Origin of the latency phase during the action of phospholipase A2 on unmodified phosphatidylcholine vesicles

The reaction progress curve for the action of pig-pancreatic phospholipase A₂ on dimyristoylphosphatidylcholine vesicles is characterized under a variety of conditions. The factors that regulate the rate of hydrolysis during the presteady-state phase determine the latency period. The results demonstrate that the accelerated hydrolysis following the latency phase of the reaction progress curve is due to the product-assisted binding of the enzyme to the substrate bilayer by chaning the number of bindings sites and therefore the binding equilibrium. A critical mole fraction of products appears to be formed in the substrate bilayers before the steady-state phase of hydrolysis begins. The latency phase shows a minimum at the phase-transition temperature of the substrate vesicles; however, we did not observe a significant binding of the enzyme to pure substrate bilayers even at the phase-transition temperature. The rate of binding of the enzyme is found to be fast and the rate of desorption of the bound enzyme is very slow compared to the latency phase. The rate of redistribution of products between substrate bilayers is rather slow. These observations demonstrate that during the latency phase of the action of phospholipase A₂, a critical mole fraction of products is formed in the substrate bilayer.     

The relationship between compositional phase separation and vesicle morphology: Implications for the regulation of phospholipase A2 by membrane structure

The action of phospholipase A₂ (PLA₂) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA₂. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.     

Induction of distinct sets of secretory phospholipase A2 in rodents during inflammation

Although the expression of the prototypic secretory phospholipase A₂ (sPLA₂), group IIA (sPLA₂-IIA), is known to be up-regulated during inflammation, it remains uncertain if other sPLA₂ enzymes display similar or distinct profiles of induction under pathological conditions. In this study, we investigated the expression of several sPLA₂s in rodent inflammation models. In lipopolysaccharide (LPS)-treated mice, the expression of sPLA₂-V, and to a lesser extent that of sPLA₂-IID, -IIE, and -IIF, were increased, whereas that of sPLA₂-X was rather constant, in distinct tissues. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, in which the expression of sPLA₂-IID, -IIF and -V was increased, was significantly reduced by YM-26734, a competitive sPLA₂-IIA inhibitor that turned out to inhibit sPLA₂-IID, -IIE, -V and -X as well. In contrast, sPLA₂-IIA was dominant in carageenin-induced pleurisy in rats, where the accumulation of exudate fluids and leukocytes was significantly ameliorated by YM-26734. These results indicate that distinct sPLA₂s can participate in inflammatory diseases according to tissues, animal species, and types of inflammation.     

Thermotropic change in phospholipid packing in microsomal membranes sensed by phospholipase A2

A reversible, temperature-dependent change in phospholipid packing occurring between 0°C and 12°C has been identified in microsomal membranes by the use of phospholipase A₂ from Crotalus atrox. It manifests itself as a drastic increase in susceptibility to the phospholipase and depends on non-lipid (presumably protein) membrane components. It is suggested that this change could underlie the change in transmembrane mobility of phospholipids which occurs in the same temperature range.     

TLR-4 mediated group IVA phospholipase A2 activation is phosphatidic acid phosphohydrolase 1 and protein kinase C dependent

Group IVA phospholipase A₂ (GIVA PLA₂) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA₂. We also studied the effect of PAP-1 and PKC on Ca^+ 2 induced and synergy enhanced GIVA PLA₂ activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo₂-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca^+ 2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA₂ synergistic activation by stimulation with Kdo₂-Lipid A and ATP.     

Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes

The activities of enzymes involved in lipid metabolism—phospholipase A₂ (PLA₂) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)—were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA₂ activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA₂ activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA₂ activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA₂ activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA₂ activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.     

Functional characterization and FTIR-based 3D modeling of full length and truncated forms of Scorpio maurus venom phospholipase A2

Background

Heterodimeric phospholipase A₂ from venom glands of Tunisian scorpion Scorpio maurus (Sm-PLGV) had been purified. It contains long and short chains linked by a disulfide bridge. Sm-PLGV exhibits hemolytic activity towards human erythrocytes and interacts with phospholipid monolayers at high surface pressure. The investigation of structure-function relationships should provide new clues to understand its activity.

