| 1. |
- Hallin, Erik Ingmar, et al.
(författare)
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Functional and structural characterization of domain truncated violaxanthin de-epoxidase
- 2016
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317. ; 157:4, s. 414-421
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Tidskriftsartikel (refereegranskat)abstract
- Photosynthetic organisms need protection against excessive light. By using non-photochemical quenching, where the excess light is converted into heat, the organism can survive at higher light intensities. This process is partly initiated by the formation of zeaxanthin, which is achieved by the de-epoxidation of violaxanthin and antheraxanthin to zeaxanthin. This reaction is catalyzed by violaxanthin de-epoxidase (VDE). VDE consists of three domains of which the central lipocalin-like domain has been the most characterized. By truncating the domains surrounding the lipocalin-like domain, we show that VDE activity is possible without the C-terminal domain but not without the N-terminal domain. The N-terminal domain shows no VDE activity by itself but when separately expressed domains are mixed, VDE activity is possible. This shows that these domains can be folded separately and could therefore be studied separately. An increase of the hydrodynamic radius of wild-type VDE was observed when pH was lowered toward the pH required for activity, consistent with a pH-dependent oligomerization. The C-terminally truncated VDE did not show such an oligomerization, was relatively more active at higher pH but did not alter the KM for ascorbate. Circular dichroism measurements revealed the presence of α-helical structure in both the N- and C-terminal domains. By measuring the initial formation of the product, VDE was found to convert a large number of violaxanthin molecules to antheraxanthin before producing any zeaxanthin, favoring a model where violaxanthin is bound non-symmetrically in VDE.
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| 2. |
- Hallin, Erik Ingmar, et al.
(författare)
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Molecular studies on structural changes and oligomerisation of violaxanthin de-epoxidase associated with the pH-dependent activation
- 2016
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Ingår i: Photosynthesis Research. - : Springer Science and Business Media LLC. - 0166-8595 .- 1573-5079. ; 129:1, s. 29-41
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Tidskriftsartikel (refereegranskat)abstract
- Violaxanthin de-epoxidase (VDE) is a conditionally soluble enzyme located in the thylakoid lumen and catalyses the conversion of violaxanthin to antheraxanthin and zeaxanthin, which are located in the thylakoid membrane. These reactions occur when the plant or algae are exposed to saturating light and the zeaxanthin formed is involved in the process of non-photochemical quenching that protects the photosynthetic machinery during stress. Oversaturation by light results in a reduction of the pH inside the thylakoids, which in turn activates VDE and the de-epoxidation of violaxanthin. To elucidate the structural events responsible for the pH-dependent activation of VDE, full length and truncated forms of VDE were studied at different pH using circular dichroism (CD) spectroscopy, crosslinking and small angle X-ray scattering (SAXS). CD spectroscopy showed the formation of α-helical coiled-coil structure, localised in the C-terminal domain. Chemical crosslinking of VDE showed that oligomers were formed at low pH, and suggested that the position of the N-terminal domain is located near the opening of lipocalin-like barrel, where violaxanthin has been predicted to bind. SAXS was used to generate models of monomeric VDE at high pH and also a presumably dimeric structure of VDE at low pH. For the dimer, the best fit suggests that the interaction is dominated by one of the domains, preferably the C-terminal domain due to the lost ability to oligomerise at low pH, shown in earlier studies, and the predicted formation of coiled-coil structure.
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| 3. |
- Hallin, Erik, et al.
(författare)
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Violaxanthin de-epoxidase disulphides and their role in activity and thermal stability.
- 2015
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Ingår i: Photosynthesis Research. - : Springer Science and Business Media LLC. - 0166-8595 .- 1573-5079. ; 124:2, s. 191-198
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Tidskriftsartikel (refereegranskat)abstract
- Violaxanthin de-epoxidase (VDE) catalyses the conversion of violaxanthin to zeaxanthin at the lumen side of the thylakoids during exposure to intense light. VDE consists of a cysteine-rich N-terminal domain, a lipocalin-like domain and a negatively charged C-terminal domain. That the cysteines are important for the activity of VDE is well known, but in what way is less understood. In this study, wild-type spinach VDE was expressed in E. coli as inclusion bodies, refolded and purified to give a highly active and homogenous preparation. The metal content (Fe, Cu, Ni, Mn, Co and Zn) was lower than 1 mol% excluding a metal-binding function of the cysteines. To investigate which of the 13 cysteines that could be important for the function of VDE, we constructed mutants where the cysteines were replaced by serines, one by one. For 12 out of 13 mutants the activity dropped by more than 99.9 %. A quantification of free cysteines showed that only the most N-terminal of these cysteines was in reduced form in the native VDE. A disulphide pattern in VDE of C9-C27, C14-C21, C33-C50, C37-C46, C65-C72 and C118-C284 was obtained after digestion of VDE with thermolysin followed by mass spectroscopy analysis of reduced versus non-reduced samples. The residual activity found for the mutants showed a variation that was consistent with the results obtained from mass spectroscopy. Reduction of the disulphides resulted in loss of a rigid structure and a decrease in thermal stability of 15 °C.
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| 4. |
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| 5. |
- Albertsson, Per-Åke, et al.
(författare)
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Chloroplast membranes retard fat digestion and induce satiety: effect of biological membranes on pancreatic lipase/co-lipase
- 2007
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Ingår i: Biochemical Journal. - 0264-6021. ; 401, s. 727-733
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Tidskriftsartikel (refereegranskat)abstract
- Human obesity is a global epidemic, which causes a rapidly increased frequency of diabetes and cardiovascular disease. One reason for obesity is the ready availability of refined food products with high caloric density, an evolutionarily new event, which makes over-consumption of food inevitable. Fat is a food product with high caloric density. The mechanism for regulation of fat intake has therefore been studied to a great extent. Such studies have shown that, as long as fat stays in the intestine, satiety is promoted. This occurs through the fat-released peptide hormones, the best known being CCK (cholecystokinin), which is released by fatty acids. Hence, retarded fat digestion with prolonged time for delivery of fatty acids promotes satiety. Pancreatic lipase, together with its protein cofactor, co-lipase, is the main enzymatic system responsible for intestinal fat digestion. We found that biological membranes, isolated from plants, animals or bacteria, inhibit the lipase/co-lipase-catalysed hydrolysis of triacylglycerols even in the presence of bile salt. We propose that the inhibition is due to binding of lipase/co-lipase to the membranes and adsorption of the membranes to the aqueous/triacylglycerol interface, thereby hindering lipase/co-lipase from acting on its lipid substrate. We also found that chloroplast membranes (thylakoids), when added to refined food, suppressed food intake in rats, lowered blood lipids and raised the satiety hormones, CCK and enterostatin. Consequently, the mechanism for satiety seems to be retardation of fat digestion allowing the fat products to stay longer in the intestine.
