, but still possess a large proportion of Q_b, while rapid rereduction of flash-oxidized cytochrome c requires an additional complement of UQ-10 (Q_Z). Extracted particles lacking Q_Z and a large amount of Q_B have been reconstituted with different UQ homologs (UQ-1, UQ-3, and UQ-10). Specific redox events have been studied in reconstituted particles. All UQ homologs act as secondary acceptors from the reaction center; UQ-3 and UQ-10, but not UQ-1, are also able to reconstitute the function of Q_Z as electron donor to cytochrome c. Only UQ-10, however, is able to restore normal rates of the overall cyclic electron transfer induced by a train of flashes, and maximal rates of the light-induced ATP synthesis. The results are interpreted in terms of Q-cycle mechanisms in which quinone and quinol at both the Q_Z and Q_b sites are in rapid equilibrium with the quinone pool.     

Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodopseudomonas capsulata and rhodopseudomonas sphaeroides

1. In Rhodopseudomonas sphaeroides the Q_x absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect.2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the α-band (λ_max at 551–551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c₂ (λ_max at 549.5 ± 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed.3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b₅₀ appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b_?90 was observed.4. Difference spectra attributed to (BChl)₂, Q^?_II, c type cytochrome and cytochrome b₅₀ were resolved in the Soret region for Rhodopseudomonas capsulata.5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.     

The role of pH and membrane potential in the reactions of Photosystem II as measured by effects on delayed fluorescence

(1) If DCMU is added to chloroplasts which have been preilluminated (0–8 flashes) the turnover of the water-splitting enzyme is limited to one further transition upon continuous illumination. (2) The intensity of millisecond delayed fluorescence measured in the presence of mediators of cyclic electron transport around Photosystem I and of DCMU added after pre-flashing is stimulated above the level in the presence of DCMU alone and varies according to the number of pre-flashes (Bowes, J.M. and Crofts, A.R. (1978) Z. Naturforsch 33c, 271–275). (3) Separate contributions of the following energetic terms to the induction kinetics and extent of millisecond delayed fluorescence under these conditions have been examined with a view to assessing their involvement in and the mechanism of the stimulation of the emission above the level in dark-adapted chloroplasts in the presence of DCMU: (a) the initial pH of the phase in equilibrium with the water-splitting enzyme; (b) the change in internal pH which occurred when Photosystem I acted as a proton pump; (c) the electrical potential difference across the membrane resulting from rapid charging of the membrane capacitance. (4) It was confirmed that delayed light was stimulated as a result of the interaction of the intrathylakoid pH (3a and b) with the equilibria of the S-states involving proton release according to the model in which this occurs on all except the transition S₁ → S₂; the stimulation was qualitatively proportional to the number of protons released. (5) There was no marked variation of the membrane potential as a function of the number of pre-flashes.     

pH dependent chlorophyll fluorescence quenching in spinach thylakoids from light treated or dark adapted leaves

The pH dependence of maximum chlorophyll fluorescence yield (F_m) was examined in spinach thylakoids in the presence of nigericin to dissipate the transthylakoid pH gradient. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was present to eliminate photochemical quenching. Thylakoids were prepared from dark adapted leaves (dark thylakoids) or preilluminated leaves (light thylakoids). In the latter there had been approximately 50% conversion of the xanthophyll violaxanthin to zeaxanthin, while no conversion had occurred in the former. In the presence of a reductant such as ascorbate, antimycin A sensitive quenching was observed (half maximal quenching at 5 M), whose pH dependence differed between the two types of thylakoid. Preillumination of leaves resulted in more quenching at pH values where very little quenching was observed in dark thylakoids (pH 5–7.6). This was similar to activation of high-energy-state quenching (qE) observed previously (Rees D, Young A, Noctor G, Britton G and Horton P (1989) FEBS Lett 256: 85–90). Thylakoids isolated from preilluminated DTT treated leaves, that contained no zeaxanthin, behaved like dark thylakoids. A second form of quenching was observed in the presence of ferricyanide, that could be reversed by the addition of ascorbate. This was not antimycin A sensitive and showed the same pH dependence in both types of thylakoid. The former type of quenching, but not the latter, showed similar low temperature fluorescence emission spectra to qE, and was considered to occur by the same mechanism.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - EDTA Ethylenediaminetetra-acetic acid - F₀ dark level fluorescence yield - F_m maximum fluorescence yield - F_v/F_m ratio of variable to total fluorescence yield - Hepes 4-(2-hydroxyethyl)1-piperazineethanesul-phonic acid - Mes 2-(N-morpholino) ethanesulfonate - pH transthylakoid pH gradient - PS I Photosystem I - PS II Photosystem II - Q_A primary stable electron acceptor of Photosystem II - qE high-energy-state fluorescence quenching     

Regulation of coupling factor in field-grown sunflower: A Redox model relating coupling factor activity to the activities of other thioredoxin-dependent chloroplast enzymes

