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BBA - Bioenergetics (v.1708, #1)

Editorial Board (pp. ii).
Contents (pp. co4).

Antenna ring around trimeric Photosystem I in chlorophyll b containing cyanobacterium Prochlorothrix hollandica by Ladislav Bumba; Ondrej Prasil; Frantisek Vacha (pp. 1-5).
Prochlorothrix hollandica is one of the three known species of an unusual clade of cyanobacteria (formerly called “prochlorophytes�) that contain chlorophyll a and b molecules bound to intrinsic light-harvesting antenna proteins. Here, we report the structural characterization of supramolecular complex consisting of Photosystem I (PSI) associated with the chlorophyll a/ b-binding Pcb proteins. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the Pcb–PSI supercomplex consists of a central trimeric PSI surrounded by a ring of 18 Pcb subunits. We conclude that the formation of the Pcb ring around trimeric PSI represents a mechanism for increasing the light-harvesting efficiency in chlorophyll b-containing cyanobacteria.

Keywords: Photosystem I; Prochlorothrix hollandica; Prochlorococcus marinus; Prochlorophyta; Chlorophyll; a; /; b; antenna protein; Electron microscopy


Weakened coupling of conserved arginine to the proteorhodopsin chromophore and its counterion implies structural differences from bacteriorhodopsin by Ranga Partha; Richard Krebs; Tamara L. Caterino; Mark S. Braiman (pp. 6-12).
In wild-type proteorhodopsin (pR), titration of the chromophore's counterion Asp97 occurs with a p Ka of 8.2±0.1. R94C mutation reduces this slightly to 7.0±0.2, irrespective of treatment with ethylguanidinium. This contrasts with the homologous archaeal protein bacteriorhodopsin (bR), where R82C mutation was previously shown to elevate the p Ka of Asp85 by ∼5 units, while reconstitution with ethylguanidinium restores it nearly to the wild-type value of 2.5. We conclude there is much weaker electrostatic coupling between Arg94 and Asp97 in the unphotolyzed state of pR, in comparison to Arg82 and Asp85 in bR. Therefore, while fast light-driven H+ release may depend on these two residues in pR as in bR, no tightly conserved pre-photolysis configuration of them is required.

Keywords: Abbreviations; pR; proteorhodopsin; bR; bacteriorhodopsin; HEPES; N; -2-hydroxyethylpiperazine-; N; ′-2-ethanesulfonic acid; OG; β-octyl-; d; -glucoside; TCM; triple cysteine mutant of pR [C(107, 156, 175)S]; QM; quadruple mutant of pR [C(107, 156, 175)S, R94C]; NpSRII; Natronobacterium pharaonis; sensory rhodopsin IISensory rhodopsin; Light-driven proton pumping; Red to purple transition; Blue membrane; Fast H; +; release; Photocycle


Transport and metabolism ofd-lactate in Jerusalem artichoke mitochondria by Anna Atlante; Lidia de Bari; Daniela Valenti; Roberto Pizzuto; Gianluca Paventi; Salvatore Passarella (pp. 13-22).
We report here initial studies ond-lactate metabolism in Jerusalem artichoke. It was found that: 1)d-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-d-lactoyl-glutathione. 2) Externally addedd-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3)d-lactate was metabolized inside mitochondria by a flavoprotein, a putatived-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally addedd-lactate by means of ad-lactate/H+ symporter investigated by measuring the rate of reduction of endogenous flavins. The action of thed-lactate translocator and of the mitochondriald-lactate dehydrogenase could be responsible for the subsequent metabolism ofd-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.

Keywords: Abbreviations; AA; antimycin A; ASC; ascorbate; β-NH; 2; -BUT; β-aminobutyrate; BUT; butyrate; CN; −; cyanide; α-CCN; −; α-cyano-4-hydroxycinnamate; DCIP; dichloroindophenol; d; -LAC; d; -lactate; d; -LDH; d; -lactate dehydrogenase; DTNB; 5,5′-dithiobis(2-nitrobenzoic acid); ΔΨ; electrical membrane potential; e.u.; enzymatic unit; FCCP; carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; G3P; glycerol-3-phosphate; GSH; reduced glutathione; JAM; Jerusalem artichoke mitochondria; Lact-GSH; S; -; d; -lactoylglutathione; l; -LAC; l; -lactate; l; -LDH; l; -lactate dehydrogenase; MERS; mersalyl; TNB; 5-thio-2-nitrobenzoic acid; OLIGO; oligomycin; PheSUCC; phenylsuccinate; PHT; phthalonate; PMS; phenazine methosulphate; PYR; pyruvate; RAC; rotenone+antimycin A+cyanide; ROT; rotenone; SUCC; succinate; TMPD; N; ,; N; ,; N; ′,; N; ′-tetramethyl-; p; -phenylenediamine; TX-100; Triton X-100; VAL; valinomycind; -lactate; Jerusalem artichoke mitochondria; d; -lactate dehydrogenase; Glyoxalase II; Mitochondrial transport; Methylglyoxal pathway


