Submembrane Fractions Research Articles

Characterization of the structural changes and photochemical activity of photosystem I under Al3+ effect

The photochemical activity of photosystem I (PSI) as affected by Al3+ was investigated in thylakoid membranes and PSI submembrane fractions isolated from spinach. Biophysical and biochemical techniques such as oxygen uptake, light induced absorbance changes at 820nm, chlorophyll fluorescence emission, SDS–polyacrylamide gel electrophoresis, and FTIR spectroscopy have been used to analyze the sites and action modes of this cation on the PSI complex. Our results showed that Al3+ above 3mM induces changes in the redox state of P700 reflected by an increase of P700 photooxidation phase and a delay of the slower rate of P700 re-reduction which reveals that Al3+ exerted an inhibitory action at the donor side of PSI especially at plastocyanin (PC). Furthermore, results of P700 photooxidation monitored in the presence of DCMU with or without MV suggested that the same range of Al3+ concentrations impairs the photochemical reaction centers (RC) of PSI, as shown by the decline in the amount of active population of P700, and disrupts the charge separation between P700 and the primary electron acceptor A0 leading to the inhibition of electron transfer at the acceptor side of PSI. These inhibitory actions were also accompanied by an impairment of the energy transfer from light harvesting complex (LHCI) to RC of PSI, following the disconnection of LHCI antenna as illustrated by an enhancement of chlorophyll fluorescence emission spectra at low temperature (77K). The above results coincided with FTIR measurements that indicated a conformational change of the protein secondary structures in PSI complex where 25% of α-helix was converted into β-sheet, β-antiparallel and turn structures. These structural changes in PSI complex proteins are closely related with the alteration photochemical activity of PSI including the inhibition of the electron transport through both acceptor and donor sides of PSI.

Mechanism of interaction of Al3+ with the proteins composition of photosystem II.

The inhibitory effect of Al3+on photosystem II (PSII) electron transport was investigated using several biophysical and biochemical techniques such as oxygen evolution, chlorophyll fluorescence induction and emission, SDS-polyacrylamide and native green gel electrophoresis, and FTIR spectroscopy. In order to understand the mechanism of its inhibitory action, we have analyzed the interaction of this toxic cation with proteins subunits of PSII submembrane fractions isolated from spinach. Our results show that Al 3+, especially above 3 mM, strongly inhibits oxygen evolution and affects the advancement of the S states of the Mn4O5Ca cluster. This inhibition was due to the release of the extrinsic polypeptides and the disorganization of the Mn4O5Ca cluster associated with the oxygen evolving complex (OEC) of PSII. This fact was accompanied by a significant decline of maximum quantum yield of PSII (Fv/Fm) together with a strong damping of the chlorophyll a fluorescence induction. The energy transfer from light harvesting antenna to reaction centers of PSII was impaired following the alteration of the light harvesting complex of photosystem II (LHCII). The latter result was revealed by the drop of chlorophyll fluorescence emission spectra at low temperature (77 K), increase of F0 and confirmed by the native green gel electrophoresis. FTIR measurements indicated that the interaction of Al 3+ with the intrinsic and extrinsic polypeptides of PSII induces major alterations of the protein secondary structure leading to conformational changes. This was reflected by a major reduction of α-helix with an increase of β-sheet and random coil structures in Al 3+-PSII complexes. These structural changes are closely related with the functional alteration of PSII activity revealed by the inhibition of the electron transport chain of PSII.

Alteration of the structure and function of photosystem I by Pb2+

The toxic effects of Pb2+ on photosynthetic electron transport were studied in photosystem I (PSI) submembrane fractions isolated from spinach. Structural and spectroscopic analysis using FTIR, fluorescence and X-ray photoelectron spectroscopy (XPS) showed that Pb2+ binds with proteins via oxygen and nitrogen atoms with an overall binding constant of KPb-PSI=4.9×103 (±0.2) M−1 and the number of bound Pb2+ cation was 0.9 per PSI complex. Pb2+ binding altered the protein conformation indicating a partial protein destabilization. Electron transport and P700 photooxidation/reduction measurements showed that the interaction of Pb2+ cations with PSI produced a donor side limitation of electron transport presumably due to Pb2+ binding to or in the vicinity of plastocyanin.

