linoleic-acid and 1-palmitoyl-2-linoleoylphosphatidylcholine

Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A2 (TdsPLA2III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA2III and shed further light on the involvement of sPLA2 isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA2III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA2III with an N-terminal 6×His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6×His-TdsPLA2III fusion protein is then refolded using a step-wise dialysis approach. About 40mg purified and active protein was obtained from 1L of cell culture. This recombinant 6×His-TdsPLA2III protein shows PLA2 activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA2s: Ca(2+)-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature.

Topics: Amino Acid Sequence; Dithiothreitol; Escherichia coli; Gene Expression; Hot Temperature; Inclusion Bodies; Linoleic Acid; Molecular Sequence Data; Phosphatidylcholines; Phospholipase A2 Inhibitors; Phospholipases A2, Secretory; Protein Folding; Recombinant Fusion Proteins; Triticum

Hydrolysis of 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) hydroperoxide (PLPC-OOH) in PLPC liposomal membrane by Crotalus adamanteus venom phospholipase A2 (PLA2) was studied by measuring the decay of PLPC and PLPC-OOH and the formation of linoleate and linoleate hydroperoxide. We demonstrate that PLA2 has a preference to hydrolyze PLPC-OOH over PLPC when more than 25 mole % of cholesterol is incorporated into the PLPC liposomal membrane. Similar results were obtained for PLPC hydroxide (PLPC-OH). These results suggest that cholesterol displaces the hydrophilic hydroperoxyl and hydroxyl moieties of PLPC-O(O)H to the surface interface of the liposomal membrane where they are more accessible to PLA2 hydrolysis.

Topics: Cholesterol; Crotalid Venoms; Hydrolysis; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Liposomes; Oxidation-Reduction; Phosphatidylcholines; Phospholipases A; Phospholipases A2

Despite repeated suggestions that antioxidant activity of conjugated linoleic acid (CLA), a collective of conjugated dienoic isomers of linoleic acid, underlies its reported anticarcinogenic and antiatherosclerotic effects, the antioxidant properties of CLA remain ill-defined. Therefore, this study was undertaken to gain more insight into the mechanism of potential CLA antioxidant activity. It was tested whether CLA could protect membranes composed of 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) from oxidative modification under conditions of metal ion-dependent or -independent oxidative stress. Progress of oxidation was determined by direct spectrophotometric measurement of conjugated diene formation and by gas chromatographic/mass spectrometric analysis of fatty acids. The oxidative susceptibility of CLA was higher than that of linoleic acid, and comparable to arachidonic acid. When oxidation of PLPC (1.0 mM) was initiated using the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) or the water-soluble 2,2'-azobis(2-amidinopropane) hydrochloride, the radical scavengers vitamin E and butylated hydroxytoluene (BHT) at 0.75 microM efficiently inhibited PLPC oxidation, as evident from a clear lag phase. In contrast, 0.75 microM CLA did not have any significant effect on PLPC oxidation. Inhibition of PLPC oxidation by higher concentrations of CLA appeared due to competition, not to an antioxidant effect. When oxidation of PLPC was initiated by hydrogen peroxide/Fe2+ (500 microM/0.05-20 microM), both vitamin E (1 microM) and ethylene glycol-bis(aminoethyl ether) tetraacetic acid (50 microM) efficiently inhibited PLPC oxidation. However, CLA (1-50 microM) did not show a clear protective effect under any of the conditions tested. We conclude that CLA, under these test conditions, does not act as an efficient radical scavenger in any way comparable to vitamin E or BHT. CLA also does not appear to be converted into a metal chelator under metal-ion dependent oxidative stress, as had previously been suggested. On the basis of our observations, a role for CLA as an antioxidant does not seem plausible.

Topics: Antioxidants; Arachidonic Acid; Butylated Hydroxytoluene; Egtazic Acid; Fatty Acids; Free Radical Scavengers; Gas Chromatography-Mass Spectrometry; Linoleic Acid; Linoleic Acids; Liposomes; Oxidation-Reduction; Oxidative Stress; Phosphatidylcholines; Spectrophotometry; Vitamin E

Kinetics of the consumption of hypochlorous acid in its reaction with double bonds of unsaturated phospholipids and fatty acids were measured using luminol chemiluminescence. Stoichiometry ratios between the consumption of HOCl/OCl- and the loss of double bonds vary from 2:1 to 1:1. Highest values were found in DMPC liposomes containing 5 mol% oleic acid or OPPC. With increasing content of double bonds or higher numbers of double bonds in a fatty acid acyl chain due to incorporated unsaturated fatty acids or phospholipids in DMPC liposomes the stoichiometry ratio falls continuously to 1:1. A ratio of about 1:1 was observed in multilamellar and unilamellar liposomes composed of egg yolk phosphatidylcholine. Products of the reaction of oleic acid with hypochlorous acid were analyses by 1H-NMR spectroscopy. Chlorohydrins were formed in both DMPC liposomes containing 5 or 40 mol% oleic acid.

Topics: Chemical Phenomena; Chemistry, Physical; Dimyristoylphosphatidylcholine; Hypochlorous Acid; Linoleic Acid; Linoleic Acids; Liposomes; Luminescent Measurements; Magnetic Resonance Spectroscopy; Oleic Acid; Oleic Acids; Phosphatidylcholines