Treatment Of Thylakoids Research Articles

Pancreatic lipase–colipase binds strongly to the thylakoid membrane surface

Isolated thylakoid membranes, i.e. the photosynthetic membranes of green leaves, inhibit the activity of pancreatic lipase and colipase during hydrolysis of fat in vitro. This inhibition has been demonstrated to cause reduced food intake and improved hormonal and lipid profile in vivo. One of the reasons suggested for the inhibiting effect is binding of lipase-colipase to the thylakoid membrane surface. This prompted a study of the binding of lipase and colipase to thylakoids. The results showed that lipase and colipase strongly bind to the thylakoid membrane surface. The dissociation constant was determined at 1.2 × 10⁻⁸ mol L⁻¹; binding decreased after treatment of thylakoids with pepsin/trypsin to 1.0 × 10⁻⁷ and to 0.6 × 10⁻⁷ mol L⁻¹ after treatment with pancreatic juice. Similarly, delipidation of thylakoids caused a decrease in binding, the dissociation constant being 2.0 × 10⁻⁷ mol L⁻¹. The binding of pancreatic lipase-colipase to the thylakoid membrane is strong and may explain the inhibition of lipase-colipase activity by thylakoids. After treatment with proteases to mimic intestinal digestion binding is decreased, but is still high enough to explain the observed metabolic effects of thylakoids in vivo.

Identification of a 2-cys peroxiredoxin as a tetramethyl benzidine-hydrogen peroxide stained protein from the thylakoids of the extreme halophyte Arthrocnemum macrostachyum L.

Tetramethylbenzidine-H(2)O(2) staining of SDS-polyacrylamide gel is a widely used method for the specific detection of proteins with heme-dependent peroxidase activity. When this method was used with thylakoids from the halophytic plant Arthrocnemum macrostachyum, besides the cytochrome f and cytochrome b6 proteins usually found in higher plants and cyanobacteria, at least four additional bands were detected. One of them, a 46-kDa protein, was shown to be an extrinsic protein, and identified by mass spectrometry and immunoblotting as a 2-cys peroxiredoxin. Peroxidase activity was insensitive to oxidizing agents such as trans-4,4-diydroxy-1,2-dithiane or hydrogen peroxide, but was inhibited by treatment of thylakoids with reducing agents such as dithiothreitol or mercaptoethanol. By immunoblotting, it was shown that loss of peroxidase activity was paralleled by disappearance of the 46-kDa band, which was converted to a 23-kDa immunoreactive form. A dimer/monomer relationship between the two proteins is suggested, with the dimeric form likely being a heme-binding protein. This possibility was further supported by anionic exchange chromatography and de novo sequencing of tryptic fragments of the protein and sequence comparison, as most of the residues previously implicated in heme binding in 2-cys peroxiredoxin from Rattus norvegicus were conserved in A. macrostachyum. The amount of this protein was modulated by environmental conditions, and increased when salt concentration in the growth medium was higher or lower than the optimal one.

Multiple sites of retardation of electron transfer in Photosystem II after hydrolysis of phosphatidylglycerol

Phosphatidylglycerol (PG), containing the unique fatty acid Delta3, trans-16:1-hexadecenoic acid, is a minor but ubiquitous lipid component of thylakoid membranes of chloroplasts and cyanobacteria. We investigated its role in electron transfers and structural organization of Photosystem II (PSII) by treating Arabidopsis thaliana thylakoids with phospholipase A(2) to decrease the PG content. Phospholipase A(2) treatment of thylakoids (a) inhibited electron transfer from the primary quinone acceptor Q(A) to the secondary quinone acceptor Q(B), (b) retarded electron transfer from the manganese cluster to the redox-active tyrosine Z, (c) decreased the extent of flash-induced oxidation of tyrosine Z and dark-stable tyrosine D in parallel, and (d) inhibited PSII reaction centres such that electron flow to silicomolybdate in continuous light was inhibited. In addition, phospholipase A(2) treatment of thylakoids caused the partial dissociation of (a) PSII supercomplexes into PSII dimers that do not have the complete light-harvesting complex of PSII (LHCII); (b) PSII dimers into monomers; and (c) trimers of LHCII into monomers. Thus, removal of PG by phospholipase A(2) brings about profound structural changes in PSII, leading to inhibition/retardation of electron transfer on the donor side, in the reaction centre, and on the acceptor side. Our results broaden the simple view of the predominant effect being on the Q(B)-binding site.