Sterol-phospholipid interactions in model membranes Effect of polar group substitutions in the cholesterol side-chain at C20 and C22

The interactions of phospholipids with four different cholesterol derivatives substituted with one OH or one keto group at position C₂₀ or C₂₂ of the side-chain were studied. The derivatives were the 22,R-hydroxy; 22,S-hydroxy; 22-keto- and 20,S-hydroxycholesterol. Two aspects of the interactions were investigated: (1) the effect of the cholesterol derivatives on the gel → liquid crystalline phase transition of dipalmitoylphosphatidylcholine (DPPC) and of dielaidoylphosphatidylethanolamine (DEPE) monitored by differential scanning calorimetry and (2) The effect on the lamellar → hexagonal H_II phase transition of DEPE monitored by DSC and by ³¹P-NMR to determine structural changes. The gel → liquid crystalline phase transition was affected by the cholesterol derivatives to a much larger extent in the case of DPPC than of DEPE. In both cases, there was a differential effect of the four derivatives, the 22,R-hydroxycholesterol being the less effective. In DPPC-sterol 1:1 systems, 22,R-hydroxycholesterol does not suppress the melting transition, the ΔH values becomes 7.1 kcal · mol^?1 as compared to 8.2 kcal · mol^?1 for the pure lipid. 22,S-OH cholesterol has a much stronger effect (ΔH = 3.1 kcal · mol^?1) and 22-ketocholesterol suppresses the transition completely. In DEPE mixtures of all these compounds, the melting transition of the phospholipid is still observable. The transition temperature was shifted to lower values (?13.5°C in the presence of 20,S-OH cholesterol). The ΔH of the transition was lowered by these compounds except in DEPE-22,R-OH cholesterol mixtures and the cooperativity of the transition (reflected by the width at half peak height) was reduced. The lamellar → hexagonal H_II phase transition was also affected by the presence of these cholesterol derivatives. The transition temperature value was depressed with all these compounds. 20,S-OH cholesterol was the most effective followed by 22,R-OH cholesterol. The ΔH of the transition was not strongly affected. The molecular interfacial properties of these derivatives were studied by the monomolecular film technique. It is most likely that 22,R-OH cholesterol due to the hydroxyl groups at the 3β- and 22,R-positions orients with the sterol nucleus lying flat at the air/water interface, since the compression isotherm of either the pure sterol or the DOPC-sterol mixture (molar ration, 1:1) monomolecular film exhibits a transition at approx. 103 Ų, corresponding to the area of revolution of the sterol nucleus. This remarkable property, due probably to the existence of a kink between the side-chain and the long axis of the steroid nucleus, might explain the smaller effect of this sterol on the melting transition of either PC or PE systems.     

Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at > 8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested.     

Transmembrane distribution of phospholipids and their involvement in electron transport,as revealed by phospholipase A2 treatment of spinach thylakoids

Thylakoid membranes were treated with either pancreatic or snake venom phospholipase A₂, and the residual phospholipid content of these membranes was determined and compared to the rates of Photosystem II and/or Photosystem I electron transports. The hydrolysis curves of both phosphatidylglycerol and phosphatidylcholine displayed a first, rapid phase which was almost temperature-insensitive, followed by a second, slower phase which depended strongly on the temperature. When pancreatic phospholipase A₂ had access either to the outer face or to both faces of the thylakoid membrane, either only part of or all the phospholipids, respectively, could be hydrolysed. These results were interpreted as indicating an asymmetric distribution of phospholipids across the thylakoid membrane, phosphatidylglycerol and phosphatidylcholine being preferentially located in the outer and the inner layer, respectively. When acting on uncoupled thylakoid membranes, phospholipase A₂ exerted an inhibitory effect on Photosystem II activity and a stimulatory effect on Photosystem I activity. The involvement of phosphatidylcholine and of phosphatidylglycerol in electron transport activities of Photosystem II and of Photosystem I are discussed with special reference to the role of the external and internal pools of these phospholipids.     

Encapsulation of [Mn(bpy)3] cations in 2D [Mn(dca)3] sheet: Synthesis, X-ray structure and EPR study

The reaction of Mn(NO₃)₂ · 4H₂O, 2,2′-bipyridine (bpy) and sodium dicyanamide (dca) in aqueous medium yielded the {[Mn(bpy)₃][Mn(dca)₃]₂}_n (1). The single-crystal X-ray analysis of 1 revealed that the anionic part of the complex, [Mn(dca)₃]⁻, features infinite 2D sheets with a honeycomb-like porous structure having a void space of ca. 12 Å in which [Mn(bpy)₃]²⁺ cations are encapsulated to yield a fascinating molecular assembly. Mn^II ions possess an octahedral geometry both in the anionic and cationic components of complex 1. In the anionic component, each Mn^II ion is bridged by three pairs of dicyanamide anions in an end-to-end fashion with two other Mn^II ions from adjacent [Mn(dca)₃]⁻ moieties. This type of linking propagates parallel to the bc crystallographic plane to form 2D sheets. [Mn(bpy)₃]²⁺ is found to have somewhat “squeezed” upon encapsulation. No measurable magnetic interaction was evidenced through variable temperature magnetic susceptibility measurements. However, in addition to the broad g ≈ 2 resonance typical of magnetically diluted [Mn(bpy)₃]²⁺ cations, EPR spectroscopy evidenced exchange narrowing of the [Mn(dca)₃]⁻ resonance at g ≈ 2 thus indicating operation of weak magnetic interactions extended over the whole 2D network through the dca⁻ bridges.     

