Abdelkarim Abousalham

Egg yolk lipoproteins as substrates for lipases.

Egg yolk emulsions containing phospholipids (about 31%, w/w) are classically used as substrates for measuring phospholipase A2 activity using the pH-stat method. Here we investigated the susceptibility of egg yolk lipoproteins to lipolysis by various highly purified lipases of animal or microbial origin. Egg yolk lipoproteins, which contain up to 65% triacylglycerols, were found to be effective substrates for all the lipases tested. The specific activities measured on egg yolk lipoproteins using the pH-stat technique were found to be 8000, 1000, 1250 and 1700 U/mg in the case of human pancreatic lipase, horse pancreatic lipase, porcine pancreatic lipase and Humicola lanuginosa lipase, respectively. No activity was detected in the absence of colipase with any of the pancreatic lipases tested. Consequently, the classical egg yolk assay cannot be considered as a specific phospholipase A2 assay.

Surface properties of unsaturated non-oxidized and oxidized free fatty acids spread as monomolecular films at an argon/water interface.

The interfacial properties of monomolecular films of stearic acid (SA) oleic acid (OA), linoleic acid (LA), ricinoleic acid (RA), 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (13-HPODE) and 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid (13-HODE) were studied by recording the changes occurring in response to monomolecular film compression in their surface pressure and surface potential at the argon/water interface. The oxidized free fatty acids are more expanded than the parent non-oxidized free fatty acids, reflecting a higher hydrophilic-lipophilic balance. The lift-off values of the molecular area of 13-HODE, 13-HPODE and RA were 68, 74 and 106 A2 molecule(-1), respectively, as compared to 47 and 40 A2 molecule(-1) in the case of LA and OA, respectively. Variations in the molecular orientation of free fatty acids can result in large changes in the dipole moment which are not accompanied by appreciable changes in the surface pressure. In the case of the oxidized free fatty acids, the spontaneous desorption into the aqueous phase was found to increase at increasing surface pressures. The desorption rates of OA and LA increased dramatically in the presence of beta-cyclodextrin (beta-CD); whereas the presence of beta-CD only slightly increased the desorption rates of the oxidized free fatty acids.

Crystallization and preliminary crystallographic study of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp)

The plant phospholipase D (PLD) is considered to be a key enzyme involved in various physiological processes such as signal transduction and membrane metabolism. Crystals of the PLD protein from Vigna unguiculata have been produced from the recombinant 768 amino-acid protein. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 157.7, b = 65.6, c = 90.2 A, beta = 111.5 degrees. There is one molecule in the asymmetric unit. Frozen crystals diffract to at least 1.94 A resolution using synchrotron radiation. A search for heavy-atom derivatives using ytterbium and tungstate is currently under way in order to solve the three-dimensional structure.

Continuous measurement of the lipoxygenase-catalyzed oxidation of unsaturated lipids using the monomolecular film technique.

PurposeThis paper presents the first detailed kinetic investigation involving the continuous measurement of the soybean lipoxygenase 1 (LOX1)-catalyzed oxidation of unsaturated lipids using the monomolecular film technique at an argon/water interface.Materials and MethodsThe presence of oxidation products in the monolayer is qualitatively detected, at a constant area, by an increase in the monolayer surface pressure. Alternatively, the rate of lipid oxidation can be measured, at a constant surface pressure, by a backward movement of the mobile barrier, due to the oxidation-dependent increase in the monolayer area.ResultsFor instance, the LOX1-catalyzed oxidation of 1,2-di[cis-9,12-octadecadienoyl]-sn-glycero-3-phosphocholine (diC18:2PC) monolayer was found to be characterized by a time dependent increase in the monolayer area, at constant surface pressure. However, the increase in the monolayer area was thought to be caused first by the penetration of the enzyme into the interface, and secondly, by the formation of hydroperoxides at the interface, due to the LOX1-catalyzed oxidation of the diC18:2PC film. The rate of the LOX1-catalyzed oxidation of diC18:2PC film was measured by subtracting the increase in the area due to the LOX1-penetration into the non-oxidizable 1,2-di[cis-9-octadecenoyl]-sn-glycero-3-phosphocholine (diC18:1PC) film from the increase in the area due to LOX penetration and oxidation of the diC18:2PC film. At a constant optimum surface pressure of 1xa0mN m−1, similar initial rates of LOX1-catalyzed oxidation are observed with both linoleic acid methyl ester (C18:2) and diC18:2PC. It is worth noting that the surface density of C18:2 acyl chains is also similar in both films. We observed that a phosphatidylcholine (PC) film with two potentially oxidizable chains (e.g., diC18:2PC) is oxidized at a rate which is twice that obtained with a PC containing a single oxidizable chain (e.g., 1-hexadecanoyl-2-[cis-9,12-octadecadienoyl]-sn-glycero-3-phosphocholine).ConclusionsThe enzymatic lipid oxidation seems to occur when the monolayer is in the expanded state. This expanded state may possibly result in vivo from the lipolysis of a biomembrane and consequently lipolysis and lipid oxidation are coupled at the membrane level.