Chloroplast Oxa1p Homolog Albino3 Is Required for Post-translational Integration of the Light Harvesting Chlorophyll-binding Protein into Thylakoid Membranes

Multiple sorting pathways operate in chloroplasts to localize proteins to the thylakoid membrane. The signal recognition particle (SRP) pathway in chloroplasts employs the function of a signal recognition particle (cpSRP) to target light harvesting chlorophyll-binding protein (LHCP) to the thylakoid membrane. In assays that reconstitute stroma-dependent LHCP integration in vitro, the stroma is replaceable by the addition of GTP, cpSRP, and an SRP receptor homolog, cpFtsY. Still lacking is an understanding of events that take place at the thylakoid membrane including the identification of membrane proteins that may function at the level of cpFtsY binding or LHCP integration. The identification of Oxa1p in mitochondria, an inner membrane translocase component homologous to predicted proteins in bacteria and to the albino3 (ALB3) protein in thylakoids, led us to investigate the potential role of ALB3 in LHCP integration. Antibody raised against a 50-amino acid region of ALB3 (ALB3-50aa) identified a single 45-kDa thylakoid protein. Treatment of thylakoids with antibody to ALB3-50aa inhibited LHCP integration, whereas the same antibody treatment performed in the presence of antigen reversed the inhibition. In contrast, transport by the thylakoid Sec or Delta pH pathways was unaffected. These data support a model whereby a distinct translocase containing ALB3 is used to integrate LHCP into thylakoid membranes.

Transmembrane topology of the Rieske Fe/S protein of the cytochrome b 6/f complex from spinach chloroplasts

The topology of the Rieske protein of the cytochrome b 6/f complex in thylakoids from spinach chloroplasts was examined by protease protection experiments as well as polypeptide extraction assays using solutions of chaotropic salts or alkaline pH. While neither thermolysin nor trypsin cleave any of the Rieske protein when added to the stromal side of the thylakoid membrane, proteinase K is capable of removing approximately four residues from its NH 2-terminus. The protein is resistant to membrane extraction by 0.1 M Na 2CO 3 or 2 M NaBr but is quantitatively released by 0.1 M NaOH. Treatment of thylakoids with 2 M NaSCN leads to extraction of variable amounts of the protein, depending on the presence or absence of sucrose in the medium which apparently stabilizes the cytochrome complex. From these results we conclude that the Rieske protein is an integral component of the cytochrome complex which spans the thylakoid membrane with a single hydrophobic segment and is anchored predominantly by electrostatic interactions.

The role of phospholipids in regulating photosynthetic electron transport activities: Treatment of thylakoids with phospholipase C

The involvement of phospholipids in the regulation of photosynthetic electron transport activities was studied by incubating isolated pea thylakoids with phospholipase C to remove the head-group of phospholipid molecules. The treatment was effective in eliminating 40-50% of chloroplast phospholipids and resulted in a drastic decrease of photosynthetic electron transport. Measurements of whole electron transport (H2O→methylviologen) and Photosystem II activity (H2O→p-benzoquinone) demonstrated that the decrease of electron flow was due to the inactivation of Photosystem II centers. The variable part of fluorescence induction measured in the absence of electron acceptor was decreased by the progress of phospholipase C hydrolysis and part of the signal could be restored on addition of 3-(3',4'-dicholorophenyl)-1,1-dimethylurea. The B and Q bands of thermoluminescence corresponding to S2S3QB (-) and S2S3QA (-) charge recombination, respectively, was also decreased with a concomitant increase of the C band, which originated from the tyrosine D(+)QA (-) charge recombination. These results suggest that phospholipid molecules play an important role in maintaining the membrane organization and thus maintaining the electron transport activity of Photosystem II complexes.

