The de-epoxidase and epoxidase reactions of Mantoniella squamata (Prasinophyceae) exhibit different substrate-specific reaction kinetics compared to spinach

In vivo the prasinophyceaen alga Mantoniella squamata Manton et Parke uses an incomplete violaxanthin (Vx) cycle, leading to a strong accumulation of antheraxanthin (Ax) under conditions of high light. Here, we show that this zeaxanthin (Zx)-depleted Vx/Ax cycle is caused by an extremely slow second de-epoxidation step from Ax to Zx, and a fast epoxidation from Ax back to Vx in the light. The rate constant of Ax epoxidation is 5 to 6 times higher than the rate constant of Zx formation, implying that Ax is efficiently converted back to Vx before it can be de-epoxidated to Zx. It is, however, only half the rate constant of the first de-epoxidation step from Vx to Ax, thus explaining the observed net accumulation of Ax during periods of strong illumination. When comparing the rate constant of the second de-epoxidation step in M. squamata with Zx formation in spinach (Spinacia oleracea L.) thylakoids, we find a 20-fold reduction in the reaction kinetics of the former. This extremely slow Ax de-epoxidation, which is also exhibited by the isolated Mantoniella violaxanthin de-epoxidase (VDE), is due to a reduced substrate affinity of M. squamata VDE for Ax compared with the VDE of higher plants. Mantoniella VDE, which has a similar Km value for Vx, shows a substantially increased Km for the substrate Ax in comparison with spinach VDE. Our results furthermore explain why Zx formation in Mantoniella cells can only be found at low pH values that represent the pH optimum of VDE. A pH of 5 blocks the epoxidation reaction and, consequently, leads to a slow but appreciable accumulation of Zx.     

Characterization of violaxanthin de-epoxidase purified in the presence of Tween 20: effects of dithiothreitol and pepstatin A

Violaxanthin de-epoxidase (VDE) was purified from thylakoid membranes of spinach by conventional column chromatography in the presence of Tween 20. The neutral detergent was necessary to prevent non-specific interaction of VDE with column resins. In anion-exchange chromatography on Mono Q, VDE appeared in two peaks. Both peaks exhibited a polypeptide of 41 kDa when fully reduced with 5 mM dithiothreitol. Re-chromatography of either peak gave rise to both peaks, suggesting that the two forms of VDE are interconvertible. VDE characteristically changed its electrophoretic mobility depending on the concentration of dithiothreitol with which the protein was treated. When non-reduced, it showed two polypeptides of 43 and 42 kDa. These polypeptides moved down to the position of 40 kDa, and then up to the position of 41 kDa, along with the increase in the dithiothreitol concentration from 0 to 2 mM. These findings suggest that VDE has more than one disulfide bond and takes multiple forms depending on the extent of the reduction. Studies with various types of protein-modifying reagent revealed that VDE is sensitive to pepstatin A, a specific inhibitor of aspartic protease. This finding suggests that the reaction center of VDE contains a reactive aspartic acid residue(s).     

Zeaxanthin epoxidation - an in vitro approach

Zeaxanthin epoxidase (ZE) is an enzyme operating in the violaxanthin cycle, which is involved in photoprotective mechanisms. In this work model systems to study zeaxanthin (Zx) epoxidation were developed. Two assay systems are presented in which epoxidation of Zx was observed. In these assays two mutants of Arabidopsis thaliana which have active only one of the two xanthophyll cycle enzymes were used. The npq1 mutant possesses an active ZE and is thus able to convert Zx to violaxanthin (Vx) but the violaxanthin de-epoxidase (VDE) is inactive, so that Vx cannot be converted to Zx. The other mutant, npq2, possesses an active VDE and can convert exogenous Vx to Zx under strong light conditions but reverse reaction is not possible. The first assay containing thylakoids from npq1 and npq2 mutants of A. thaliana gave positive results and high efficiency of epoxidation reaction was observed. The amount of Zx was reduced by 25%. To optimize high efficiency of epoxidation reaction additional factors facilitating both fusion of the two types of thylakoids and incorporation of Zx to their membranes were also studied. The second kind of assay contained npq1 mutant thylakoids of A. thaliana supplemented with exogenous Zx and monogalactosyldiacylglycerol (MGDG). Experiments with different proportions of Zx and MGDG showed that their optimal ratio is 1:60. In such system, due to epoxidation, the amount of Zx was reduced by 38% of its initial level. The in vitro systems of Zx epoxidation described in this paper enable analysis some properties of the ZE without necessity of its isolation.     