Divergence of the mitochondrial electron transport chains from the green alga Chlamydomonas reinhardtii and its colorless close relative Polytomella sp. by Robert van Lis; Diego González-Halphen; Ariane Atteia (pp. 23-34).
Compelling evidence exists that the colorless algae of the genus Polytomella arose from a green Chlamydomonas-like ancestor by losing its functional photosynthetic apparatus. Due to the close relationship between the colorless and the green chlorophyte, Polytomella sp. appeared as a useful indicative framework for structural studies of Chlamydomonas reinhardtii mitochondria. However, comparative studies reported here unexpectedly revealed significant differences between the mitochondrial respiratory systems of the two algae. Two-dimensional blue native/SDS-PAGE of isolated mitochondria indicated that cytochrome-containing respiratory complexes III and IV in the two chlorophytes contrast in size, subunit composition and relative abundance. Complex IV in Polytomella is smaller than its counterpart in C. reinhardtii and occurs in two forms that differ presumably in the presence of subunit COXIII. The cytochrome c and the iron–sulfur Rieske protein of both chlorophytes revealed structural differences on the amino acid sequence level. Under comparable culture conditions, the colorless alga exhibits lower levels of cytochrome c and complex IV but a higher respiratory activity than the green alga. Cytochrome c levels were also found to be differently regulated by the growth conditions in both algae. The divergence between the respiratory systems in the two related chlorophytes can be viewed as a consequence of the loss of photosynthetic activity and/or of the adaptation to the environment via the acquisition of a more flexible, heterotrophic metabolism. Our understanding of mitochondrial function and evolution is expected to be greatly enhanced via further parallel studies of photosynthetic/non-photosynthetic algae, for which this study forms an incentive.

Keywords: Abbreviations; AOX; alternative oxidase; BN-PAGE; blue native polyacrylamide gel electrophoresis; Cyt; cytochrome; ISP; iron–sulfur protein; MPP; mitochondrial processing peptidase; OXPHOS; oxidative phosphorylation; TMPD; N; ,; N; ,; N′; ,; N′; -tetramethyl-; p; -phenylenediamine dihydrochlorideBlue native PAGE; Chlorophyte; Heterotrophic lifestyle; Mitochondrial cytochrome; Photosynthesis; Respiratory chain


EPR kinetic studies of the S−1 state in spinach thylakoids by M. Reza Razeghifard; Damien Kuzek; Ron J. Pace (pp. 35-41).
The YZ decay kinetics in a formal S−1 state, regarded as a reduced state of the oxygen evolving complex, was determined using time-resolved EPR spectroscopy. This S−1 state was generated by biochemical treatment of thylakoid membranes with hydrazine. The steady-state oxygen evolution of the sample was used to optimize the biochemical procedure for performing EPR experiments. A high yield of the S−1 state was generated as judged by the two-flash delay in the first maximum of oxygen evolution in Joliot flash-type experiments. We have shown that the YZ re-reduction rate by the S−1 state is much slower than that of any other S-state transition in hydrazine-treated samples. This slow reduction rate in the S−1 to S0 transition, which is in the order of the S3 to S0 transition rate, suggests that this transition is accompanied by some structural rearrangements. Possible explanations of this unique, slow reduction rate in the S−1 to S0 transition are considered, in light of earlier observations by others on hydrazine/hydroxylamine reduced PS II samples.

Keywords: Abbreviations; Chl; chlorophyll; EPR; electron paramagnetic resonance; HEPES; N; -2-hydroxyethyl piperazine-; N; ′-; 2-; ethanesulfonic acid; OEC; oxygen evolving complex; PPBQ; phenyl-; p; -benzoquinone; PSII; photosystem II; MSP; Manganese stabilizing protein; Y; Z; redox-active tyrosine of D1 polypeptide; Y; D; redox-active tyrosine of D2 polypeptidePhotosystem II; Oxygen evolving complex; Tyrosine Z reduction kinetics; Super-reduced state; Hydrazine


EPR properties of a g=2 broad signal trapped in an S1 state in Ca2+-depleted Photosystem II by Hiroyuki Mino; Shigeru Itoh (pp. 42-49).
We investigated a new EPR signal that gives a broad line shape around g=2 in Ca2+-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of YZ. The ratio of the intensities between the g=2 broad signal and the YZ signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the YZ signal decayed together in parallel with the appearance of the S2 state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S1 X state in the transition from the S1 to the S2 state, where X represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with YZ. Possible reactions in the S state transitions in Ca2+-depleted PS II were discussed.