Electro-optical investigation of thylakoids and PS II fragments treated with trypsin

The surface electric properties of thylakoids and Photosystem II-enriched particles subjected to proteolytic treatment were investigated by electric light scattering and microelectrophoresis. The observed variation of the charge asymmetry (permanent dipole moment) and electric polarizability upon treatment of thylakoids with trypsin in a wide concentration range and different time of enzyme action, associated with the removal of positively charged polypeptides from the thylakoid membranes, correlated with the increase of the electrokinetic charge. The higher transversal charge asymmetry of trypsin-treated Photosystem II fragments could be connected with destacking of the paired membranes upon treatment with trypsin and formation of unpaired single membrane fragments.

On the function of subunit PsaE in chloroplast Photosystem I

Treatment of thylakoids from spinach with NaSCN removes extrinsic stroma-exposed subunits of the Photosystem I complex in addition to CF 1 and some other surface proteins. By increasing the NaSCN concentration, PsaE is released first, followed by PsaD and PsaC. At 0.5 M NaSCN, about 80% of PsaE is resolved without significant loss of other PS I polypeptides. Time-resolved fluorescence spectroscopy showed no significant alteration of PS I isolated from membranes thus treated with regard to energy transfer within the antennas as well as primary charge separation. Washing of thylakoids with NaSCN results in inhibition of electron transport from an artificial electron donor (ascorbate/DAD) to either methylviologen or NADP. Although higher NaSCN concentrations are required for inhibition than for resolution of PsaE, electron transport is restored by reconstitution with isolated PsaE from Synechococcus. We conclude that inhibition is due to dislocation of PsaC as a consequence of PsaE resolution, impeding efficient electron transfer from F x to F A F B . An antibody raised against PsaC inhibits methylviologen reduction only when PsaE has been removed previously. An antibody raised against PsaE inhibits electron transport to NADP, but not to methylviologen. We conclude that binding of this antibody sterically hinders the access of ferredoxin either to the F A F B center or the catalytic site of ferredoxin: NADP oxidoreductase (FNR). Our results suggest an essential role of PsaE in stabilization of the acceptor side of PS I, in particular in maintenance of the functional integrity between the F A F B protein and the membrane-integral sector of the PS I core.

Zero-length crosslinking between subunits σ and I of the H +-translocating ATPase of chloroplasts

Treatment of spinach thylakoids with 1-ethyl-3-(dimethylaminopropyl)-carbdiimide (EDC) / N-hydroxysulfosuccinimide (sulfo-NHS) induced formation of a zero-length crosslink of an apparent molecular mass of 38 kDa. This product was shown, by immunodetection, to consist of subunit σ of CF 1 and subunit I of CF 0. The crosslink was isolated by preparative SDS gel electrophoresis and subjected to cyanogen bromide cleavage. Electrophoretic and immunological analysis of the resulting peptides suggested that the crosslink was formed between a glutamyl or aspartyl residue at the C-terminal end of subunit I and a basic amino acid of subunit δ in the range between Val-1 to Met-165. Treatment of thylakoids with EDC/Sulfo-NHS resulted in inhibition of photophosphorylation and CF 0CF 1-catalyzed ATP hydrolysis without affecting formation of a proton gradient related to phenazine methosulfate mediated cyclic electron transport. Inhibition of H + transport-coupled ATP hydrolysis was more pronounced than non-coupled methanol-stimulated ATP hydrolysis. The results suggest that subunits δ and I form a connection between the partial complexes CF 1 and CF 0 in situ. Crosslinking of the subunits may impede the translocation of protons through CF 0CF 1.