A Dithiothreitol-Sensitive Tetrameric Protease from Spinach Thylakoids Has Polyphenol Oxidase Activity

Polyclonal antibody raised against a dithiothreitol-sen-sitivetetrameric protease (DSTP) from PSII membranes specificallyinhibited the polyphenol oxidase (PPO) activity of spinach thylakoids.DSTP was copurified with PPO activity on an affinity columnprepared with antibody against DSTP. These results suggest thatDSTP and PPO are the same protein. During purification of DSTP,Tween 20 was essential for stabilization of the protein, whichwas degraded in the absence of the detergent. Gel-filtrationchromatog-raphy of the purified DSTP revealed the presence of230-kDa (tetramer) and 60-kDa (monomer) species. The coppercontent of monomer species was determined to be 0.4 Cu atomper protein molecule, when the molecular weight of the proteinwas calculated to be 62,243, which is the value reported forspinach PPO [Hind et al. (1995) Biochemistry 34: 8157]. PurifiedDSTP caused the degradation as well as the dimerization of theextrinsic 23-kDa protein of PSII. The degradation of the proteinwas suppressed under anaerobic conditions induced by the presenceof glucose oxidase and glucose together. This fact suggeststhat oxygen molecules are involved in the proteolytic reactionand that the proteolytic activity and PPO activity may be correlatedwith each other. (Received September 27, 1996; Accepted December 4, 1996)     

Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.     

Role of histidines in the binding of violaxanthin de-epoxidase to the thylakoid membrane as studied by site-directed mutagenesis

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.     

Substrate specificity of the violaxanthin de-epoxidase of the primitive green alga<Emphasis Type="Italic"> Mantoniella squamata</Emphasis> (Prasinophyceae)

The substrate specificity of the enzyme violaxanthin de-epoxidase (VDE) of the primitive green alga Mantoniella squamata (Prasinophyceae) was tested in in vitro enzyme assays employing the following xanthophyll mono-epoxides: antheraxanthin (Ax), diadinoxanthin (Ddx), lutein-epoxide (LE), cryptoxanthin-epoxide (CxE), 9- cis neoxanthin (cNx), all- trans neoxanthin (Nx), and xanthophyll di-epoxides: 9- cis violaxanthin (cVx), all- trans violaxanthin (Vx), cryptoxanthin-di-epoxide (CxDE). The data presented in this study show that the VDE of M. squamata not only exhibits a low affinity for the mono-epoxide Ax, as has been reported by R. Frommolt et al. (2001, Planta 213:446-456), but has a reduced substrate affinity for the mono-epoxides Ddx, LE, CxE, and Nx as well. On the other hand, xanthophylls with a second epoxy-group (Vx, CxDE) can be de-epoxidized with a higher efficiency. Such a preference for xanthophyll di-epoxides cannot be observed for the higher-plant VDE, where, in general, no marked differences in the pigment de-epoxidation rates between xanthophyll mono- and di-epoxides are visible. Despite this substantial difference between the VDEs of M. squamata and S. oleracea there are also features common to both enzymes. Neither VDE is able to convert xanthophylls with a 9- cis configuration in the acyclic polyene chain and both rely on substrates in the all- trans configuration. Both enzymes furthermore exhibit a dependence of enzyme activity on the polarity of the substrate. Highly polar (Nx) or non-polar (CxE) xanthophylls are de-epoxidized with greatly reduced rates in comparison to substrates with an intermediate polarity (Vx, Ax, LE, Ddx). This dependence on substrate polarity becomes more obvious when the higher-plant VDE is examined, as the substrate affinity of the VDE of M. squamata is more strongly influenced by the existence or absence of a second epoxy-group. In summary, the data presented in this study underline the fact that different VDEs, although in general catalyzing the same reaction sequence, are functionally diverse.     

Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of diatoxanthin in the diatom Phaeodactylum tricornutum

Based on our recent findings that in the diatom Phaeodactylum tricornutum, chlororespiration in periods of prolonged darkness leads to the accumulation of diatoxanthin (DT), we have elaborated in detail the interdependence between the chlororespiratory proton gradient and the activation of diadinoxanthin de-epoxidase (DDE). The data clearly demonstrates that activation of DDE in Phaeodactylum occurs at higher pH-values compared to activation of violaxanthin de-epoxidase (VDE) in higher plants. In thylakoid membranes as well as in enzyme assays with isolated DDE, the de-epoxidation of diadinoxanthin (DD) is efficiently catalyzed at pH 7.2. In comparison, de-epoxidation of violaxanthin (Vx) in spinach thylakoids is observed below pH 6.5. Phaeodactylum thylakoids isolated from high light grown cells, that also contain the pigments of the violaxanthin cycle, show violaxanthin de-epoxidation at higher pH-values, thus suggesting that in Phaeodactylum, one de-epoxidase converts both diadinoxanthin and violaxanthin. We conclude that the activation of DDE at higher pH-values can explain how the low rates of chlororespiratory electron flow, that lead to the build-up of a rather small proton gradient, can induce the observed accumulation of diatoxanthin in the dark. Furthermore, we show that dark activation of diadinoxanthin de-epoxidation is not restricted to Phaeodactylum tricornutum but was also found in another diatom, Cyclotella meneghiana     

Functional roles of the major chloroplast lipids in the violaxanthin cycle

Monogalactosyldiacylglyceride (MGDG) and digalactosyldiacylglyceride (DGDG) are the major membrane lipids of chloroplasts. The question of the specialized functions of these unique lipids has received limited attention. One function is to support violaxanthin de-epoxidase (VDE) activity, an enzyme of the violaxanthin cycle. To understand better the properties of this system, the effects of galactolipids and phosphatidylcholines on VDE activity were examined by two independent methods. The results show that the micelle-forming lipid (MGDG) and bilayer forming lipids (DGDG and phosphatidylcholines) support VDE activity differently. MGDG supported rapid and complete de-epoxidation starting at a threshold lipid concentration (10 μM) coincident with complete solubilization of violaxanthin. In contrast, DGDG supported slow but nevertheless complete to nearly complete de-epoxidation at a lower lipid concentration (6.7 μM) that did not completely solubilize violaxanthin. Phosphotidylcholines showed similar effects as DGDG except that de-epoxidation was incomplete. Since VDE requires solubilized violaxanthin, aggregated violaxanthin in DGDG at low concentration must become solubilized as de-epoxidation proceeds. High lipid concentrations had lower activity possibly due to formation of multilayered structures (liposomes) that restrict accessibility of violaxanthin to VDE. MGDG micelles do not present such restrictions. The results indicate VDE operates throughout the lipid phase of the single bilayer thylakoid membrane and is not limited to putative MGDG micelle domains. Additionally, the results also explain the differential partitioning of violaxanthin between the envelope and thylakoid as due to the relative solubilities of violaxanthin and zeaxanthin in MGDG, DGDG and phospholipids. The violaxanthin cycle is hypothesized to be a linked system of the thylakoid and envelope for signal transduction of light stress.     

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using (31)P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L beta) to liquid-crystalline (L alpha) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L alpha to the inverted hexagonal (HII) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L alpha phase compared to those in the presence of HII phase forming lipids. Our data furthermore imply that the HII phase is better suited to maintain high VDE activities at lower temperatures.     