NHE-1 Relocation Outside Cholesterol-rich Membrane Microdomains is Associated with its Benzo[a]pyrene-related Apoptotic Function

Background: Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are ubiquitous toxic environmental pollutants capable of inducing cell death. Intracellular pH plays a key role in the regulation of cell survival and death. Our previous works have demonstrated that intracellular alkalinization mediated by Na⁺/H⁺ exchanger 1 (NHE-1) is a critical event involved in B[a]P-induced apoptosis. The aim of this study was to further elucidate the mechanisms of NHE-1 activation upon B[a]P exposure. Methods: We tested the effects of plasma membrane cholesterol enrichment or depletion on B[a]P-induced NHE-1 activation related to apoptosis. We isolated cholesterol-rich plasma membrane microdomains to assess NHE-1 submembrane location and immunoprecipitated NHE-1 from the different sub-membrane fractions obtained to examine NHE-1 protein interactions during B[a]Pinduced apoptosis. Results: We found that NHE-1 is preferentially located in cholesterol-rich microdomains and that B[a]P activates NHE-1 via its relocation and binding of calmodulin outside these specialized plasma membrane microstructures; these events are necessary for the execution of the apoptosis-related intracellular alkalinization. Conclusion: Plasma membrane location of NHE-1 affects its protein interactions and apoptotic function.

Methylamine interaction with proteins of photosystem II: A comparison with biogenic polyamines

Biogenic polyamines are essential for cell growth and differentiation. The interaction of polyamines with protein of photosystem II (PSII) are well investigated, while there has been no report on the effect of monoamines complexation on photosynthetic oxygen evolution. This study was designed to investigate the interaction of methylamine with proteins of PSII, using PSII-enriched submembrane fractions with various concentrations of methylamine. Fourier transformed infrared (FTIR) and fluorescence spectroscopic methods were used in order to determine the methylamine binding mode, the protein conformational changes, and the effect of amine interaction on photosynthetic oxygen evolution. Spectroscopic evidence showed that methylamine interacts with protein (H-bonding) through polypeptide C O, C–N and NH groups with major perturbations of protein secondary structure. Major reduction of α-helix from 50% (free PSII) to 35% with an increase of β-sheet from 10% (free PSII) to 16% was observed in methylamine-PSII complexes. At very low methylamine concentration, no inhibition of oxygen-evolution occurred, while at higher amine content (12 mM), 100% inhibition was observed. Chlorophyll (Chl) fluorescence measurements indicated the inhibition mainly affects the oxygen evolving complex (OEC) of PSII. Comparisons of the effects of methylamine with biogenic polyamine spermine, spermidine and putrescine showed a similar mode of binding with protein (H-bonding) through polypeptide C O, C–N and NH groups. However, major alterations of the protein secondary structure are induced by monoamine and not by polyamines.

Phenotypic Correction of Hemophilia A by Ectopic Expression of Activated Factor VII in Platelets

Platelets are receiving much attention as novel target cells to secrete a coagulation factor for hemophilia gene therapy. In order to extend the application of platelet-directed gene therapy, we examined whether ectopic expression of activated factor VII (FVIIa) in platelets would result in an efficient bypass therapy to induce sufficient thrombin generation on platelet surfaces in mice with hemophilia A. Transduction of bone marrow cells with a simian immunodeficiency virus (SIV)-based lentiviral vector harboring the platelet-specific GPIb alpha promoter resulted in efficient transgene expression in platelets. FVIIa antigen was expressed in platelets by this SIV system; FVII transgene products were found to localize in the cytoplasm and translocate toward the sub-membrane zone and cell surface after activation. Although FVII antigen levels in platelets did not reach the therapeutic levels seen with FVIIa infusion therapy, whole-blood coagulation, as assessed by thromboelastography, was significantly improved in mice with hemophilia A. Further, we observed correction of the bleeding phenotype in mice with hemophilia A after transplantation, even in the presence of FVIII-neutralizing antibodies. Our results demonstrate that FVIIa-expressing platelets can strengthen hemostatic function and may be useful in treating hemophilia and other inherited bleeding disorders. These findings are comparable to the proven therapeutic effects of FVIIa infusion.