Keywords: Abbreviations; CW; continuous-wave; DCMU; 3-(3,4-dichlorophenyl)-1,1-dimethylurea; EDTA; ethylene diamine tetraacetic acid; EPR; electron paramagnetic resonance; ENDOR; electron nuclear double resonance; MES; 2-morpholinoethanesulfonic acid; MOPS; 2-(; N; -morpholino)ethanesulfonic acid; OEC; oxygen-evolving complex; PS; photosystem; Y; Z; tyrosine-Z, the D1-Tyr161; Y; D; tyrosine-D, the D2-Tyr161Ca; 2+; depletion; Manganese cluster; Oxygen evolving complex; Photosystem II; Split EPR signal


An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis by Anna Atlante; Sergio Giannattasio; Antonella Bobba; Sara Gagliardi; Vito Petragallo; Pietro Calissano; Ersilia Marra; Salvatore Passarella (pp. 50-62).
Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine andl-lactate in cells undergoing apoptosis during the commitment phase (0–8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level ofl-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP]2. When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase ofl-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease inl-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.

Keywords: Abbreviations; Act D; actinomycin D; AQ; ADK; ADK quotient activity; DIV; days in vitro; BME; basal medium Eagle; CGCs; cerebellar granule cells; CITR; citrate; cyt; c; cytochrome; c; FCS; fetal calf serum; HPLC; high-performance liquid chromatography; l; -LACT; l; -lactate; MK801; (+)-5methyl-10,11dihydro-5; H; -dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate; NMDA; N; -methyl-; d; -aspartate; O; 2; molecular oxygen; OLIGO; oligomycin; PBS; phosphate buffer saline medium; PFK; phosphofructokinase; S-K25 cells; control cells; S-K5 cells; apoptotic cells; ROS; reactive oxygen speciesCerebellar granule cell; Apoptosis; ATP content; l; -lactate; Mitochondria


Function of the 23 kDa extrinsic protein of Photosystem II as a manganese binding protein and its role in photoactivation by Natallia Bondarava; Peter Beyer; Anja Krieger-Liszkay (pp. 63-70).
The function of the extrinsic 23 kDa protein of Photosystem II (PSII) was studied with respect to Mn binding and its ability to supply Mn to PSII during photoactivation, i.e. the light-dependent assembly of the tetramanganese cluster. The extrinsic proteins and the Mn cluster were removed by TRIS treatment from PSII-enriched membrane fragments and purified by anion exchange chromatography. Room temperature EPR spectra of the purified 23 kDa protein demonstrated the presence of Mn. Photoactivation was successful with low Mn concentrations when the 23 kDa protein was present, while in its absence a higher Mn concentration was needed to reach the same level of oxygen evolution activity. In addition, the rate of photoactivation was significantly accelerated in the presence of the 23 kDa protein. It is proposed that the 23 kDa protein plays an important role in providing Mn during the process of PSII assembly and that it acquires Mn during the light-induced turnover of D1 in the PSII damage–repair cycle and delivers Mn to repaired PSII.

Keywords: Abbreviations; Chl; chlorophyll; DCPIP; 2,6-dichlorophenol-indophenol; EDTA; ethylenediaminetetraacetic acid; EPR; electron paramagnetic resonance; MES; 4-morpholineethanesulfonic acid; PSII; Photosystem II; SDS-PAGE; sodium dodecyl sulfate polyacrylamid gel electrophoresis; TRIS; Tris(hydroxymethyl)aminomethane; TyrZ; redox active amino acid residue of the D1 proteinPhotosynthesis; Photosystem II; Photoactivation; 23 kDa protein; Manganese


Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP by Wieslawa Jarmuszkiewicz; Malgorzata Czarna; Francis E. Sluse (pp. 71-78).
In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the KMox values were around 4–5 μM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KKMox values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized ( KMox was around 1.2 μM for succinate and around 11 μM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in KMox. However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the KMox increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.