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| 6. |
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| 7. |
- Arvidsson, Per-Ola, et al.
(författare)
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Purification and identification of the violaxanthin de-epoxidase as a 43 kDa protein
- 1996
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Ingår i: Photosynthesis Research. - 0166-8595. ; 49:2, s. 119-129
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Tidskriftsartikel (refereegranskat)abstract
- Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH4)2SO4 fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g–1 s–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b 6 and an internal sequence of polyphenol oxidase.
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| 8. |
- Arvidsson, Per-Ola, et al.
(författare)
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Violaxanthin accessibility and temperature dependency for de-epoxidation in spinach thylakoid membranes
- 1997
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Ingår i: Photosynthesis Research. - 0166-8595. ; 52:1, s. 39-48
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Tidskriftsartikel (refereegranskat)abstract
- Using DTT and iodoacetamide as a novel irreversible method to inhibit endogenous violaxanthin de-epoxidase, we found that violaxanthin could be converted into zeaxanthin from both sides of the thylakoid membrane provided that purified violaxanthin de-epoxidase was added. The maximum conversion was the same from both sides of the membrane. Temperature was found to have a strong influence both on the rate and degree of maximal violaxanthin to zeaxanthin conversion. Thus only 50% conversion of violaxanthin was detected at 4 degreesC, whereas at 25 degreesC and 37 degreesC the degree of conversion was 70% and 80%, respectively. These results were obtained with isolated thylakoids from non-cold acclimated leafs. Pigment analysis of sub-thylakoid membrane domains showed that violaxanthin was evenly distributed between stroma lamellae and grana partitions. This was in contrast to chlorophyll a and beta-carotene which were enriched in stroma lamellae fractions while chlorophyll b, lutein and neoxanthin were enriched in the grana membranes. In combination with added violaxanthin de-epoxidase we found almost the same degree of conversion of violaxanthin to zeaxanthin (73-78%) for different domains of the thylakoid membrane. We conclude that violaxanthin de-epoxidase converts violaxanthin in the lipid matrix and not at the proteins, that violaxanthin does not prefer one particular membrane region or one particular chlorophyll protein complex, and that the xanthophyll cycle pigments are oriented in a vertical manner in order to be accessible from both sides of the membrane when located in the lipid matrix.
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| 9. |
- Bornman, Janet, et al.
(författare)
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Action spectrum for inhibition by ultraviolet-radiation of photosystem-II activities in spinach thylakoids
- 1984
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Ingår i: Photobiochemistry and Photobiophysics. - 0165-8646. ; 8:5-6, s. 305-313
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Tidskriftsartikel (refereegranskat)abstract
- The effect of UV radiation (half-band width 10 nm) in the wavelength range 248-340 nm on chlorophyll fluorescence from a thin layer of spinach thylakoid suspension was investigated. The parameter most sensitive to UV radiation was the rise time of variable fluorescence. The increase in rise time was proportional to UV photon fluence and was used for the determination of an action spectrum. The action spectrum falls off from a maximum at .apprx. 275 nm towards longer wavelengths and rises from a minimum at 260 nm towards shorter wavelengths. The UV inhibition apparently is mainly on the PS II oxidizing side. Possibly damage is also inflicted to the PS II reaction center.
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| 10. |
- Bratt, C E, et al.
(författare)
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Isolation of pigment-free bulk lipids from thylakoids
- 1993
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002. ; 1165:3, s. 288-290
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Tidskriftsartikel (refereegranskat)abstract
- Lipids from spinach thylakoids were extracted with chloroform/methanol and separated from pigments in a single chromatographic step run at 5 degrees C using silicic acid adjusted to pH 8. The isolated lipid fraction contained essentially the same amounts of individual lipids as in the initial extract. It contained less than 0.1% of the initial chlorophylls and carotenoids.
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| 11. |
- Bratt, Charlotte Eva, et al.
(författare)
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Regulation of violaxanthin de-epoxidase activity by pH and ascorbate concentration
- 1995
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Ingår i: Photosynthesis Research. - 0166-8595. ; 45:2, s. 169-175
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Tidskriftsartikel (refereegranskat)abstract
- The activity of violaxanthin de-epoxidase has been studied both in isolated thylakoids and after partial purification, as a function of pH and ascorbate concentration. We demonstrate that violaxanthin de-epoxidase has a Km for ascorbate that is strongly dependent on pH, with values of 10, 2.5, 1.0 and 0.3 mM at pH 6.0, 5.5, 5.0 and 4.5, respectively. These values can be expressed as a single Km±0.1±0.02 mM for the acid form of ascorbate. Release of the protein from the thylakoids by sonication was also found to be strongly pH dependent with a cooperativity of 4 with respect to protons and with an inflexion point at pH 6.7. These results can explain some of the discrepancies reported in the literature and provide a more consistent view of zeaxanthin formation in vivo.
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| 12. |
- Bunea, Ada-Ioana, et al.