Inhibition of the oxygen-evolving complex of photosystem II and depletion of extrinsic polypeptides by nickel

The toxic effect of Ni(2+) on photosynthetic electron transport was studied in a photosystem II submembrane fraction. It was shown that Ni(2+) strongly inhibits oxygen evolution in the millimolar range of concentration. The inhibition was insensitive to NaCl but significantly decreased in the presence of CaCl(2). Maximal chlorophyll fluorescence, together with variable fluorescence, maximal quantum yield of photosystem II, and flash-induced fluorescence decays were all significantly declined by Ni(2+). Further, the extrinsic polypeptides of 16 and 24 kDa associated with the oxygen-evolving complex of photosystem II were depleted following Ni(2+) treatment. It was deduced that interaction of Ni(2+) with these polypeptides caused a conformational change that induced their release together with Ca(2+) from the oxygen-evolving complex of photosystem II with consequent inhibition of the electron transport activity.

Photoinhibitory Light-induced Changes in the Composition of Chlorophyll-Protein Complexes and Photochemical Activity in Photosystem-I Submembrane Fractions¶

The effects of irradiation on photosystem (PS)-I submembrane particles using intense white light (2000 μE·m−2·s−1) at chilling temperature (4°C) were studied. PSI-dependent oxygen uptake activity was stable during the first 3 h of photoinhibitory illumination in the presence of added superoxide dismutase (SOD). Without added SOD, the oxygen uptake almost doubled during this period, presumably due to the denaturation of native membrane-bound SOD or its release from the PSI membranes. The total chlorophyll (Chl) content and the magnitude of light-induced absorbance changes at 830 nm (ΔA830) were also barely affected during the first 3–3.5 h of photoinhibitory treatment. However, further exposure to strong light markedly accelerated Chl breakdown followed by a decline in oxygen uptake rate and ΔA830. This corresponded with the disappearance of the bands attributed to PsaA/B polypeptides on electrophoretic gels. Despite the invariant maximum magnitude of ΔA830 during the first 3–3.5 h of photoinhibitory treatment, the light–response curves of P700 oxidation gradually altered, demonstrating a several-fold increase in the ability of weak actinic light to oxidize P700. The major Chl a–protein 1 (CP1) band gradually disappeared during the first 4 h of light exposure with a corresponding increase in the Chl content of a band with lower electrophoretic mobility ascribed to the formation of oligomers containing CP1, light-harvesting complex I (LHCI)-680 and LHCI-730. This aggregation of Chl–protein complexes, likely caused by photoinhibitory-induced cross-linking favoring light harvesting, is proposed to explain the enhanced capacity of weak light to oxidize P700 in photoinhibited PSI submembrane fractions compared with untreated ones.

Spermine and spermidine inhibition of photosystem II: Disassembly of the oxygen evolving complex and consequent perturbation in electron donation from Tyr Z to P680 + and the quinone acceptors Q A− to Q B

Polyamines are implicated in plant growth and stress response. However, the polyamines spermine and spermidine were shown to elicit strong inhibitory effects in photosystem II (PSII) submembrane fractions. We have studied the mechanism of this inhibitory action in detail. The inhibition of electron transport in PSII submembrane fractions treated with millimolar concentrations of spermine or spermidine led to the decline of plastoquinone reduction, which was reversed by the artificial electron donor diphenylcarbazide. The above inhibition was due to the loss of the extrinsic polypeptides associated with the oxygen evolving complex. Thermoluminescence measurements revealed that charge recombination between the quinone acceptors of PSII, Q A and Q B, and the S 2 state of the Mn-cluster was abolished. Also, the dark decay of chlorophyll fluorescence after a single turn-over white flash was greatly retarded indicating a slower rate of Q A − reoxidation.

Regulation of Energy Dissipation in Photosystem I by the Redox State of the Plastoquinone Pool

The effect of exogenous plastoquinone (PQ) on the different deexcitation pathways of photosystem I (PSI) was investigated. Addition of oxidized decyl-plastoquinone (dPQ) and PQ-2 strongly quenched the chlorophyll (Chl) emission spectra of PSI submembrane fractions over all wavelengths. This quenching increased with the concentration of exogenous PQ added and followed the modified Stern-Volmer law. The Stern-Volmer constants found for dPQ and PQ-2 were 1.25 x 10(6) M-1 and 0.55 x 10(6) M-1, respectively, and the fraction of fluorescence accessible to the quencher was 0.7 for both exogenous PQ. dPQ and PQ-2 also retarded the P700 photooxidation measured under limiting actinic light irradiances. Photoacoustic measurements showed that addition of dPQ increased the heat dissipation and decreased the photochemical capacity of PSI. From these results, exogenous oxidized PQ were shown to efficiently quench the Chl excited state in the PSI antenna and change the balance between Chl deexcitation pathways. Moreover, reduction of the endogenous PQ pool in whole thylakoid membranes by NADPH increased PSI fluorescence by 65%, indicating the importance of the redox state of the PQ pool on PSI energy dissipation.