Characterization of nucleotide binding sites on membrane-bound chloroplast ATPase by modification with pyridoxal 5′-phosphate

Treatment of chloroplast thylakoids with pyridoxal 5′-phosphate (PLP)in the light or in the presence of Mg 2+ causes inhibition of photophosphorylation which is partially competitive to ADP and to inorganic phosphate (P i ), suggesting that PLP may modify essential lysine residues in the catalytic nucleotide binding site of the thylakoid H + -ATPase. Treatment of thylakoids with PLP and NaB 3 H 4 results in incorporation of about 4 mol [ 3 H]PLP/mol CF 1 almost equally distributed between α- and β-subunits. ADP plus P i , prevents incorporation of one PLP per three α-subunits and one per three β-subunits, but causes almost full protection against PLP inactivation, suggesting that PLP modification of only one α- and one β-subunit is sufficient for inactivation of the enzyme. As PLP modification of the ATPase is largely excluded in the absence of Mg 2+ , modification of the active site may require ionic fixation of PLP with the help of the phosphate side-chain via Mg 2+ , similar to the interaction of P β and P γ of ATP with the protein in order to facilitate the covalent attack to the vicinal lysine residue.

Probing the events of photoinhibition by altering electron‐transport activity and light‐harvesting capacity in chloroplast thylakoids

Abstract. The effect of photoinhibition on the activity of photosystem II (PSII) in spinach chloroplasts was investigated. Direct light‐induced absorbance change measurements at 320 nm (ΔA320) provided a measure of the PSII charge separation reaction and revealed that photoinhibition prevented the stable photoreduction of the primary quinone acceptor QA. Sensitivity to photoinhibition was substantially enhanced by treatment of thylakoids with NH2OH which extracts manganese from the H2O‐splitting enzyme and prevents electron donation to the reaction centre. Incubation with 3‐(3,4,‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) during light exposure did not affect the extent of photoinhibitory damage. The chlorophyll (Chl) b‐less chlorina (2 mutant of barley displayed a significantly smaller light‐harvesting antenna size of PSII (about 20% of that in wild type chloroplasts) and, simultaneously, a lower sensitivity to photoinhibition. These observations suggest that photoinhibition depends on the amount of light absorbed by PSII and that the process of photoinhibition is accelerated when electron donation to the reaction centre is prevented. It is postulated that the probability of photoinhibition is greater when excitation energy is trapped by P680+, the oxidized form of the PSII reaction centre. The results are discussed in terms of the D1/D2 heterodimer which contains the functional PSII components P680, pheophytin, QA and QB.

Probing for the interaction of the 32 kDa-Q B protein with its environment by use of bifunctional cross-linking reagents

Chlamydomonas reinhardtii thylakoid polypeptides were cross-linked with hydrophobic or hydrophilic homoor hetero-bifunctional reagents, including cleavable cross-linkers containing a dithiol group (3,3-dithiobis(succinimidylpropionate), N-succinimidyl-3-(2-pyridyldithio)propionate and 3,3-dithiobis(sulfosuccinimidylpropionate), abbreviated to DTSP, SPDP and DTSSP, respectively) or glutaraldehyde. None of these reagents cross-linked the 32 kDa-Q B-protein, as identified by specific radioactive labeling and reaction with antibodies. Treatment of thylakoids with β- d-octylglucoside prior to addition of all the cross-linkers used, resulted in effective cross-linking of the 32 kDa-Q B-protein. Treatment of thylakoids with cross-linkers, followed by removal of the non-reacted reagents and addition of β- d-octylglucoside, caused extensive cross-linking of the 32 kDa-Q B-protein by SPDP and, to a lesser extent, by DTSP, indicating a possible formation of monovalent links with the 32 kDa-Q B-protein in the native membrane. The 32 kDa-Q B-protein was not cleaved by trypsin in thylakoids treated with DTSP, DTSSP or glutaraldehyde, while SPDP did not have a similar protective effect. Photosystem II activity and the affinity for Diuron binding and/or the number of binding sites were also affected by cross-linkers. None of the cross-linkers prevented the light-induced degradation of the 32 kDa-Q B-protein in isolated thylakoids exposed to high light intensity. It is suggested that the 32 kDa-Q B-protein is confined within a specific hydrophobic environment which might be essential for its function and prevent the cross-linking of the 32 kDa-Q B-proteins −SH or −HN 2 groups with similar reactive groups of neighboring proteins. Alteration of the membrane lipid phase by detergent treatment allows cross-linking to occur.

