Montserrat Busquets

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

BackgroundCarotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts.ResultsA method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way.ConclusionsWe have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.

Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa

Lipoxygenases (LOXs), which are essential in eukaryotes, have no confirmed function in prokaryotes that are devoid of polyunsaturated fatty acids. The structure of a secretable LOX from Pseudomonas aeruginosa (Pa_LOX), the first available from a prokaryote, presents significant differences with respect to eukaryotic LOXs, including a cluster of helices acting as a lid to the active center. The mobility of the lid and the structural variability of the N‐terminal region of Pa_LOX was confirmed by comparing 2 crystal forms. The binding pocket contains a phosphatidylethanolamine phospholipid with branches of 18 (sn‐1) and 14/16 (sn‐2) carbon atoms in length. Carbon atoms from the sn‐1 chain approach the catalytic iron in a manner that sheds light on how the enzymatic reaction might proceed. The findings in these studies suggest that Pa_LOX has the capacity to extract and modify unsaturated phospholipids from eukaryotic membranes, allowing this LOX to play a role in the interaction of P. aeruginosa with host cells.—Garreta, A., Val‐Moraes, S. P., García‐Fernández, Q., Montserrat Busquets, C. J., Oliver, A., Ortiz, A., Gaffney, B. J., Fita, I., Manresa, A., Carpena, X., Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa. FASEB J. 27, 4811‐4821 (2013). www.fasebj.org

Bacterial lipoxygenases, a new subfamily of enzymes? A phylogenetic approach

Lipoxygenases (EC. 1.13.11.12) are a non-heme iron enzymes consisting of one polypeptide chain folded into two domains, the N-terminal domain and the catalytic moiety β-barrel domain. They catalyze the dioxygenation of 1Z,4Z-pentadiene moieties of polyunsaturated fatty acids obtaining hydroperoxy fatty acids. For years, the presence of lipoxygenases was considered a eukaryotic feature, present in mammals, plants, small marine invertebrates, and fungi, but now, some lipoxygenase sequences have been detected on prokaryotic organisms, changing the idea that lipoxygenases are exclusively a eukaryotic affair. Lipoxygenases are involved in different types of reactions on eukaryote organisms where the biological role and the structural characteristics of these enzymes are well studied. However, these aspects of the bacterial lipoxygenases have not yet been elucidated and are unknown. This revision discusses biochemical aspects, biological applications, and some characteristics of these enzymes and tries to determine the existence of a subfamily of bacterial lipoxygenases in the context of the phylogeny of prokaryotic lipoxygenases, supporting the results of phylogenetic analyzes with the comparison and discussion of structural information of the first prokaryotic lipoxygenase crystallized and other eukaryotic lipoxygenases structures.

Interaction of aminoglycosides and colistin with model membranes: Liposomes and monolayers

Abstract The interaction of gentamicin (G), kanamycin (K), spectinomycin (Sp), streptomycin (St) and a polymixin, colistin (C), with lipids was studied by using liposomes and monomolecular layers as membrane models. The lipids used were phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol disphosphate (PIP 2 ), gangliosides (Gan) and cholesterol (Chol). The results show that only colistin is able to induce the leakage of entrapped carboxyfluorescein (CF) from the liposomes after incubation. Moreover, this interaction is not clearly related to a single type of phospholipid, is dependent on the PL/colistin ratio and is slightly modified by the pH or the temperature of the incubation media. The interactions of these molecules with the polar heads of phospholipids were studied using 1-anilino-8-napthalenesulphonic acid (ANS) as fluorescent probe. The polarization values indicate that these antibiotics induce rigidification of the membrane. Monolayers having the same lipid composition of liposomes were spread on aqueous subphases and pressure increases, after injection of drug molecules, were recorded. In this set of experiments all molecules exhibit interaction, colistin and kanamycin being those with the maximum activity. Moreover, no specific differences could be assigned to the different lipids used except for kanamycin and gangliosides. The mathematical equations of the penetration kinetics for all the assays carried out were determined. The Linewe aver-Burk equation gave the best fit to the experimental values, the regression coefficient being higher than 0.98 in all cases. Compression isotherms of the same phospholipid mixtures were recorded, the antibiotic molecules being dissolved in the subphase. The compressibility and areaJmolecule were slightly affected by the presence of aminoglycosides in the aqueous phase. In contrast, the presence of colistin induced expansion of the monolayer especially at low pressures, thus indicating the existence of interactions. Comparing these results with those in the literature, it appears clear that the membrane model used exerts a strong influence on the results obtained.