Antisense suppression of violaxanthin de-epoxidase in tobacco does not affect plant performance in controlled growth conditions

Violaxanthin de-epoxidase (VDE) catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Tobacco was transformed with an antisense VDE construct under control of the cauliflower mosaic virus 35S promoter to determine the effect of reduced levels of VDE on plant growth. Screening of 40 independent transformants revealed 18 antisense lines with reduced levels of VDE activity with two in particular (TAS32 and TAS39) having greater than 95% reduction in VDE activity. Northern analysis demonstrated that these transformants had greatly suppressed levels of VDE mRNA. De-epoxidation of violaxanthin was inhibited to such an extent that no zeaxanthin and only very low levels of antheraxanthin could be detected after exposure of leaves to high light (2000 μmol m⁻² s⁻¹ for 20 min) with no observable effect on levels of other carotenoids and chlorophyll. Non-photochemical quenching was greatly reduced in the antisense VDE tobacco, demonstrating that a significant level of the non-photochemical quenching in tobacco requires de-epoxidation of violaxanthin. Although the antisense plants demonstrated a greatly impaired de-epoxidation of violaxanthin, no effect on plant growth or photosynthetic rate was found when plants were grown at a photon flux density of 500 or 1000 μmol m⁻² s⁻¹ under controlled growth conditions as compared to wild-type tobacco. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Structural Basis for the pH-Dependent Xanthophyll Cycle in Arabidopsis thaliana

Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent K_m that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDE_cd) at acidic and neutral pH. At neutral pH, VDE_cd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin β-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate.     

The Kinetics of Zeaxanthin Formation Is Retarded by Dicyclohexylcarbodiimide

The de-epoxidation of violaxanthin to antheraxanthin (Anth) and zeaxanthin (Zeax) in the xanthophyll cycle of higher plants and the generation of nonphotochemical fluorescence quenching in the antenna of photosystem II (PSII) are induced by acidification of the thylakoid lumen. Dicyclohexylcarbodiimide (DCCD) has been shown (a) to bind to lumen-exposed carboxy groups of antenna proteins and (b) to inhibit the pH-dependent fluorescence quenching. The possible influence of DCCD on the de-epoxidation reactions has been investigated in isolated pea (Pisum sativum L.) thylakoids. The Zeax formation was found to be slowed down in the presence of DCCD. The second step (Anth → Zeax) of the reaction sequence seemed to be more affected than the violaxanthin → Anth conversion. Comparative studies with antenna-depleted thylakoids from plants grown under intermittent light and with unstacked thylakoids were in agreement with the assumption that binding of DCCD to antenna proteins is probably responsible for the retarded kinetics. Analyses of the DCCD-induced alterations in different antenna subcomplexes showed that Zeax formation in the PSII antenna proteins was predominantly influenced by DCCD, whereas Zeax formation in photosystem I was nearly unaffected. Our data support the suggestion that DCCD binding to PSII antenna proteins is responsible for the observed alterations in xanthophyll conversion.     

Functional and structural characterization of domain truncated violaxanthin de‐epoxidase

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 K_M 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.     

Multiple sources of carbonic anhydrase activity in pea thylakoids: soluble and membrane-bound forms

Carbonic anhydrase (CA) activity of pea thylakoids, thylakoid membranes enriched with photosystem I (PSI-membranes), or photosystem II (PSII-membranes) as well as both supernatant and pellet after precipitation of thylakoids treated with detergent Triton X-100 were studied. CA activity of thylakoids in the presence of varying concentrations of Triton X-100 had two maxima, at Triton/chlorophyll (triton/Chl) ratios of 0.3 and 1.0. CA activities of PSI-membranes and PSII-membranes had only one maximum each, at Triton/Chl ratio 0.3 or 1.0, respectively. Two CAs with characteristics of the membrane-bound proteins and one CA with characteristics of the soluble proteins were found in the medium after thylakoids were incubated with Triton. One of the first two CAs had mobility in PAAG after native electrophoresis the same as that of CA residing in PSI-membranes, and the other CA had mobility the same as the mobility of CA residing in PSII-membranes, but the latter was different from CA situated in PSII core-complex (Ignatova et al. 2006 Biochemistry (Moscow) 71:525–532). The properties of the “soluble” CA removed from thylakoids were different from the properties of the known soluble CAs of plant cell: apparent molecular mass was about 262 kD and it was three orders more sensitive to the specific CA inhibitor, ethoxyzolamide, than soluble stromal CA. The data are discussed as indicating the presence of, at least, four CAs in pea thylakoids.     