Crystallization of the light-harvesting chlorophyll a/b complex within thylakoid membranes.

We have found that treatment of the photosynthetic membranes of green plants, or thylakoids, with the nonionic detergent Triton X-114 at a 10:1 ratio has three effects: (a) photosystem I and coupling factor are solubilized, so that the membranes retain only photosystem II (PS II) and its associated light-harvesting apparatus (LHC-II); (b) LHC-II is crystallized, and so is removed from its normal association with PS II; and (c) LHC-II crystallization causes a characteristic red shift in the 77 degrees K fluorescence from LHC-II. Treatment of thylakoids with the same detergent at a 20:1 ratio results in an equivalent loss of photosystem I and coupling factor, with LHC-II and PS II being retained by the membranes. However, no LHC-II crystals are formed, nor is there a shift in fluorescence. Thus, isolation of a membrane protein is not required for its crystallization, but the conditions of detergent treatment are critical. Membranes with crystallized LHC-II retain tetrameric particles on their surface but have no recognizable stromal fracture face. We have proposed a model to explain these results: LHC-II is normally found within the stromal half of the membrane bilayer and is reoriented during the crystallization process. This reorientation causes the specific fluorescence changes associated with crystallization. Tetrameric particles, which are not changed in any way by the crystallization process, do not consist of LHC-II complexes. PS II appears to be the only other major complex retained by these membranes, which suggests that the tetramers consist of PS II.

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane

The transverse heterogeneity of the polypeptides associated with the Photosystem I (PSI) complex in spinach thylakoid membranes and in a highly resolved PSI preparation has been studied using the impermeant chemical modifier, 2,4,6-trinitrobenzenesulfonate (TNBS) and the proteolytic enzyme, Pronase E. The present study has shown that the PSI reaction center polypeptide of approximately 62 kilodaltons and the 22 and 20 kilodalton polypeptides of the PSI light-harvesting chlorophyll protein (LHCPI) complex are not labeled by [(14)C]TNBS in unfractionated thylakoids. On the other hand, the 23 kilodalton polypeptide of the PSI LHCP and the 19 and 14 kilodalton polypeptides associated with the PSI primary electron acceptor complex are readily labeled by [(14)C]TNBS and are exposed to the stromal side of the thylakoid. Differences and similarities in the labeling of polypeptides associated with the PSI complex in thylakoids and in the isolated PSI complex are also noted. Treatment of thylakoids with pronase had no effect on the organization of the polypeptides in the LHCPI or the reaction center core complex, as manifested by the separation of these two subcomplexes from pronase-treated membranes. The 62, 19, and 14 kilodalton polypeptides associated with the reaction center core complex and the 23 and 22 kilodalton polypeptides associated with LHCPI are sensitive to pronase treatment while the 20 kilodalton polypeptide of LHCPI was inaccessible to the protease. The proteolysis of the 62 kilodalton polypeptide generated first a single immunodetectable fragment at about 48 kilodaltons, and further proteolytic digestion generated two other fragments at 30 and 17 kilodaltons respectively. These results are discussed in relation to the organization of the PSI complex in spinach thylakoids. A model for the transmembrane topography of the polypeptide constituents of PSI has been developed.