Group IIA and group V secretory phospholipase A2: quantitative analysis of expression and secretion and determination of the localization and routing in rat mesangial cells

Mesangial cells can be induced to express group IIA and group V secretory phospholipase A₂ (sPLA₂) at the mRNA level and at the protein level. In this report we quantitatively analyze the expression of both proteins in stimulated cells by Western blot techniques. We found that 75–80% of the total amount of synthesized group IIA sPLA₂ was secreted. The synthesized group V sPLA₂, however, was present almost exclusively intracellularly. The amount of group V present in the cell was comparable to the intracellular amount of group IIA sPLA₂. We furthermore studied the localization and routing of both proteins. Using fusion proteins of the group IIA or group V pre-sPLA₂ with green fluorescent protein it was established that both presequences are able to direct the proteins to the Golgi system. In immunofluorescence studies group V sPLA₂ expressed by rat mesangial cells was located in a punctate pattern in the cytosol with an enrichment near the nucleus. Immunofluorescent confocal laser scanning microscopy revealed that the group V and IIA sPLA₂ show partial colocalization in a Golgi-like structure in the inner part in the cell, but no colocalization was seen in the vesicles in the cytoplasm. The images also showed that group IIA sPLA₂ was located throughout the cell while group V was mainly present in the inner part of the cell. After treatment of the cells with brefeldin A or monensin the group IIA enzyme could no longer be detected, while group V sPLA₂ was still present although its localization was somewhat dependent on the treatment. Collectively, these results indicate that the two enzymes differ in both localization and routing in the cell, which underscores the hypothesis that the enzymes might have different functions.     

Synthesis, characterization and complexation properties of N-phosphorylated thioureas RNHC(S)NHP(O)(OiPr)2 (R = 2-Py, 3-Py) towards Ni(II)

Reaction of O,O′-diisopropylphosphoric acid isothiocyanate (iPrO)₂P(O)NCS with 2- or 3-aminopyridine leads to the new N-phosphorylated thioureas RNHC(S)NHP(O)(OiPr)₂ (R = 2-Py, HL^I; 3-Py, HL^II). Reaction of the potassium salt KL^I with Ni(II) in aqueous EtOH leads to the new complex Ni[2-PyNHC(S)NP(O)(OiPr)₂-N(Py),N(P),O]₂, where the metal cation is coordinated by two deprotonated ligands L^I through the pyridine and phosphorylamide nitrogen atoms, and the PO oxygen atoms. Using KL^II leads to an oligomeric (or polymeric) structure, where the Ni(II) cation is coordinated by two anionic ligands L^II through the CS sulfur atoms and the P-N nitrogen atoms, and the pyridine nitrogen atoms of neighboring molecules. The new compounds were investigated by ¹H and ³¹P{¹H} NMR spectroscopy, and microanalysis. Single crystal X-ray diffraction studies showed HL^I forms both intra- and intermolecular hydrogen bonds, which in turn lead to the formation of a polymeric chain. Moreover, π?π stacking interactions were observed between molecules of two neighboring chains.     

Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A2: Regulation of fat and cholesterol homeostasis

Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62-66)-loop deleted ΔPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric k_cat^?-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E^?B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.     

Identification and characterization of alkenyl hydrolase (lysoplasmalogenase) in microsomes and identification of a plasmalogen-active phospholipase A2 in cytosol of small intestinal epithelium