Interaction of colistin with lipids in liposomes and monolayers

Abstract Colistin is an antibiotic member of the polymixin family, showing a high amphipathic character. Its mechanism of action has been related to its ability to disrupt phospholipid bilayers of the bacterial membrane. Due to its hydrophobic properties colistin can interact both with the polar heads and the alkyl chains of the phospholipids. Moreover, it has a polycationic structure, so its interactions with phospholipids should be highly dependent on the electric charge of the lipid and the ionisation state of polymixin molecule. In the present paper we report on physicochemical studies to define the type and characteristics of these interactions. The surface activity of colistin has been shown to be highly dependent on the pH of the medium, the less protonated forms being more stable at the air–water interface. Interaction with phospholipid monolayers shows the same tendency. Colistin is also able to form mixed monolayers with phospholipids with small deviations from ideality. Physicochemical studies carried out with fluorescent probes indicate the presence of pure colistin aggregates that can coexist with mixed micelles composed of colistin/phospholipids. In no case did the interaction cause significative changes in the transition temperature of phospholipids.

Study of the inhibition capacity of an 18-mer peptide domain of GBV-C virus on gp41-FP HIV-1 activity

The peptide sequence (175-192) RFPFHRCGAGPKLTKDLE (P59) of the E2 envelope protein of GB virus C (GBV-C) has been proved to decrease cellular membrane fusion and interfere with the HIV-1 infectivity in a dose-dependent manner. Based on these previous results, the main objective of this study was to deepen in the physicochemical aspects involved in this interaction. First, we analyzed the surface activity of P59 at the air-water interface as well as its interaction with zwitterionic or negatively charged lipid monolayers. Then we performed the same experiments with mixtures of P59/gp41-FP. Studies on lipid monolayers helped us to understand the lipid-peptide interaction and the influence of phospholipids on peptide penetration into lipid media. On another hand, studies with lipid bilayers showed that P59 decreased gp41-FP binding to anionic Large Unilamellar Vesicles. Results can be attributed to the differences in morphology of the peptides, as observed by Atomic Force Microscopy. When P59 and gp41-FP were incubated together, annular structures of about 200 nm in diameter appeared on the mica surface, thus indicating a peptide-peptide interaction. All these results confirm the gp41-FP-P59 interaction and thus support the hypothesis that gp41-FP is inhibited by P59.

Effect of several anions on the activity of mitochondrial malate dehydrogenase from pig heart

Abstract Mitochondrial malate dehydrogenase (mMDH) shows a complex dependence upon ionic environment that includes kinetic and structural effects. We measured mMDH activity in several buffers (phosphate, MOPS, and MES) at pH 6.5 and 7.5, and in the presence of a number of anions, at highly diluted enzyme concentrations where mMDH showed significant loss of activity. Under these conditions, mMDH activity shows a non-linear dependence on enzyme concentration, in agreement with the existence of a dimer–monomer equilibrium, where only the dimeric form is active. According to this hypothesis, the dissociation constant of mMDH dimer has been determined to be 5.4 nM in the MES buffer at pH 6.5. Either the presence of a small anion like phosphate, or an increase of the pH from 6.5 to 7.5 shifts the equilibrium in favor of the dimeric form with the two effects appearing to be additive. To extend the study, we analysed the effect of a number of anions on the mMDH activity in 50 mM MOPS buffer at pH 7.5. All the anions had a dual effect: at low concentrations, they increased the activity of mMDH, while at high concentrations, they inhibited it. A more accurate analysis of the data revealed that the activation capacity of all the anions tested was similar, although they differed in their inhibitory influence. To show these differences more clearly, the experiment was repeated in 50 mM phosphate buffer at pH 7.5, under conditions where almost all activations were due to the buffer. The analysis of the results obtained under these conditions revealed the following sequence of inhibition potency: phosphate

Miscibility of dipalmitoylphosphatidylcholine, oleic acid and cholesterol measured by DSC and compression isotherms of monolayers

Abstract The miscibility of dipalmitoylphosphatidylcholine and oleic acid (DPPC/OA) and of dipalmitoylphosphatidylcholine/oleic acid/cholesterol (DPPC/OA/Chol) in mixed monolayers was determined. According to the thermodynamic calculations, the energy involved in the process is very low, suggesting the lack of strong interactions. Moreover, the presence of oleic acid or cholesterol has little influence on the transition temperature of DPPC.

Interaction of analgesics with lecithin and ganglioside monolayers

Abstract Using monolayers as a membrane model, the interaction of dextrometorphan, codeine and phentanyle with lecithin and ganglioside was studied. The penetration kinetics of these drugs into the monolayers were measured at 5 and 20 mN · m−1 and the results show that the interaction of phentanyl is maximal with lecithin, but not with ganglioside. This fact seems to exclude GM1 as a μ-opioid receptor.

Separation and kinetic properties of the molecular forms of chicken liver cytoplasmic aspartate aminotransferase

A method is proposed for the separation of the five molecular forms, alpha, beta, gamma, delta and epsilon, of chicken liver cytoplasmic aspartate aminotransferase free from lactate dehydrogenase activity. These molecular forms varied in isoelectric point, but no differences were observed either in their Michaelis constants or in the degree of their inhibition by excess of 2-oxoglutarate or L-aspartate.