Effects of physical and chemical treatments on chloroplast manganese. NMR and ESR studies

Measurements were made of the water proton relaxation rate ( T −1 2 = R 2), electron spin resonance (ESR) six-line signal of ‘free’ Mn 2+, and O 2-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in O 2-evolution activity, in R 2 and in the content of bound Mn, suggesting that R 2 may be related to the loosely bound Mn involved in O 2 evolution. (2) Treatment of thylakoids with tetraphenylboron (TPB) at [TPB] > 2 mM produces a 2-fold increase in R 2, without release of Mn 2+. The titration curve exhibits three sharp end points. The first end point occurs at a [ TPB] [ chlorophyll] of 1.25, at which the O 2 evolution is completely inhibited. (3) Treatment of thylakoids with NH 2OH also increases R 2 by nearly 2-fold, either by the reduction of the higher oxidation states of Mn to Mn 2+ and / or by exposing the Mn to solvent protons. Also, progressive release of bound Mn occurs at [NH 2OH] ≥ 1 mM as shown by an increase increase in the Mn 2+ ESR signal and a decrease in R 2. (4) Addition of H 2O 2 (0.1–1.0%) to thylakoids causes an enhancement of R 2 similar to that by NH 2OH, but without the release of Mn 2+. (5) Heat treatment of thylakoids at 40–50°C releases Mn 2+ and increases R 2. Conversely, pH values of 7 to 4 release Mn 2+ without changing R 2 while pH values of 7–9 increase R 2 without releasing Mn 2+. Thus, both high and low pH values as well as the heat treatment cause structural changes enhancing the relaxivity of the bound Mn or of other paramagnetic species.

Uncoupling and energy transfer inhibition of photophosphorylation by sulfhydryl reagents.

The action of the sulfhydryl reagents, o-iodosobenzoate and 2,2’-dithiobis(5-nitropyridine), on photophosphorylation by spinach chloroplast thylakoids has been reevaluated. Both of these compounds were previously reported to be energy transfer inhibitors of photophosphorylation, provided the thylakoids were illuminated in their presence prior to assay. We show here that the treatment of thylakoids in the light with iodosobenzoate uncouples phosphorylation from electron flow. This treatment enhances nonphosphorylating electron transport and markedly decreases the efficiency of photophosphorylation. The light-induced transmembrane pH gradient is also diminished by exposure of thylakoids to iodosobenzoate in the light. Dithiobisnitropyridine has been found to act either as a light-dependent uncoupler or energy transfer inhibitor. At low concentrations of this reagent, illumination elicits uncoupling, whereas at higher concentrations, energy transfer inhibition is induced. The uncoupling by iodosobenzoate and by low concentrations of dithiobisnitropyridine is largely prevented by the prior incubation of thylakoids with N-ethylmaleimide in the dark. Under these conditions, N-ethylmaleimide was previously shown to react with a group on the y subunit and with a group on the E subunit of coupling factor 1. The effects of these sulfhydryl reagents on photophosphorylation are compared to those of maleimides, and a model for the inhibition of phosphorylation by these reagents is proposed. Cross-linking two sufhydryl groups within the y subunit of coupling factor 1, either directly by disulfide bond formation or by bifunctional maleimides, causes thylakoids to become proton-leaky and phosphorylation is uncoupled from electron flow. In contrast, modification of a sulfhydryl, which becomes exposed only in the light, by monofunctional reagents elicits energy transfer inhibition.

Nachweis der Zucker in den Thylakoiden von Rhodospirillum rubrum und Rhodopseudomonas viridis

The thylakoids (chromatophores) of the sulfur-free purple bacteria Rhodospirillum rubrum contain 30% lipids soluble in methanol-chloroform, 46% protein, and 14% carbohydrates. 90% of the total sugar content was glucose, 4% fucose, 5% rhamnose. In the thylakoids of Rhodopseudomonas viridis 3,3% sugar was demonstrable (50% glucose, 17% galactose, 15% rhamnose and 17% mannose). 2-keto-3-desoxy-octonate is a structure component in both organisms. After treatment of thylakoids with phenol/water, the main sugar fraction was in the water phase, although in both organisms 3 to 4% of the protein fraction in the phenol phase consists of sugar.