A lysoplasmalogenase (EC 3.3.2.2; EC 3.3.2.5) that liberates free aldehyde from 1-alk-1′-enyl-sn-glycero-3-phospho-ethanolamine or -choline (lysoplasmalogen) was identified and characterized in rat gastrointestinal tract epithelial cells. Glycerophosphoethanolamine was produced in the reaction in equimolar amounts with the free aldehyde. The microsomal membrane associated enzyme was present throughout the length of the small intestines, with the highest activity in the jejunum and proximal ileum. The rate of alkenyl ether bond hydrolysis was dependent on the concentrations of microsomal protein and substrate, and was linear with respect to time. The enzyme hydrolyzed both ethanolamine- and choline-lysoplasmalogens with similar affinities; the K_m values were 40 and 66 μM, respectively. The enzyme had no activity with 1-alk-1′-enyl-2-acyl-sn-glycero-3-phospho-ethanolamine or -choline (intact plasmalogen), thus indicating enzyme specificity for a free hydroxyl group at the sn-2 position. The specific activities were 70 nmol/min/mg protein and 57 nmol/min/mg protein, respectively, for ethanolamine- and choline-lysoplasmalogen. The pH optimum was between 6.8 and 7.4. The enzyme required no known cofactors and was not affected by low mM levels of Ca²⁺, Mg²⁺, EDTA, or EGTA. The detergents, Triton X-100, deoxycholate, and octyl glucoside inhibited the enzyme. The chemical and physical properties of the lysoplasmalogenase were very similar to those of the enzyme in liver and brain microsomes. In developmental studies the specific activities of the small intestinal and liver enzymes increased markedly, 11.1- and 3.4-fold, respectively, in the first ～40 days of postnatal life. A plasmalogen-active phospholipase A₂ activity was identified in the cytosol of the small intestines (3.3 nmol/min/mg protein) and liver (0.3 nmol/min/mg protein) using a novel coupled enzyme assay with microsomal lysoplasmalogenase as the coupling enzyme.     

Transformations of the Vaska-type complex trans-[RhCl(CO)(PTA)2] (PTA = 1,3,5-triaza-7-phosphaadamantane) during stepwise addition of HCl: Synthesis, characterization and crystal structure of trans-[RhCl2(PTA)(PTAH)]

The new aqua-soluble rhodium(I) complex trans-[RhCl₂(PTA)(PTAH)] (1) {PTAH = N-protonated form of 1,3,5-triaza-7-phosphaadamantane (PTA)} has been synthesized via the reaction of trans-[RhCl(CO)(PTA)₂] with aqueous HCl or N-chlorosuccinimide, or by the treatment of RhCl₃ with PTA. Compound 1 has been characterized by IR, ¹H and ³¹P{H} NMR spectroscopies, ESI-MS(+), elemental and single crystal X-ray diffraction analyses, the latter showing a square planar {RhCl₂P₂} geometry. Besides, the stepwise addition of diluted HCl to an aqueous solution of trans-[RhCl(CO)(PTA)₂] has been monitored by ³¹P{¹H} NMR and ESI-MS(+) techniques, allowing to detect a number of intermediate Rh(I) species.     

The abasic site lesions in the human telomeric sequence d[TA(G3T2A)3G3]: A thermodynamic point of view

Background

The abasic sites represent one of the most frequent lesions of DNA and most of the events able to generate such modifications involve guanine bases. G-rich sequences are able to form quadruplex structures that have been proved to be involved in several important biological processes.

Methods

In this paper, we report investigations, based on calorimetric, UV, CD and electrophoretic techniques, on 12 oligodeoxynucleotides analogues of the quadruplex forming human telomere sequence d[TA(G₃T₂A)₃G₃], in which each guanine has been replaced, one at a time, by an abasic site mimic.

Results

Although all data show that the modified sequences preserve their ability to form quadruplex structures, the thermodynamic parameters clearly indicate that the presence of an abasic site decreases their thermal stability compared to the parent unmodified sequence, particularly if the replacement concerns one of the guanosines involved in the formation of the central G-tetrad.

Conclusions

The collected data indicate that the effects of the presence of abasic site lesions in telomeric quadruplex structures are site-specific. The most dramatic consequences come out when this lesion involves a guanosine in the centre of a G-run.

General significance

Abasic sites, by facilitating the G-quadruplex disruption, could favour the formation of the telomerase primer. Furthermore they could have implications in the pharmacological approach targeting telomere.     

Lysophosphatidylethanolamine is – in contrast to – choline – generated under in vivo conditions exclusively by phospholipase A2 but not by hypochlorous acid

Inflammatory liver diseases are associated with oxidative stress mediated particularly by neutrophilic granulocytes. At inflammatory loci, hypochlorous acid (HOCl) is generated by myeloperoxidase. HOCl reacts with a large variety of molecules and induces (among other reactions) the formation of lysophosphatidylcholine (LPC) from polyunsaturated phosphatidylcholines (PC).As liver tissue contains huge amounts of polyunsaturated PC species enhanced LPC concentrations are detectable under these conditions. However, human liver contains also major amounts of polyunsaturated phosphatidylethanolamine (PE). It is so far widely unknown, if PE oxidation by HOCl leads to the generation of LPE in a similar way as observed in the case of PC. Using MALDI-TOF mass spectrometry (MS) and ³¹P NMR spectroscopy, LPC generation from unsaturated PC could be verified in the presence of HOCl. In contrast, unsaturated PE led exclusively to chlorohydrins and other oxidation products but not to LPE.Although these data were obtained with a quite simple model system, it is obvious that LPC is a much more suitable biomarker of oxidative stress than LPE: LPC is more readily generated and also more sensitively detectable by means of mass spectrometry and other spectroscopic methods. Nevertheless, it will also be shown that the nitrile of LPE is also generated. However, this compound is exclusively detectable as negative ion.     