Irradiation-induced in vivo re-localization of NADPH-protochlorophyllide oxidoreductase from prolamellar body to stroma of barley etioplast

Distribution of NADPH-protochlorophyllide oxidoreductase (POR) in etioplast of etiolated barley leaf was studied by using Western blot analyses of etioplast fractions isolated on a sucrose gradient. When the leaf was exposed to light, POR content decreased in the etioplast inner membrane and prolamellar body sub-membrane fraction while it was simultaneously increased in the stroma. By using 77 K fluorescence spectroscopy analyzes, we found for irradiated etiolated leaf that the POR protein in the stroma was co-localized with chlorophyllide (Chlide) emitting at 678 nm. Relocalization of the POR-Chlide complex induced by irradiation suggests that POR participates in the pigment transport processes during early stages of the thylakoid membrane development.

Interaction of polyamines with proteins of photosystem II: Cation binding and photosynthetic oxygen evolution

Polyamines are organic cations that function in diverse physiological processes that share as a common thread a close relationship to cell proliferation and growth. Polyamines also affect photosynthetic oxygen evolution and therefore, this study was designed to investigate the interaction of 1,3-diaminopropane, 1,4-diaminobutane (putrescine), and 1,5-diaminopentane (cadaverine) cations with proteins of photosystem II (PSII) using PSII-enriched submembrane fractions with diamine concentrations between 0.01 and 20 mM. Fourier transformed infrared (FTIR) difference spectroscopy with its self-deconvolution and second derivative resolution enhancement, as well as curve-fitting procedures were applied in order to determine the diamine binding mode, the protein conformational changes, and the structural properties of diamine-protein complexes. Spectroscopic evidence showed that diamines interact with proteins (H-bonding) through polypeptide C O groups with no major perturbations of protein secondary structure. At very low diamine concentration (0.01 mM), no inhibition of oxygen-evolution occurred, while at higher diamine content (5–10 mM), 100% inhibition was observed. Chorophyll fluorescence measurements demonstrated that the inhibition mainly affects the oxygen evolving complex of PSII. Comparisons of the effects of these dipositive organic cations with divalent metal cations on one hand and with polyvalent spermine and spermidine on the other hand, show major alterations of the protein secondary structure as positive charge increases.

Presence of ‘PSI free’ LHCI and monomeric LHCII and subsequent effects on fluorescence characteristics in lincomycin treated maize

The cause of the strong non-photochemical fluorescence quenching was examined in maize (Zea mays L.) plants that were treated with lincomycin during the 72 h period of greening. They were deficient in core complexes but seemed to contain the full complement of antennae. The following results were obtained: (1) High F(o) could not be attributed to the dark reduction of Q(A) but to the presence of a high amount of not properly organized antenna complexes due to the inhibited synthesis of reaction centres. (2) On illumination fluorescence intensity dropped considerably below F(o) within 20 s, and reached a steady state still below F(o). (3) Slowly relaxing part of non-photochemical quenching was significantly higher than in control plants. (4) De-epoxidation state was constant, and corresponded to the maximal value of the control. (5) Free Lhca1/4 dimers could be detected in all submembrane fractions, including the grana, obtained by digitonin fractionation. (6) Increase in the 679 and 700 nm fluorescence emissions could be attributed to the monomerisation of part of LHCII and to the presence of free Lhca2 or LHCII aggregates, respectively. (7) LHCII or PSII+LHCII and Lhca1/4 interaction may contribute to the increase of long-wavelength fluorescence in the granal fraction. We assume that the elevated fluorescence quenching of monomeric LHCII as well as the interaction between LHCII or PSII+LHCII and Lhca1/4 can be considered as an explanation for the extensive non-photochemical fluorescence quenching in lincomycin treated plants. The permanent presence of zeaxanthin may have contributed to the fast formation of quenching.