Keywords: Abbreviations; AOX; alternative oxidase; BHAM; benzohydroxamate; K; Mox; apparent Michaelis constant of oxygen; V; max; apparent maximal velocity; Q or Q; ox; ubiquinone; Q; red; or QH; 2; ubiquinol; Q; tot; total endogenous pool of ubiquinone in the inner mitochondrial membrane (Q; ox; +Q; red; ); Q; red; /Q; tot; reduction level of the ubiquinone poolMitochondria; Alternative oxidase; Oxygen affinity; GMP stimulation; Acanthamoeba castellanii


Control of cytochrome b6 f at low and high light intensity and cyclic electron transport in leaves by Agu Laisk; Hillar Eichelmann; Vello Oja; Richard B. Peterson (pp. 79-90).
The light-dependent control of photosynthetic electron transport from plastoquinol (PQH2) through the cytochrome b6 f complex (Cyt b6 f) to plastocyanin (PC) and P700 (the donor pigment of Photosystem I, PSI) was investigated in laboratory-grown Helianthus annuus L., Nicotiana tabaccum L., and naturally-grown Solidago virgaurea L., Betula pendula Roth, and Tilia cordata P. Mill. leaves. Steady-state illumination was interrupted (light–dark transient) or a high-intensity 10 ms light pulse was applied to reduce PQ and oxidise PC and P700 (pulse-dark transient) and the following re-reduction of P700+ and PC+ was recorded as leaf transmission measured differentially at 810–950 nm. The signal was deconvoluted into PC+ and P700+ components by oxidative (far-red) titration (V. Oja et al., Photosynth. Res. 78 (2003) 1–15) and the PSI density was determined by reductive titration using single-turnover flashes (V. Oja et al., Biochim. Biophys. Acta 1658 (2004) 225–234). These innovations allowed the definition of the full light response curves of electron transport rate through Cyt b6 f to the PSI donors. A significant down-regulation of Cyt b6 f maximum turnover rate was discovered at low light intensities, which relaxed at medium light intensities, and strengthened again at saturating irradiances. We explain the low-light regulation of Cyt b6 f in terms of inactivation of carbon reduction cycle enzymes which increases flux resistance. Cyclic electron transport around PSI was measured as the difference between PSI electron transport (determined from the light–dark transient) and PSII electron transport determined from chlorophyll fluorescence. Cyclic e− transport was not detected at limiting light intensities. At saturating light the cyclic electron transport was present in some, but not all, leaves. We explain variations in the magnitude of cyclic electron flow around PSI as resulting from the variable rate of non-photosynthetic ATP-consuming processes in the chloroplast, not as a principle process that corrects imbalances in ATP/NADPH stoichiometry during photosynthesis.

Keywords: Abbreviations; Chl; chlorophyll; Cyt; cytochrome; e; −; electron(s); FeS; Rieske FeS complex; FRL; far red light; NPQ; non-photochemical quenching of excitation; PC; plastocyanin; PAD, PFD; photon flux density, absorbed and incident; PQ; PQH; 2; plastoquinone, oxidised and reduced; PSI; Photosystem I; PSII; Photosystem II; P700; donor pigment of PSI; WL; white lightCytochrome; b; 6; f; Control; Leaf


15N-labeling to determine chlorophyll synthesis and degradation in Synechocystis sp. PCC 6803 strains lacking one or both photosystems by Dmitrii Vavilin; Daniel C. Brune; Wim Vermaas (pp. 91-101).
Rates of chlorophyll synthesis and degradation were analyzed in Synechocystis sp. PCC 6803 wild type and mutants lacking one or both photosystems by labeling cells with (15NH4)2SO4 and Na15NO3. Pigments extracted from cells were separated by HPLC and incorporation of the15N label into porphyrins was subsequently examined by MALDI-TOF mass spectrometry. The life time ( τ) of chlorophyll in wild-type Synechocystis grown at a light intensity of 100 μmol photons m−2 s−1 was determined to be about 300 h, much longer than the cell doubling time of about 14 h. Slow chlorophyll degradation ( τ ∼200–400 h) was also observed in Photosystem I-less and in Photosystem II-less Synechocystis mutants, whereas in a mutant lacking both Photosystem I and Photosystem II chlorophyll degradation was accelerated 4–5 fold ( τ ∼50 h). Chlorophyllide and pheophorbide were identified as intermediates of chlorophyll degradation in the Photosystem I-less/Photosystem II-less mutant. In comparison with the wild type, the chlorophyll synthesis rate was five-fold slower in the Photosystem I-less strain and about eight-fold slower in the strain lacking both photosystems, resulting in different chlorophyll levels in the various mutants. The results presented in this paper demonstrate the presence of a regulation that adjusts the rate of chlorophyll synthesis according to the needs of chlorophyll-binding polypeptides associated with the photosystems.