(författare)
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Micropatterned Carbon-on-Quartz Electrode Chips for Photocurrent Generation from Thylakoid Membranes
- 2018
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Ingår i: ACS Applied Energy Materials. - : American Chemical Society (ACS). - 2574-0962. ; 1:7, s. 3313-3322
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Tidskriftsartikel (refereegranskat)abstract
- Harvesting the energy generated by photosynthetic organisms through light-dependent reactions is a significant step toward a sustainable future energy supply. Thylakoid membranes are the site of photosynthesis, and thus particularly suited for developing photo-bioelectrochemical cells. Novel electrode materials and geometries could potentially improve the efficiency of energy harvesting using thylakoid membranes. For commercial applications, electrodes with large surface areas are needed. Photolithographic patterning of a photoresist, followed by pyrolysis, is a flexible and fast approach for the fabrication of carbon electrodes with tailored properties. In this work, electrode chips consisting of patterned carbon supported on quartz were designed and fabricated. The patterned electrode area is 1 cm2, and the measurement chamber footprint is 0.5 cm2, 1 order of magnitude larger than previously tested electrodes for thylakoid membrane immobilization. The use of a transparent substrate allows back-side illumination, protecting the bioelectrochemical system from the environment and vice versa. Two different mediators, monomeric ([Ru(NH3)6]3+) and polymeric ([Os(2,2′-bipyridine)2-poly(N-vinylimidazole)10Cl]+/2+), are used for evaluating photocurrent generation from thylakoid membranes with different electrode geometries. Current densities up to 71 μA cm–2 are measured upon illumination through the transparent electrode chip with solar simulated irradiance (1000 W m–2).
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| 13. |
- Clausén, Maria, et al.
(författare)
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Post harvest improvement of zeaxanthin content of vegetables
- 2010
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Ingår i: Journal of Food Engineering. - : Elsevier BV. - 0260-8774. ; 98:2, s. 192-197
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Tidskriftsartikel (refereegranskat)abstract
- Zeaxanthin is a carotenoid produced by plants and has been associated with protection of the photosynthetic machinery under light stress and, together with lutein, in protection of the central retina of the eye. Zeaxanthin levels in blood plasma have been negatively correlated to the development of AMD (age-related macular degeneration) (Gale et al, 2003). Under normal conditions, plants have a low content of zeaxanthin. The aim of this study was to increase the zeaxanthin content in green vegetables by post harvest treatments. Efficient conditions for activation of the endogenous enzyme system generating zeaxanthin was established and included incubation at low pH (2.5-5.5), with the membrane permeable acetic acid/acetate buffer at room temperature or above for 30 min or more. Typically more than 20-fold increase in zeaxanthin content was obtained for spinach, corn salad, parsley, basil, lemon balm and peas. For spinach up to 4 mg/100 g fresh weight of leaves were obtained. In consequence less amount of vegetables would be needed in the diet to provide the same amount of zeaxanthin for the eye. (C) 2010 Elsevier Ltd. All rights reserved.
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| 14. |
- Emek, Sinan Cem, et al.
(författare)
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Pancreatic lipase-colipase binds strongly to the thylakoid membrane surface.
- 2013
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Ingår i: Journal of the Science of Food and Agriculture. - : Wiley. - 1097-0010 .- 0022-5142. ; 93:9, s. 2254-2258
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: 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. RESULTS: 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) . CONCLUSION: 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. © 2013 Society of Chemical Industry.
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| 15. |
- Emek, Sinan Cem, et al.
(författare)
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Pigments protect the light harvesting proteins of chloroplast thylakoid membranes against digestion by gastrointestinal proteases
- 2011
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Ingår i: Food Hydrocolloids. - : Elsevier BV. - 0268-005X. ; 25:6, s. 1618-1626
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Tidskriftsartikel (refereegranskat)abstract
- Chloroplast thylakoid membranes inhibit pancreatic lipase/colipase activity in vitro and, when included in food, induce satiety signals. As thylakoid membranes themselves are nutrients, containing lipids and proteins, it is of interest to study the digestion of thylakoids by enzymes of the gastrointestinal tract. Thylakoid membranes were treated with pepsin, trypsin, gastric and pancreatic juice at 37 degrees C and the resulting enzymatic breakdown was analyzed by gel electrophoresis, electron microscopy and mass spectroscopy. In all cases, several of the proteins were degraded within half an hour, while the main parts of the pigment-protein complexes were resistant for hours. Oil emulsified thylakoids were more resistant towards the enzymatic breakdown. Electron microscopy demonstrated that, after treatments, the thylakoids still remained in a membrane vesicular form. The capacity of thylakoid membranes to inhibit the lipase/colipase activity was partly reduced in all cases. About 50% of the inhibition capacity remained after treatment with pancreatic juice when the thylakoids were present in an oil emulsion. Delipidated thylakoids and plasma membranes, which lack the photosynthetic pigments, were degraded rapidly by pancreatic juice. Conclusion: The pigments, closely bound to the trans-membrane helices of thylakoid membrane proteins protect these from digestion by pepsin, trypsin, gastric and pancreatic juice. This supports the notion that a substantial inhibition of lipase/colipase takes place during the first 2 h in the intestine resulting in a retardation and prolongation of lipolysis in vivo. (C) 2010 Elsevier Ltd. All rights reserved.
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| 16. |
- Eskling, Marie, et al.
(författare)
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Changes in the quantities of violaxanthin de-epoxidase, xanthophylls and ascorbate in spinach upon shift from low to high light
- 1998
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Ingår i: Photosynthesis Research. - 0166-8595. ; 57:1, s. 41-50
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Tidskriftsartikel (refereegranskat)abstract
- Zeaxanthin, a carotenoid in the xanthophyll cycle, has been suggested to play a role in the protection against photodestruction. We have studied the importance of the parameters involved in zeaxanthin formation by comparing spinach plants grown in low light (100 to 250 mol m-2 s-1) to plants transferred to high light (950 mol m-2 s-1). Different parameters were followed for a total of 11 days. Our experiments show that violaxanthin de-epoxidase decreased between 15 and 30%, the quantity of xanthophyll cycle pigments doubled to 100 mmol (mol Chl)-1, corresponding to 27 mol m-2, and the rate of violaxanthin to zeaxanthin conversion was doubled. Lutein and neoxanthin increased from 50 to 71 mol m-2 and from 16 to 23 mol m-2, respectively. On a leaf area basis, chlorophyll and -carotene levels first decreased and then after 4 days increased. The chlorophyll a/b ratio was unchanged. The quantity of ascorbate was doubled to 2 mmol m-2, corresponding to an estimated increase in the chloroplasts from 25 to 50 mM. In view of our data, we propose that the increase in xanthophyll cycle pigments and ascorbate only partly explain the increased rate of conversion of violaxanthin to zeaxanthin, but the most probable explanation of the faster conversion is an increased accessibility of violaxanthin in the membrane.