Protective effect of active oxygen scavengers on protein degradation and photochemical function in photosystem I submembrane fractions during light stress

The protective role of reactive oxygen scavengers against photodamage was studied in isolated photosystem (PS) I submembrane fractions illuminated (2000 microE x m(-2) x s(-1)) for various periods at 4 degrees C. The photochemical activity of the submembrane fractions measured as P700 photooxidation was significantly protected in the presence of histidine or n-propyl gallate. Chlorophyll photobleaching resulting in a decrease of absorbance and fluorescence, and a blue-shift of both absorbance and fluorescence maximum in the red region, was also greatly delayed in the presence of these scavengers. Western blot analysis revealed the light harvesting antenna complexes of PSI, Lhca2 and Lhca1, were more susceptible to strong light when compared to Lhca3 and Lhca4. The reaction-center proteins PsaB, PsaC, and PsaE were most sensitive to strong illumination while other polypeptides were less affected. Addition of histidine or n-propyl gallate lead to significant protection of reaction-center proteins as well as Lhca against strong illumination. Circular dichroism (CD) spectra revealed that the alpha-helix content decreased with increasing period of light exposure, whereas beta-strands, turns, and unordered structure increased. This unfolding was prevented with the addition of histidine or n-propyl gallate even after 10 h of strong illumination. Catalase or superoxide dismutase could not minimize the alteration of PSI photochemical activity and structure due to photodamage. The specific action of histidine and n-propyl gallate indicates that 1O2 was the main form of reactive oxygen species responsible for strong light-induced damage in PSI submembrane fractions.

Profiling and comprehensive expression analysis of ABC transporter solute-binding proteins of Bacillus subtilis membrane based on a proteomic approach.

We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).

Control of energy dissipation and photochemical activity in photosystem I by NADP-dependent reversible conformational changes.

Addition of NADP(+) to thylakoid membranes or isolated photosystem I (PSI) submembrane fractions quenched chlorophyll fluorescence by up to 40% at low or room temperature. This quenching was reversed by NADPH. Similar quenching was also observed with the addition of heparin or thenoyltrifluoroacetone (TTFA), inhibitors that bind ferredoxin:NADP(+) reductase (FNR) and prevent reduction of NADP(+). The NADP(+)-induced quenching coincided with a reversible conformational change of the secondary protein structure in the PSI submembrane fractions where 20% of the alpha-helix conformations were transformed mainly into beta-sheet-like structures. Further, P700 photooxidation was retarded due to this conformational change, and about 25% of the centers could not be photooxidized, these changes being also reversible with addition of NADPH. The above modifications in the presence of NADP(+) also increased photodamage processes under strong illumination, and NADPH protected it. Conformational modification of FNR upon binding of NADP(+) or NADPH is proposed to trigger the macromolecular changes in a larger part of the protein complex of PSI. The conformational changes must increase the intermolecular distances and change the mutual orientation between the various cofactors in the PSI complex. This new control mechanism of energy dissipation and photochemical activity by NADP(+)/NADPH is proposed to increase the turnover rate of PSI under conditions when both linear and cyclic electron transport activities must be supported.

Quenching of excited states of chlorophyll molecules in submembrane fractions of Photosystem I by exogenous quinones

The ability of three substituted quinones, 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB), 2,6-dichloro- p-benzoquinone (DCBQ), and tetramethyl- p-benzoquinone (duriquinone) to quench the excited states of chlorophyll (Chl) molecules in Photosystem I (PSI) was studied. Chl fluorescence emission measured with isolated PSI submembrane fractions was reduced following the addition of exogenous quinones. This quenching progressively increased with rising concentrations of the exogenous quinones according to the Stern–Volmer law. The values of Stern–Volmer quenching coefficients were found to be 3.28×10 5 M −1 (DBMIB), 1.31×10 4 M −1 (DCBQ), and 3.7×10 3 M −1 (duroquinone). The relative quenching capacities of the various exogenous quinones in PSI thus strictly coincided to those found for the quenching of Fo level of Chl fluorescence in isolated thylakoids, which is emitted largely by Photosystem II (PSII) [Biochim. Biophys. Acta (2003) 1604, 115–123]. Quenching of Chl excited states in PSI submembrane fractions by exogenous quinones slowed down the rate of P700, primary electron donor of PSI, photooxidation measured at limiting actinic light irradiances thus revealing a reduced photochemical capacity of absorbed quanta. The possible involvement of non-photochemical quenching of excited Chl states by oxidized phylloquinones, electron acceptors of PSI, and oxidized plastoquinones, mobile electron carriers between PSII and the cytochrome b 6/ f complex, into the control of photochemical activity of PSI is discussed.