Simultaneous, non-invasive measurements were made of the rate of photosynthetic CO₂ fixation and the state of activation of the chloroplast CF₁CF₀-ATP synthase (CF) in field-grown sunflower (Helianthus annuus L.) during the dark-to-light transition at sunrise. CO₂ fixation showed a linear response with light intensity from zero to about 500–700 E m^-2 s^-1. However, at light intensities of only 5–22 E m^-2 s^-1, the energetic threshold for activation of the CF was found to be significantly lowered (as compared to the pre-dawn state), presumably through reduction of the regulatory sulfhdryl groups of the -subunit of the CF. When these studies were extended to chamber-grown plants, it was found that as little as 5 seconds of illumination at 4 E m^-2 s^-1 caused apparently full CF reduction. It is clear, therefore, that the catalytic activation of CF is not rate limiting to the induction of carbon assimilation under field conditions during a natural dark-to-light transition at sunrise. A model, based on the redox properties of the regulatory sulfhydryls, was developed to examine the significance of sulfhydryl midpoint potential in explaining the differences in light sensitivity and oxidation and reduction kinetics, between the CF and other thioredoxin-modulated chloroplast enzymes. Computer simulations of the light-induced regulation of three representative thioredoxin-modulated enzymes are presented.Abbreviations CF chloroplast CF₁-CF₀ ATP synthease or coupling factor - E_a active form of CF - E_a ⁰ active, oxidized form of CF - E_a ^r active, reduced form of CF - E_i inactive form of CF - E_i ⁰ inactive, oxidized form of CF - E_i ^r inactive, reduced form of CF - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin oxidoreductase - G6PDH glucose-6-phosphate dehydrogenase - MDH NADP-malate dehydrogenase - pmf protonmotive force - pmf_T threshold pmf required to activate CF - pmf_T ⁰ threshold pmf required to active the oxidized form of CF - pmf_T ^r threshold pmf required to activate the reduced form of CF - TR thioredoxin     

Demonstration of a highly-sensitive portable double-flash kinetic spectrophotometer for measurement of electron transfer reactions in intact plants

A highly sensitive, portable spectrophotometer for use in measuring flash-induced absorbance changes in intact leaves is demonstrated. The design of the instrument is modified for whole plant use from that suggested by Joliot and Joliot (Biochim. Biophys. Acta 765, 210–218). The spectrophotometer uses trifurcated light guides to deliver measuring and actinic beams to two comparable areas of the leaf. The measuring beam is provided by a series of short, relatively intense light pulses from a xenon flashlamp in place of the constant weak measuring beam used in conventional machines. The use of a flash measuring beam and differential detection allows for a high signal-to-noise ratio (noise levels of 10^-5A) without significant actinic effects. The time resolution of the instrument is 2 sec and the noise level is independent of the experimental time range. The instrument is battery or mains powered, computer operated, and has a liquid crystal display for computer-user interface and dialogue, and to show the kinetic traces graphically. Wavelength selection is provided by interchangeable interference filters. The instrument can communicate with a laboratory-based computer, receiving programming information and sending experimental data to be processed and plotted. The instrument is demonstrated by following the kinetics of the electrochromic shift, the change in redox states of cytochrome f and the b cytochromes in an intact cucumber leaf, and in the same leaf after infiltration with DCMU.     

The liver cell plasma membrane Ca2+ inflow systems exhibit a broad specificity for divalent metal ions.

1. The inflow of Mn2+ across the plasma membranes of isolated hepatocytes was monitored by measuring the quenching of the fluorescence of intracellular quin2, by atomic absorption spectroscopy and by the uptake of 54Mn2+. The inflow of other divalent metal ions was measured using quin2. 2. Under ionic conditions which resembled those present in the cytoplasmic space, Mn2+, Zn2+, Co2+, Ni2+ and Cd2+ each quenched the fluorescence of a solution of Ca2(+)-quin2. 3. The addition of Mn2+, Zn2+, Co2+, Ni2+ or Cd2+ to cells loaded with quin2 caused a time-dependent decrease in the fluorescence of intracellular quin2. Plots of the rate of decrease in fluorescence as a function of the concentration of Mn2+ reached a plateau at 100 microM-Mn2+. 4. The rate of decrease in fluorescence induced by Mn2+ was stimulated by 20% in the presence of vasopressin. The effect of vasopressin was completely inhibited by 200 microM-verapamil. Adrenaline, angiotensin II and glucagon also stimulated the rate of decrease in the fluorescence of intracellular quin2 induced by Mn2+. 5. The rate of decrease in fluorescence induced by Zn2+, Co2+, Ni2+ or Cd2+ was stimulated by between 20 and 190% in the presence of vasopressin or angiotensin II. 6. The rates of uptake of Mn2+ measured by atomic absorption spectroscopy or by using 54Mn2+ were inhibited by about 20% by 1.3 mM-Ca2+o and stimulated by 30% by vasopressin. 7. Plots of Mn2+ uptake, measured by atomic absorption spectroscopy or with 54Mn2+, as a function of the extracellular concentration of Mn2+ were biphasic over the range 0.05-1.0 mM added Mn2+ and did not reach a plateau at 1.0 mM-Mn2+. 8. It is concluded that (i) hepatocytes possess both a basal and a receptor-activated divalent cation inflow system, each of which has a broad specificity for metal ions, and (ii) the receptor-activated divalent cation inflow system is the receptor-operated Ca2+ channel.     

A thermodynamic characterisation of the cytochromes of chromatophores from Rhodopseudomonas capsulata

1. The cytochromes of chromatophores from photosynthetically grown Rhodopseudomonas capsulata have been characterised both spectrally, using the carotenoid free mutant Ala Pho⁺, and thermodynamically, using the technique of redox titrations. Five cytochromes were present; two cytochromes b, E′₀ = 60 mV at pH 7.0; and three cytochromes c, E′₀ = 340 mV, $\text{E}\text{t}\text{?}{}_0\text{= 120}\text{mV}$, E′₀ = 0 mV at pH 7.0.2. Redox titrations at different values of pH indicated that the mid point potentials of all the cytochromes varied with pH over some parts of the range between pH 6 and 9, with the possible exception of cytochrome c₃₄₀.3. The effects of succinate and NADH on the steady state reduction of the cytochromes are reported. Succinate could reduce cytochromes c₃₄₀, c₁₂₀ and b₆₀; NADH could reduce cytochromes c₃₄₀, c₁₂₀, b₆₀ and b_?25. Cytochrome c₀ could be reduced by dithionite but not by the other substrates tested.