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| 17. |
- Eskling, Marie, et al.
(författare)
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Enzymes and mechanisms for violaxanthin-zeaxanthin conversion
- 2001
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Ingår i: Regulation of photosynthesis. - Dordrecht : Springer Netherlands. - 0792363329 ; , s. 433-452
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The xanthophyll cycle is of great importance in relation to light stress. Particularly, interest has been focused on the possible photoprotective role of zeaxanthin. In higher plants under light stress, zeaxanthin is formed from violaxanthin in a reaction catalyzed by violaxanthin de-epoxidase (VDE). The reverse reaction is catalyzed by zeaxanthin epoxidase (ZE) under low light or in darkness. VDE has been purified from spinach and lettuce as a 43-kDa protein. The gene has been cloned and sequenced from several species, and a few mutants have been isolated. The gene is nuclear encoded and the transit peptide is characteristic for targeting to the thylakoid lumen. The activity of VDE is affected by factors such as a pH-dependent binding to the thylakoid membrane, concentration of ascorbic acid, temperature and availability of violaxanthin in relation to amount, type and distribution of pigment-protein complexes in the membrane. The information about ZE is more limited. The enzyme has not yet been isolated but its gene has been cloned and sequenced and a number of mutants have been isolated. The role of the xanthophyll cycle in the dissipation of excess light energy will be discussed particularly in relation to the recent progress in studies on various mutants. The possible role of the xanthophyll cycle in other processes, such as protection against oxidative stress of lipids, regulation of membrane fluidity, participation in blue light responses, and regulation of abscisic acid synthesis will also be presented.
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| 18. |
- Eskling, Marie, et al.
(författare)
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The xanthophyll cycle, its regulation and components
- 1997
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 100:4, s. 806-816
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Forskningsöversikt (refereegranskat)abstract
- During the last few years much interest has been focused on the photoprotective role of zeaxanthin. In excessive light zeaxanthin is rapidly formed in the xanthophyll cycle from violaxanthin, via the intermediate antheraxanthin, a reaction reversed in the dark. The role of zeaxanthin and the xanthophyll cycle in photoprotection, is based on fluorescence quenching measurements, and in many studies a good correlation to the amount of zeaxanthin (and antheraxanthin) has been found. Other suggested roles for the xanthophylls involve, protection against oxidative stress of lipids, participation in the blue light response, modulation of the membrane fluidity and regulation of abscisic acid synthesis. The enzyme violaxanthin de-epoxidase has recently been purified from spinach and lettuce as a 43-kDa protein. It was found as 1 molecule per 20-100 electron-transport chains. The gene has been cloned and sequenced from Lactuca sativa, Nicotiana tabacum and Arabidopsis thaliana. The transit peptide was characteristic of nuclear-encoded and lumen-localized proteins. The activity of violaxanthin de-epoxidase is controlled by the lumen pH. Thus, below pH 6.6 the enzyme binds to the thylakoid membrane. In addition ascorbate becomes protonated to ascorbic acid (pKa= 4.2) the true substrate (Km= 0.1 mM) for the violaxanthin de-epoxidase. We present arguments for an ascorbate transporter in the thylakoid membrane. The enzyme zeaxanthin epoxidase requires FAD as a cofactor and appears to use ferredoxin rather than NADPH as a reductant. The zeaxanthin epoxidase has not been isolated but the gene has been sequenced and a functional protein of 72.5 kDa has been expressed. The xanthophyll cycle pigments are almost evenly distributed in the thylakoid membrane and at least part of the pigments appears to be free in the lipid matrix where we conclude that the conversion by violaxanthin de-epoxidase occurs.
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| 19. |
- Forsberg, Jens, et al.
(författare)
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Protease activities in the chloroplast capable of cleaving an LHCII N-terminal peptide
- 2005
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 123:1, s. 21-29
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Tidskriftsartikel (refereegranskat)abstract
- Two protease activities of pea chloroplasts, one located in the stroma and the other associated to the thylakoid membrane, are described. Both proteases catalyse the endo-proteolytic cleavage of a peptide corresponding to the N-terminal loop and the first turn in helix-B of light-harvesting complex II (Lhcb1 from pea). The stromal protease cleaves preferentially on the carboxy-side of glutamic acid residues. Inhibitor studies indicate that this protease is a serine-type protease. The protease was partially purified and could be correlated to a 95-kDa polypeptide band on SDS-polyacrylamide gels. The 95 kDa protein was partially sequenced and showed similarity to an to an 'unknown protein' from A. thaliana (in the NCBI public database) as well as to a glutamyl endopeptidase purified from crude extract of cucumber leaves. It is concluded that the stromal protease is a chloroplast glutamyl endopeptidase (cGEP). The protease localized in the thylakoid membrane, cleaved the peptide at only one site, close to its N terminus. The activity of the thylakoid-associated protease was found to be drastically increased in the presence of the reducing agent 1,4-dithiothreitol. Inhibitor studies suggest that this protease is a cysteine- or serine-type protease. The possible roles of these proteases in the regulation of photosynthetic electron transport and in the chloroplast homeostasis are discussed.
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| 20. |
- Gisselsson, Anna, et al.
(författare)
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Chemical and mutational modification of histidines in violaxanthin de-epoxidase from Spinachcia oleracea.
- 2003
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317. ; 119:1, s. 97-104
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Tidskriftsartikel (refereegranskat)abstract
- The violaxanthin de-epoxidase (VDE) gene from spinach (Spinacia oleracea) was cloned, sequenced (GenBank AJ 250433), and expressed in Escherichia coli. The highest obtained conversion rate of violaxanthin was 86 nmol s-1 per litre of growth medium, corresponding to an amount of active enzyme of 0.4 mg l-1. Sequence comparison between VDE from different species were made and particular interest was focused on four highly conserved histidines (H121,124,167,173) and their possible involvement in enzymatic activity. Chemical modification of the histidines using DEPC or by site-directed mutations resulted in partial or total inactivation of the enzyme. The chemical modification could be reversed by hydroxylamine treatment, regenerating a large percentage of the original activity. The histidine residues, which are located in pairs close to each other, were pairwise substituted for either alanine or arginine. This resulted in one inactive mutant (H121,124R) and three mutants with very different activities and decreased binding of ascorbic acid, as reflected by an up to four-fold increase in Km. A substitution of all four histidines for either alanine or arginine resulted in inactive enzymes. Based on these results it is suggested that the histidine residues are important for the activity of VDE.