Photoinhibitory light-induced changes in the composition of chlorophyll-protein complexes and photochemical activity in photosystem-I submembrane fractions.

The effects of irradiation on photosystem (PS)-I submembrane particles using intense white light (2000 micoE x m(-2) x S(-1)) at chilling temperature (4 degrees C) were studied. PSI-dependent oxygen uptake activity was stable during the first 3 h of photoinhibitory illumination in the presence of added superoxide dismutase (SOD). Without added SOD, the oxygen uptake almost doubled during this period, presumably due to the denaturation of native membrane-bound SOD or its release from the PSI membranes. The total chlorophyll (Chl) content and the magnitude of light-induced absorbance changes at 830 nm (deltaA830) were also barely affected during the first 3-3.5 h of photoinhibitory treatment. However, further exposure to strong light markedly accelerated Chl breakdown followed by a decline in oxygen uptake rate and deltaA830. This corresponded with the disappearance of the bands attributed to PsaA/B polypeptides on electrophoretic gels. Despite the invariant maximum magnitude of deltaA830 during the first 3-3.5 h of photoinhibitory treatment, the light-response curves of P700 oxidation gradually altered, demonstrating a several-fold increase in the ability of weak actinic light to oxidize P700. The major Chl a-protein 1 (CP1) band gradually disappeared during the first 4 h of light exposure with a corresponding increase in the Chl content of a band with lower electrophoretic mobility ascribed to the formation of oligomers containing CP1, light-harvesting complex I (LHCI)-680 and LHCI-730. This aggregation of Chl-protein complexes, likely caused by photoinhibitory-induced cross-linking favoring light harvesting, is proposed to explain the enhanced capacity of weak light to oxidize P700 in photoinhibited PSI submembrane fractions compared with untreated ones.

ＰＶＤＦ膜を利用した高感度・ハイスループットＭＡＬＤＩ−ＴＯＦ ＭＳによるプロテオーム解析法の開発と細菌細胞膜タンパク質解析への応用

To improve sample preparation method for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) with the aim of proteomics, proteins separated by Tris/tricine SDS-PAGE were electroblotted onto PVDF membrane by a semi-dry discontinuous buffer system, visualized by staining with Coomassie Brilliant Blue, excised, digested with trypsin or lysC in the presence of 80% acetonitrile. Digested proteins were analyzed with MALDI-TOF MS. This method has several advantages over “in gel digestion” in terms of simple handling, sensitivity and time. We analyzed 105 fmol of Bacillus subtilis SecA protein and 300 to 500 fmol of standard proteins. We also analyzed three submembrane fractions of B. subtilis which had been solubilized stepwise using different mixtures of detergents from a washed cell membrane that was insoluble in 134 mM non-detergent sulfobetain solution. After two different 1D- and three different 2D-PAGEs followed by MALDI-TOF MS analysis, we identified 637 different proteins. Among them, 357 were predicted to be membrane proteins having certain or putative transmembrane segments (TMS) using the analysis by TopPred II algorithm, but 280 were soluble proteins having no TMS. On the basis of a bioinformatics analysis on B. subtilis genome data-base, 38 ABC trasnporter solute-binding proteins were predicted and 30 of the 38 were identified by this analysis. However, this improved method still have a limitation for the analysis of cell membrane proteins having more than 4 TMSs in their amino acid sequences.

Potential for the use of photosystem II submembrane fractions immobilised in poly(vinylalcohol) to detect heavy metals in solution or in sewage sludge

Photosystem II submembrane fractions were immobilised by entrapment in poly(vinylalcohol) bearing styrylpyridinium groups (PVA-SbQ). The properties of the immobilised material, in a single-compartment micro-photoelectrochemical cell using platinum electrodes in potentiostatic mode, were compared with native (free) samples. The optimal operating conditions were investigated (electron acceptor concentration, pH, temperature, time contact and chlorophyll concentration). The photocurrent of the immobilised fractions could be inhibited by pollutants such as heavy metals (mercury, copper, lead, cadmium, chromium, nickel, and zinc) in solution. The potential for use of this system to evaluate the toxicity of sewage sludges was shown.