Keywords: Abbreviations; MALDI-TOF MS; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; OD; 730; optical density at 730 nm; PS; photosystem; SCP; small Cab-like proteinChlorophyll turnover; Mass spectrometry; Metabolic flux; Photosystem I; Photosystem II; Stable isotope labeling


Towards elucidating the energy of the first excited singlet state of xanthophyll cycle pigments by X-ray absorption spectroscopy by W.I. Gruszecki; H. Stiel; D. Niedzwiedzki; M. Beck; J. Milanowska; H. Lokstein; D. Leupold (pp. 102-107).
The first excited singlet state (S1) of carotenoids (also termed 2Ag−) plays a key role in photosynthetic excitation energy transfer due to its close proximity to the S1 (Q y) level of chlorophylls. The determination of carotenoid 2Ag− energies by optical techniques is difficult; transitions from the ground state (S0, 1Ag−) to the 2Ag− state are forbidden (“optically dark�) due to parity (g � //→ g) as well as pseudo-parity selection rules (− � //→ −). Of particular interest are S1 energies of the so-called xanthophyll-cycle pigments (violaxanthin, antheraxanthin and zeaxanthin) due to their involvement in photoprotection in plants. Previous determinations of S1 energies of violaxanthin and zeaxanthin by different spectroscopic techniques vary considerably. Here we present an alternative approach towards elucidation of the optically dark states of xanthophylls by near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The indication of at least one π* energy level (about 0.5 eV below the lowest 1Bu+ vibronic sublevel) has been found for zeaxanthin. Present limitations and future improvements of NEXAFS to study optically dark states of carotenoids are discussed. NEXAFS combined with simultaneous optical pumping will further aid the investigation of these otherwise hardly accessible states.

Keywords: Abbreviations; Chl; chlorophyll; EET; excitation energy transfer; IP; ionization potential; LHC II; light-harvesting complex II; NEXAFS; near-edge X-ray absorption fine structure spectroscopy; XAS; X-ray absorption spectroscopy; XUV; extreme ultravioletCarotenoid; Xanthophyll cycle; NEXAFS; Violaxanthin; Zeaxanthin; X-ray absorption spectroscopy


Multiple proteins with single activities or a single protein with multiple activities: The conundrum of cell surface NADH oxidoreductases by Debbie-Jane G. Scarlett; Patries M. Herst; Michael V. Berridge (pp. 108-119).
Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.

Keywords: Abbreviations; BN-PAGE; blue native polyacrylamide gel electrophoresis; capsaicin; 8-methyl-; N; -vanillyl-; trans; -6-nonenamide; CNOX; constitutive NADH oxidase; ECTO-NOX; external NADH oxidase; EP; pH 5 eluted cell surface proteins; FBS; foetal bovine serum; HBSS; Hanks' Balanced Saline Solution; KCN; potassium cyanide; m; PMS; 1-methoxy-5-methylphenazinium methylsulfate; NBT; nitro blue tetrazolium; PBS; phosphate buffered saline; pCMBS; p; -chloromercuriphenylsulfonic acid; PI; preimmune; PM; plasma membrane; PMOX; plasma membrane oxidoreductase; PMSF; phenylmethylsulfonyl fluoride; PVDF; polyvinylidene difluoride; RT; room temperature; SDS-PAGE; SDS polyacrylamide gel electrophoresis; tNOX; tumour NADH oxidase; tPMET; trans; -plasma membrane electron transport; WST-1; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2; H; -tetrazolium monosodium saltCNOX; gp96; NADH oxidase; NADH oxidoreductase; Trans; -plasma membrane electron transport; WST-1


Towards a spin coupling model for the Mn4 cluster in Photosystem II by Marie-France Charlot; Alain Boussac; Geneviève Blondin (pp. 120-132).
The X-band EPR spectra of the IR sensitive untreated PSII and of MeOH- and NH3-treated PSII from spinach in the S2-state are simulated with collinear and rhombic g- and Mn-hyperfine tensors. The obtained principal values indicate a 1Mn(III)3Mn(IV) composition for the Mn4 cluster. The four isotropic components of the Mn-hyperfine tensors are found in good agreement with the previously published values determined from EPR and55Mn-ENDOR data. Assuming intrinsic isotropic components of the Mn-hyperfine interactions identical to those of the Mn-catalase, spin density values are calculated. A Y-shape 4 J-coupling scheme is explored to reproduce the spin densities for the untreated PSII. All the required criteria such as a S=1/2 ground state with a low lying excited spin state (30 cm−1) and an easy conversion to a S=5/2 system responsible for the g=4.1 EPR signal are shown to be satisfied with four antiferromagnetic interactions lying between −290 and −130 cm−1.

Keywords: Photosystem II; Manganese; EPR spectroscopy; Hyperfine interaction; Spin coupling
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