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| 21. |
- Gisselsson, A, et al.
(författare)
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Role of histidines in the binding of violaxanthin de-epoxidase to the thylakoid membrane as studied by site-directed mutagenesis
- 2004
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 122:3, s. 337-343
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Tidskriftsartikel (refereegranskat)abstract
- Regulation of violaxanthin de-epoxidase (VDE) involves a conformational change at low lumenal pH, followed by binding of the enzyme to the thylakoid membrane. The role of histidine residues in this process was studied by release of unbound enzyme from thylakoids upon sonication, on a pH scale from 4.7 to 7.1. The co-operativity for binding of spinach VDE (four histidines) to the membrane was found to be 3.8, with respect to protons, and had an inflexion point at pH 6.6, whereas VDE from wheat (three histidines) showed a co-operativity of 2.9 and had an inflexion point at pH 6.2. Mutant forms of VDE were constructed and probed for their binding to the outside of thylakoid membranes. With one or two histidines substituted for alanine or arginine, a lower co-operativity (1.6-2.3) was found, compared with the wild type. Based on these findings, and that the pKa value for histidine is within the range where the VDE binding takes place, we propose that protonation of the histidine residues at low pH induces the conformational change of VDE, and hence indirectly regulates binding of the enzyme to the thylakoid membrane.
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| 22. |
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| 23. |
- Hall, Michael, 1980-, et al.
(författare)
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Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function
- 2010
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Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 10:5, s. 987-1001
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Tidskriftsartikel (refereegranskat)abstract
- The light-dependent regulation of stromal enzymes by thioredoxin (Trx)-catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx-mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol-dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx-linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin deepoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox-controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.
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| 24. |
- Hamidi, Hassan, et al.
(författare)
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Photocurrent Generation from Thylakoid Membranes on Osmium-Redox-Polymer-Modified Electrodes.
- 2015
-
Ingår i: ChemSusChem. - : Wiley. - 1864-564X .- 1864-5631. ; 8:6, s. 990-993
-
Tidskriftsartikel (refereegranskat)abstract
- Thylakoid membranes (TMs) are uniquely suited for photosynthesis owing to their distinctive structure and composition. Substantial efforts have been directed towards use of isolated photosynthetic reaction centers (PRCs) for solar energy harvesting, however, few studies investigate the communication between whole TMs and electrode surfaces, due to their complex structure. Here we report on a promising approach to generate photosynthesis-derived bioelectricity upon illumination of TMs wired with an osmium-redox-polymer modified graphite electrode, and generate a photocurrent density of 42.4 μA cm(-2) .
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| 25. |
- Hasan, Kamrul, et al.
(författare)
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Photoelectrochemical Communication between Thylakoid Membranes and Gold Electrodes through Different Quinone Derivatives
- 2014
-
Ingår i: ChemElectroChem. - : Wiley. - 2196-0216. ; 1:1, s. 131-139
-
Tidskriftsartikel (refereegranskat)abstract
- Photosynthesis is a sustainable process for the conversion of light energy into chemical energy. Thylakoids in energy-transducing photosynthetic membranes are unique in biological membranes because of their distinguished structure and composition. The quantum trapping efficiency of thylakoid membranes is appealing in photobioelectrochemical research. In this study, thylakoid membranes extracted from spinach are shown to communicate with a gold-nanoparticle-modified solid gold electrode (AuNP-Au) through a series of quinone derivatives. Among these, para-benzoquinone (PBQ) is found to be the best soluble electron-transfer mediator, generating the highest photocurrent of approximately 130 mu Acm(-2) from water oxidation under illumination. In addition, the photocurrent density is investigated as a function of applied potential, the effect of light intensity, quinone concentration, and amount of thylakoid membrane. Finally, the source of photocurrent is confirmed by using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (known by its trade name, Diuron), an inhibitor of photosystem II, which decreases the total photocurrent by 50%.
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| 26. |
- Latowski, D, et al.
(författare)
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Violaxanthin de-epoxidase, the xanthophyll cycle enzyme, requires lipid inverted hexagonal structures for its activity
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:15, s. 4417-4420
-
Tidskriftsartikel (refereegranskat)abstract
- Bilayer-forming lipids were shown to be ineffective in sustaining the enzymatic activity of violaxanthin de-epoxidase. On the other hand, non-bilayer-forming lipids, regard-less of their different chemical character, ensured high activity of violaxanthin de-epoxidase, resulting in conversion of violaxanthin to zeaxanthin. Our data indicates that the presence of lipids forming reversed hexagonal structures is necessary for violaxanthin de-epoxidase activity and this activity is dependent on the degree of unsaturation of the fatty acids. The significance of the reversed hexagonal phase domains in the conversion of violaxanthin into zeaxanthin in model systems and in the native thylakoid membranes is discussed.
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| 27. |
- Liu, Yunjun, et al.