On-line EPR study of free radicals induced by peroxidase/H2O2 in human low-density lipoprotein

The aim of this study was to use direct electron paramagnetic resonance (EPR) spectroscopy at 37 °C and spin trapping techniques to study radical species formed during horseradish peroxidase/H₂O₂-initiated low-density lipoprotein (LDL) oxidation. Using direct EPR, we obtained evidence for the formation not only of the α-tocopheroxyl radical but also of a protein radical(s), assigned to a tyrosyl radical(s) of apolipoprotein B-100 (apo B-100). Spin trapping with 2-methyl-2-nitrosopropane revealed (i) the formation of a mobile adduct with β-hydrogen coupling assigned to a lipid radical and (ii) a partially immobilised adduct detected in LDL as well as in apo B-100, assigned after proteolytic digestion to the trapping of a radical centred on a tertiary carbon atom of an aromatic residue, probably tyrosine. Our results support the hypothesis that radicals are initiators of the oxidative process, and show that their formation is an early event in peroxidase-mediated oxidation. We also tested the effects of resveratrol (RSV), a polyphenolic antioxidant present in red wine. Our data indicate that 1–10 μM RSV is able to accelerate α-tocopherol consumption, conjugated dienes formation and the decay kinetics of LDL-centred radicals. Since phenols are substrates for peroxidases, this result may be ascribed to a RSV-mediated catalysis of peroxidase activity.     

Modelling O2 and O2 unidirectional fluxes in plants. III: Fitting of experimental data by a simple model

Photosynthetic assimilation of CO₂ in plants results in the balance between the photochemical energy developed by light in chloroplasts, and the consumption of that energy by the oxygenation processes, mainly the photorespiration in C₃ plants. The analysis of classical biological models shows the difficulties to bring to fore the oxygenation rate due to the photorespiration pathway. As for other parameters, the most important key point is the estimation of the electron transport rate (ETR or J), i.e. the flux of biochemical energy, which is shared between the reductive and oxidative cycles of carbon. The only reliable method to quantify the linear electron flux responsible for the production of reductive energy is to directly measure the O₂ evolution by ¹⁸O₂ labelling and mass spectrometry. The hypothesis that the respective rates of reductive and oxidative cycles of carbon are only determined by the kinetic parameters of Rubisco, the respective concentrations of CO₂ and O₂ at the Rubisco site and the available electron transport rate, ultimately leads to propose new expressions of biochemical model equations. The modelling of ¹⁸O₂ and ¹⁶O₂ unidirectional fluxes in plants shows that a simple model can fit the photosynthetic and photorespiration exchanges for a wide range of environmental conditions. Its originality is to express the carboxylation and the oxygenation as a function of external gas concentrations, by the definition of a plant specificity factor Sp that mimics the internal reactions of Rubisco in plants. The difference between the specificity factors of plant (Sp) and of Rubisco (Sr) is directly related to the conductance values to CO₂ transfer between the atmosphere and the Rubisco site. This clearly illustrates that the values and the variation of conductance are much more important, in higher C₃ plants, than the small variations of the Rubisco specificity factor. The simple model systematically expresses the reciprocal variations of carboxylation and oxygenation exchanges illustrated by a “mirror effect”. It explains the protective sink effect of photorespiration, e.g. during water stress. The importance of the CO₂ compensation point, in classical models, is reduced at the benefit of the crossing points Cx and Ox, concentration values where carboxylation and oxygenation are equal or where the gross O₂ uptake is half of the gross O₂ evolution. This concept is useful to illustrate the feedback effects of photorespiration in the atmosphere regulation. The constancy of Sp and of Cx for a great variation of P under several irradiance levels shows that the regulation of the conductance maintains constant the internal CO₂ and the ratio of photorespiration to photosynthesis (PR/P). The maintenance of the ratio PR/P, in conditions of which PR could be reduced and the carboxylation increased, reinforces the hypothesis of a positive role of photorespiration and its involvement in the plant-atmosphere co-evolution.