High Irradiance Effects on the Xanthophyll Cycle Pigments and the Activity of Violaxanthin De-Epoxidase in Soybean Callus

High irradiance (HI) effects on xanthophyll cycle pigments (XCP) and activity of violaxanthin de-epoxidase (VDE) in terms of de-epoxidation index (DEI) were studied in soybean calli. The calli from the hypocotyl segments of 5-d seedlings were induced on a solid (1.1 % agar) MS medium (pH 5.8) supplemented with 4.52 M 2,4-dichloro-phenoxyacetic acid, 2.32 M kinetin, and 3 % sucrose. After a 30 d cultivation, the green calli were irradiated for 24 h with white light (HI, 1 300 mol m^–2 s^–1) and VDE was isolated from the photosystem 2 (PS2) particles. In the control (0 h irradiation) callus, the reaction of PS2 particles with VDE in the presence or absence of Tween 20 resulted in the decrease of VIO content and the increase of ZEA content. In the 24 h HI-callus, the reaction of PS2 particles in the absence of VDE led to the decrease of VIO and ANT contents and increase of ZEA content. In the control, DEIs in the presence of VDE with or without 0.1 %Tween 20 (1.04 and 1.06, respectively) were significantly higher than the DEI (0.76) in the absence of VDE. In the HI-callus, DEIs in the presence of VDE with or without 0.1 %Tween 20 (0.98 and 0.96, respectively) were similar to that (1.03) in the absence of VDE.     

Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using ³¹P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L_β) to liquid-crystalline (L_α) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L_α to the inverted hexagonal (H_II) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L_α phase compared to those in the presence of H_II phase forming lipids. Our data furthermore imply that the H_II phase is better suited to maintain high VDE activities at lower temperatures.     

Cyclic electron flow within PSII protects PSII from its photoinhibition in thylakoid membranes from spinach chloroplasts

Cyclic electron flow within PSII (CEF-PSII) was proven to alleviate the photoinhibition of PSII. We set the conditions where CEF-PSII functioned or did not, by adding nigericin to the reaction mixture for the dissipation of DeltapH across thylakoid membranes, and then the thylakoids were illuminated. When CEF-PSII did not function and the activity of linear electron flow (LEF) was low, light-treated thylakoid membranes largely lost the activity of LEF. The inactivation of LEF was due to the loss of the activity of PSII, but not that of PSI. The inactivation of PSII was suppressed, when CEF-PSII functioned or LEF was enhanced. These results imply that CEF-PSII contributes to the protection of PSII from its photoinhibition with LEF, as an electron sink.     

Extrinsic photosystem II carbonic anhydrase in maize mesophyll chloroplasts

One form of carbonic anhydrase (CA) has been observed in maize (Zea mays) thylakoids and photosystem II (PSII)-enriched membranes. Here, we show that an antibody produced against a thylakoid lumen-targeted CA found in Chlamydomonas reinhardtii reacts with a single 33-kD polypeptide in maize thylakoids. With immunoblot analysis, we found that this single polypeptide could be identified only in mesophyll thylakoids and derived PSII membranes, but not in bundle sheath thylakoids. Likewise, a CA activity assay confirmed a large amount of activity in mesophyll, but not in bundle sheath membranes. Immunoblot analysis and CA activity assay showed that the maximum CA can be obtained in the supernatant of the PSII-enriched membranes washed with 1 M CaCl(2), the same procedure used to remove all extrinsic lumenal proteins from PSII. Because this CA reacts with an antibody to lumen-directed CA in C. reinhardtii, and because it can be removed with 1 M CaCl(2) wash, we refer to it tentatively as extrinsic CA. This is to distinguish it from another form of CA activity tightly bound to PSII membranes that remains after CaCl(2) wash, which has been described previously. The function of extrinsic CA is not clear. It is unlikely to have the same function as the cytoplasmic CA, which has been proposed to increase the HCO(-)(3) concentration for phosphoenolpyruvate carboxylase and the C(4) pathway. We suggest that because the extrinsic CA is associated only with thylakoids doing linear electron flow, it could function to produce the CO(2) or HCO(-)(3) needed for PSII activity.