(författare)
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The mitochondrial external NADPH dehydrogenase modulates the leaf NADPH/NADP+ ratio in transgenic Nicotiana sylvestris
- 2008
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Ingår i: Plant and Cell Physiology. - : Oxford University Press (OUP). - 1471-9053 .- 0032-0781. ; 49:1, s. 251-263
-
Tidskriftsartikel (refereegranskat)abstract
- Plant mitochondria contain alternative external NAD(P)H dehydrogenases, which oxidise cytosolic NADH or NADPH and reduce ubiquinone without inherent linkage to proton pumping and ATP production. In potato, St-NDB1 is an external Ca2+-dependent NADPH dehydrogenase. The physiological function of this enzyme was investigated in homozygous Nicotiana sylvestris lines overexpressing St-ndb1 and co-suppressing St-ndb1 and an N. sylvestris ndb1. In leaf mitochondria isolated from the overexpressor lines, higher activity of alternative oxidase (AOX) was detected. However, the AOX induction was substantially weaker than in the complex I deficient CMSII mutant, previously shown to contain elevated amounts of NAD(P)H dehydrogenases and AOX. An aox1b and an aox2 gene were up-regulated in CMSII, but only aox1b showed a response, albeit smaller, in the transgenic lines, indicating differences in AOX activation between the genotypes. As in CMSII, the increase of AOX in the overexpressing lines was not due to a general oxidative stress. The lines overexpressing St-ndb1 had consistently lowered leaf NADPH/NADP+ ratios in the light and variably decreased levels in darkness, but unchanged NADH/NAD+ ratios. CMSII instead had similar NADPH/NADP+ and lower NADH/NAD+ ratios than wildtype. These results demonstrate that St-NDB1 is able to modulate the cellular balance of NADPH and NADP+ at least in the day and that reduction of NADP(H) and NAD(H) is independently controlled. Similar growth rates, chloroplast malate dehydrogenase activation and xanthophyll ratios indicate that the change in reduction does not communicate to the chloroplast, and that the cell tolerates significant changes in NADP(H) reduction without deleterious effects.
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| 28. |
- Malmström, Susanna, et al.
(författare)
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Regulatory role of the N terminus of the vacuolar calcium-ATPase in cauliflower
- 2000
-
Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 122:2, s. 517-526
-
Tidskriftsartikel (refereegranskat)abstract
- The vacuolar calmodulin (CaM)-stimulated Ca2+-ATPase, BCA1p, in cauliflower (Brassica oleracea) has an extended N terminus, which was suggested to contain a CaM-binding domain (S. Malmstrom, P. Askerlund, M.G. Palmgren [1997] FEBS Lett 400: 324328). The goal of the present study was to determine the role of the N terminus in regulating BCA1p. Western analysis using three different antisera showed that the N terminus of BCA1p is cleaved off by trypsin and that the N terminus contains the CaM-binding domain. Furthermore, the expressed N terminus binds CaM in a Ca2+dependent manner. A synthetic peptide corresponding to the CaM-binding domain of BCA1p (Ala-19 to Leu-43) strongly inhibited ATP-dependent Ca2+ pumping by BCA1p in cauliflower low-density membranes, indicating that the CaM-binding region of BCA1p also has an autoinhibitory function. The expressed N terminus of BCA1p and a synthetic peptide (Ala-19 to Met-39) were good substrates for phosphorylation by protein kinase C. Sequencing of the phosphorylated fusion protein and peptide suggested serine-16 and/or serine-28 as likely targets for phosphorylation. Phosphorylation of serine-28 had no effect on CaM binding to the alanine-19 to methionine-39 peptide. Our results demonstrate the regulatory importance of the N terminus of BCA1p as a target for CaM binding, trypsin cleavage, and phosphorylation, as well as its importance as an autoinhibitory domain.
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| 29. |
- Matic, Sandra, et al.
(författare)
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Interaction between phosphofructokinase and aldolase from Saccharomyces cevisiae studied by aqueous two-phase partitioning
- 2001
-
Ingår i: Journal of Chromatography. B. - 1387-2273. ; 751:2, s. 341-348
-
Tidskriftsartikel (refereegranskat)abstract
- Phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13) have been highly purified from Saccharomyces cerevisiae by improved protocols. Partitioning of the enzymes in aqueous polymer two-phase systems was used to detect complex formation. The partition of each enzyme was found to be affected by the presence of the other enzyme. AMP affected the partition of the individual enzymes as well as the mixture of the two. The activities of the respective enzymes were stimulated in the putative complex in an AMP-dependent manner. Two strictly conserved residues belonging to an acidic surface loop of class II aldolases, are a potential site for electrostatic interaction with the positively charged regions close to the active site in phosphofructokinase. Ó 2001 Elsevier Science B.V. All rights reserved.
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| 30. |
- Matic, Sandra, et al.
(författare)
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Sucrose synthase isoforms in cultured tobacco cells
- 2004
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Ingår i: Plant Physiology and Biochemistry. - : Elsevier BV. - 1873-2690 .- 0981-9428. ; 42:4, s. 299-306
-
Tidskriftsartikel (refereegranskat)abstract
- The plant enzyme sucrose synthase (SuSy; EC 2.4.1.13) catalyzes the reversible conversion of sucrose and UDP into UDP-glucose (UDP-Glc) and fructose. The enzyme exists in different isoforms and is both located in the cytosol, membrane-bound and associated to the actin cytoskeleton. We here investigate sucrose synthase from tobacco (Nicotiana tabacum L.) BY-2 heterotrophic cell suspensions. Two different isoforms of sucrose synthase SuSy1 and SuSy2, could be purified from cytosolic extracts of these cells using a combination of poly(ethylene glycol) (PEG) precipitation, gel filtration, ion-exchange chromatography and affinity chromatography. They were clearly distinct. both with regard to the binding to the ion-exchange column and with regard to their kinetic and regulatory properties. SuSy 1, the more abundant species, showed lower V-max and K-m for sucrose and UDP compared to the less abundant SuSy2. The activity of SuSy2 in the breakdown direction was stimulated by 60% by actin, in contrast to that of SuSy 1, which showed a 17% inhibition. An indication of interaction between SuSy I and actin was obtained by partitioning in aqueous Dextran-PEG two-phase systems. Furthermore, fructose 2,6-bisphosphate (F26BP) at micromolar concentrations stimulated SuSy2 in the presence of actin while SuSy I was strongly inhibited by fructose. Possible roles of these two isoforms in the sucrose turnover in BY-2 cells are discussed. (C) 2004 Elsevier SAS. All rights reserved.
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| 31. |
- Miranda, Helder, 1981-
(författare)
-
Stress response in the cyanobacterium Synechocystis sp. PCC 6803
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Adaptation to environmental changes is important for the survival of living organisms. Under extreme abiotic conditions, organic molecules (such as lipids, proteins and nucleic acids) are prone to damage. Under these conditions stress response mechanisms are activated, either to prevent the source of damage or to promote the rapid turnover of damaged molecules. Like all photoautotrophic organisms, cyanobacteria are sensitive to high light intensity and oxidative stress, which induces damage to the photosynthetic apparatus. My thesis is divided in two subjects related to particular stress responses in the cyanobacterium Synechocystis sp. PCC 6803: 1) the role of Deg/HtrA proteases and 2) investigations on the small CAB-like proteins. Deg/HtrA proteases are ATP-independent serine endopeptidases with a characteristic C-terminal PDZ domain. These proteases are largely dispersed among living organisms, with many different functions, mostly involved in protein quality control. The genome of Synechocystis sp. PCC 6803 contains three genes coding for Deg/HtrA proteases: HtrA, HhoA and HhoB. These proteases are essential for survival under high light and heat stress, and may overlap in their functions. During my Ph.D. studies I focused on the identification of the precise localization of the Deg/HtrA proteases in the cyanobacterial cell, analyzed the biochemical properties of recombinant Synechocystis Deg/HtrA proteases in vitro and adopted proteomic and metabolomic approaches to study the physiological importance of these proteases. My data show that Deg/HtrA proteases are not only important in stress response mechanisms under adverse conditions, but are also involved in the stabilization of important physiological processes, such as polysaccharides biosynthesis and peptidoglycan turnover. The small CAB-like proteins (SCPs) belong to the light harvesting-like family of stress induced proteins and are thought to be involved in the photoprotection of the photosynthetic apparatus. Five small CAB-like proteins where identified in Synechocystis sp. PCC 6803 (ScpA-E). In my studies I identified another relative to the SCPs, LilA, which I found to be co-transcribed with ScpD. I also focused on the subcellular localization and identification of potential interaction partners of the SCPs.
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| 32. |
- Pankratov, Dmitry, et al.
(författare)
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Supercapacitive Biosolar Cell Driven by Direct Electron Transfer between Photosynthetic Membranes and CNT Networks with Enhanced Performance
- 2017
-
Ingår i: ACS Energy Letters. - : American Chemical Society (ACS). - 2380-8195. ; 2:11, s. 2635-2639
-
Tidskriftsartikel (refereegranskat)abstract
- Integrating photosynthetic cell components with nanostructured materials can facilitate the conversion of solar energy into electric power for creating sustainable carbon-neutral energy sources. With the aim at exploring efficient photoinduced biocatalytic energy conversion systems, we have used an amidated carbon nanotube (aCNT) networked matrix to integrate thylakoid membranes (TMs) for construction of a direct electron transfer-driven biosolar cell. We have evaluated the resulting photobioelectrochemical cells systematically. Compared to the carboxylated CNT (cCNT)-TMs system, the aCNT-TMs system enabled a 1.5-fold enhancement in photocurrent density. This system offers more advantages including a reduced charge-transfer resistance, a lower open-circuit potential, and an improved cell stability. More remarkably, the average power density of the optimized cells was 250 times higher than that of reported analogue systems. Our results suggest the significance of physical and electronic interactions between the photosynthetic components and the support nanomaterials and may offer new clues for designing improved biosolar cells.
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| 33. |
- Pankratova, Galina, et al.
(författare)
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Supercapacitive Photo-Bioanodes and Biosolar Cells : A Novel Approach for Solar Energy Harnessing
- 2017
-
Ingår i: Advanced Energy Materials. - : John Wiley & Sons. - 1614-6832 .- 1614-6840. ; 7:12
-
Tidskriftsartikel (refereegranskat)abstract
- The concept of supercapacitive photo-bioanode and biosolar cell (photo-biosupercapacitor) for simultaneous solar energy conversion and storage is demonstrated for the first time. Exploiting the capacitive component significantly improves the electron transfer processes and allows the achievement of a current density of 280 µA cm−2 in the pulse mode.
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| 34. |
- Polivka, Tomas, et al.
(författare)
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Direct observation of the (forbidden) S-1 state in carotenoids
- 1999
-
Ingår i: Proceedings of the National Academy of Sciences. - 1091-6490. ; 96:9, s. 4914-4917
-
Tidskriftsartikel (refereegranskat)abstract
- Carotenoids are involved in a variety of biological functions, yet the underlying mechanisms are poorly understood, in part because of the long-standing difficulty in assigning the location of the first excited (S-1) state. Here, we present a method for determining the energy of the forbidden S-1 state, on the basis of ultrafast spectroscopy of the short lived level. Femtosecond transient absorption spectra and kinetics of the S-1 --> S-2 transition revealed the location of the intermediate level in two carotenoid species involved in the xanthophyll cycle, zeaxanthin and violaxanthin, and yielded surprising implications regarding the mechanism of photoregulation in photosynthesis.
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| 35. |
- Szilagyi, Anna, et al.
(författare)
-
Laurdan fluorescence spectroscopy in the thylakoid bilayer : The effect of violaxanthin to zeaxanthin conversion on the galactolipid dominated lipid environment.
- 2008
-
Ingår i: Biochimica et Biophysica Acta. - : Elsevier. - 0006-3002 .- 1878-2434 .- 0005-2736. ; 1778:1, s. 348-355
-
Tidskriftsartikel (refereegranskat)abstract
- Laurdan (6-lauroyl-2-dimethylaminonaphthalene) fluorescence spectroscopy has been applied to probe the physical status of the thylakoid membrane upon conversion of violaxanthin to zeaxanthin. So far, only phospholipid-dominated membranes have been studied by this method and hereby we report the first use of laurdan in mono- and digalactosyldiacylglycerol-dominated membrane systems. The generalised polarisation (GP) of laurdan was used as a measure of the structural effect of xanthophyll cycle pigments in isolated spinach (Spinacia oleracea) thylakoids and in model membrane vesicles composed of chloroplast galactolipids. Higher GP values indicate a membrane in a more ordered structure, whereas lower GP values point to a membrane in a less ordered fluid phase. The method was used to probe the effect of violaxanthin and zeaxanthin in thylakoid membranes at different temperatures. At 4, 25 and 37 °C the GP values for dark-adapted thylakoids in the violaxanthin-form were 0.55, 0.28 and 0.26. After conversion of violaxanthin to zeaxanthin, at the same temperatures, the GP values were 0.62, 0.36 and 0.34, respectively. GP values increased gradually upon conversion of violaxanthin to zeaxanthin. Similar results were obtained in the liposomal systems in the presence of these xanthophyll cycle pigments. We conclude from these results that the conversion of violaxanthin to zeaxanthin makes the thylakoid membrane more ordered.
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| 36. |
- Szilagyi, Anna, et al.
(författare)
-
Membrane curvature stress controls the maximal conversion of violaxanthin to zeaxanthin in the violaxanthin cycle - influence of alpha-tocopherol, cetylethers, linolenic acid, and temperature
- 2007
-
Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 0006-3002 .- 1878-2434. ; 1768:9, s. 2310-2318
-
Tidskriftsartikel (refereegranskat)abstract
- Zeaxanthin, an important component in protection against overexcitation in higher plants, is formed from violaxanthin by the enzyme violaxanthin de-epoxidase. We have investigated factors that may control the maximal degree of conversion in the violaxanthin cycle. The conversion of violaxanthin to zeaxanthin in isolated spinach thylakoids was followed at different temperatures and in the presence of lipid packing modifiers. The maximum degree of conversion was found to be 35%, 70% and 80% at 4 'C, 25 'C and 37 'C respectively. In the presence of membrane modifying agents, known to promote non-lamellar structures (Hit), such as linolenic acid the conversion increased, and the maximal level of violaxanthin deepoxidation obtained was close to 100%. In contrast, substances promoting lamellar phases (L.), such as alpha-tocopherol and 8-cetylether (C16EO8), only 55% and 35% of the violaxanthin was converted at 25 degrees C, respectively. The results are interpreted in light of the lipid composition of the thylakoid membrane, and we propose a model where a negative curvature elastic stress in the thylakoid lipid bilayer is required for violaxanthin deepoxidase activity. In this model zeaxanthin with its longer hydrophobic stretch is proposed to promote lamellar arrangements of the membrane. As a result, zeaxanthin relieves the curvature elastic stress, which in turn leads to inactivation of violaxanthin de-epoxidase. (c) 2007 Elsevier B.V. All rights reserved.
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| 37. |
- Tapia, Cristina, et al.
(författare)
-
Wiring of Photosystem I and Hydrogenase on an Electrode for Photoelectrochemical H2 Production by using Redox Polymers for Relatively Positive Onset Potential
- 2017
-
Ingår i: ChemElectroChem. - : Wiley. - 2196-0216. ; 4:1, s. 90-95
-
Tidskriftsartikel (refereegranskat)abstract
- Photosystem I (PSI) is combined with Desulfovibrio gigas hydrogenase for the bioelectrocatalytic photosynthesis of hydrogen at an electrode surface. The activity of these two biocatalysts is linked by two redox polymers; a redox polymer with a relatively positive potential (loaded with an Os complex) is able to reduce PSI and thus facilitates the production of photoexcited electrons, whereas redox polymers of relatively low potential are able to transfer electrons to the hydrogenase. Two negative-potential redox polymers are tested, with either a viologen pendant (4-methyl-4′-bromopropylviologen functionalized linear polyethylenimine) or a cobaltocene pendant (cobaltocene-functionalized branched polyethylenimine, Cc-BPEI). Both are able to protect hydrogenase from O2 inactivation, but only the use of Cc-BPEI yields significant photocurrents for H+ reduction, likely due to its lower redox potential. The photocurrents obtained are found to be proportional to the quantity of H2 produced, reaching a maximum of −30 μA cm−2 for the system incorporating Cc-BPEI and showing a relatively positive onset potential at +0.38 V versus SHE.
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| 38. |
- Åkerlund, Hans-Erik, et al.
(författare)
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Isolation of inside-out thylakoid vesicles
- 2000
-
Ingår i: Aqueous Two-Phase Systems: Methods and Protocols. - New Jersey : Humana Press. - 0896035417 ; , s. 167-175
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- To understand the function of a biological membrane like that of chloroplast thylakoids, it is important to understand the arrangement of its different protein and lipid components. Preparations that have proven to be particularly suited for such studies are those consisting of membrane vesicles that are turned inside-out. Inside-out vesicles from the thylakoid membrane were first obtained from spinach chloroplasts by a combination of mechanical fragmentation and separation by aqueous two-phase partition (1,2). By the same or very similar procedures, inside-out thylakoid vesicles have now also been obtained from other plant sources such as pea (3), barley (4), mangrove (Avicennia marina) (5), lettuce (6), Euglena gracilis (7), cyanobacteria (8,9) and the photosynthetic bacteria Rhodopseudomonas viridis (10). Because the isolation procedure does not involve the use of detergents, the inside-out thylakoids have a preserved membrane structure and are ideally suited for structure-function studies. They have been used extensively in studies on thylakoid membrane topography (11-14) and for the identification of proteins associated with oxygen evolution (15,16). An important finding was that the inside-out vesicles are only formed from appressed (not exposed to stroma) thylakoid membranes, whereas right side-out vesicles derive from nonappressed (stroma exposed) membranes (17). As a result, the usefulness of inside-out vesicles could be extended to include studies on the lateral organization of the thylakoid membrane. The use of inside-out thylakoids in studies on structure and function of the thylakoid membrane has been reviewed by Andersson et al. (18).
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| 39. |
- Åkerlund, Hans-Erik, et al.
(författare)
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Partition by countercurrent distribution (CCD)
- 2000
-
Ingår i: Aqueous two-phase systems : mehods and protocols. - New Jersey : Humana Press. - 0896035417 ; , s. 55-64
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- A crucial step in many biochemical studies is the separation and purification of the individual structures of interest. For cells, cell organelles, and membrane fragments, the use of aqueous two-phase partition has become an important tool. The basis for separation in these phase systems is the surface properties of the material, and the method, therefore, nicely complements the more traditional centrifugation techniques. Excellent separations have sometimes been obtained in just one or a few partition steps. However, in most instances, a single partition step only gives a partial or incomplete separation of components. By a multiple-extraction procedure, the resolving power can be drastically increased and components with only small differences in surface properties can be separated.
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| 40. |
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