- Phospholipase A2 Catalyses in Organic Media
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Phospholipase A2 catalyses in organic solvents with low water content are described.First, the effect of the different organic media on the hydrolysis of phosphatidylcholine catalyzed by phospholipase A2 is discussed.Second, the effect of temperature on the hydrolysis of phosphatidylcholine catalyzed by phospholipase A2 in chloroform with low water content is discussed.Finally, a novel transacylation of lysophosphatidylcholine catalyzed by phsopholipase A2 with long-chain acyl donors in chloroform with low water content is reported.
- Lin, Gialih,Wu, Fang-Chen,Liu, Shih-Huang
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- Optically trapping confocal Raman microscopy of individual lipid vesicles: Kinetics of phospholipase A2-catalyzed hydrolysis of phospholipids in the membrane bilayer
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Phospholipase A2 (PLA2)-catalyzed hydrolysis at the sn-2 position of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in optically trapped liposomes is monitored in situ using confocal Raman microscopy. Individual optically trapped liposomes (0.6 μm in diameter) are exposed to PLA2 isolated from cobra (Naja naja naja) venom at varying enzyme concentrations. The relative Raman scattering intensities of C-C stretching vibrations from the trans and gauche conformers of the acyl chains are correlated directly with the extent of hydrolysis, allowing the progress of the reaction to be monitored in situ on a single vesicle. In dilute vesicle dispersions, the technique allows the much higher local concentration of lipid molecules in a single vesicle to be detected free of interferences from the surrounding solution. Observing the local composition of an optically trapped vesicle also allows one to determine whether the products of enzyme-catalyzed hydrolysis remain associated with the vesicle or dissolve into solution. The observed reaction kinetics exhibited a time lag prior to the rapid hydrolysis. The lag time varied inversely with the enzyme concentration, which is consistent with the products of enzyme-catalyzed lipid hydrolysis reaching a critical concentration that allows the enzyme to react at a much faster rate. The turnover rate of membrane-bound enzyme determined by Raman microscopy during the rapid, burst-phase kinetics was 1200 s-1. Based on previous measurements of the equilibrium for PLA2 binding to lipid membranes, the average number of enzyme molecules responsible for catalyzing the hydrolysis of lipid on a single optically trapped vesicle is quite small, only two PLA2 molecules at the lowest enzyme concentration studied.
- Cherney, Daniel P.,Myers, Grant A.,Horton, Robert A.,Harris, Joel M.
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p. 6928 - 6935
(2008/02/12)
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- γ-Ray irradiation of liposomes of polymerizable phospholipids containing octadeca-2,4-dienoyl groups and characterization of the irradiated liposomes
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The synthesis of a variety of polymerizable phospholipids containing the octadeca-2,4-dienoyl moiety on 2-acyl chains and the characteristics of liposomes containing those phospholipids of the γ-irradiation are described. We synthesized three different polymerizable phosphocholines that have different 1-acyl chain lengths with the octadeca-2,4-dienoyl moiety on the 2- acyl chain: myristoyl (MODPC), palmitoyl (PODPC) and stearoyl (SODPC). The liposomes were prepared by extrusion through polycarbonate filters with a pore size of 0.2 μm, and were polymerized by γ-irradiation with various dose rates. The polymerization rate increased in the order SODPC>MODPC>PODPC. The mechanism of the polymerization of SODPC was the same as that of 1,2-bis- [(E,E)-octadeca-2,4-dienoyl]-sn-glycero-3-phosphocholine (DODPC), but differed from that of MODPC and PODPC. Freeze-thaw testing was used to evaluate the stability of the polymerizable liposomes. The MODPC liposome was more stable than other monofunctional liposomes. For similar irradiation, the polymerization behavior of the liposomes was significantly affected by the 1- acyl length.
- Akama, Kazuhiro,Yano, Yoshihiro,Tokuyama, Satoru,Hosoi, Fumio,Omichi, Hideki
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p. 1047 - 1059
(2007/10/03)
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- Studies of the topography of biomembranes: the four-step synthesis of a photoactivatable transmembrane phospholipidic probe and its dideuterated analogue
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A convenient method for the synthesis of a dipolar transmembrane phospholipid 1a, which would be a useful photolabelling probe for the study of the topography of sterols of proteins in biomembranes, is described.Starting from 4,4'-dihydroxybenzophenone 13, 1a was synthesized in 4 steps in 31percent total yield.The final, key step, acylation of lysophosphatidylcholine-cadmium chloride complex (4), was achieved using cesium fluoride as a catalyst in DMF.The preparation of the dideuterated 1b or diiodinated 1c analogues is also described.The reaction scheme presented here can also be used for the synthesis of a spin label, a fluorescence label, or a label carrying a high specific radioactivity.Keywords: photolabelling / transmembrane probe / topography / biomembrane / phospholipid bilayer
- Yamamoto, Masakuni,Dolle, Valerie,Warnock, William,Diyizou, Yvonne,Yamada, Masashi,et al.
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p. 317 - 329
(2007/10/02)
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- NMR Studies of Micellar Aggregates in 1-Acyl-sn-glycerophosphocholine Systems. The Formation of a Cubic Liquid Crystalline Phase
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Measurements of the amphiphile diffusion coefficient, using the NMR pulsed-field gradient technique, have been performed on micellar solutions and cubic liquid crystalline phases of the following lysophosphatidylcholines: 1-lauroyl-sn-glycero-3-phosphocholine (LaLPC), 1-myristoyl-sn-glycero-3-phosphocholine (MyLPC), 1-palmitoyl-sn-glycero-3-phosphocholine (PaLPC), 1-stearoyl-sn-glycero-3-phosphocholine (StLPC), 1-oleoyl-sn-glycero-3-phosphocholine (OlLPC), and 1-linoleoyl-sn-glycero-3-phosphocholine (LiLPC).The 2H spin relaxation rates at two magnetic fields have been measured in micellar solutions of PaLPC.The phase equilibria at 25 and 35 deg C in the aqueous binary systems of LaLPC and MyLPC have been studied by 31P NMR and by optical techniques for lipid concentrations up to 50percent (w/w) 2H2O. 2H and 14N quadrupole splittings have been measured in anisotropic phases of PaLPC, OlLPC, and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).A cubic liquid crystalline phase, I1, located between the micellar and hexagonal phase regions, is formed by LaLPC and MyLPC between approximately 40percent and 45percent (w/w) lipid in 2H2O.As previously shown the I1 phase is also formed by PaLPC while not by StLPC, OlLPC, and LiLPC for which a concentrated micellar solution is in equilibrium with the hexagonal phase (Arvidson et al.Eur.J.Biochem. 1985, 152, 753).For LaLPC, amphiphile diffusion measurements show that the cubic phase I1 consists of closed micellar aggregates.Amphiphile diffusion and 2H spin relaxation studies of the micellar phases show that the aggregates formed by LaLPC, MyLPC, PaLPC, and StLPC remain small and globular over the whole micellar phase region, while the micelles of OlLPC and LiLPC are large and polydispersed.Micelle diffusion measurements provide information about aggregate interactions, which is shown to be compatible with magnitudes of the hydration force and the van der Waals interactions previously obtained for corresponding bilayers.The formation of the cubic face I1 is discussed in terms of the packing of interacting micellar aggregates in a newly proposed structure for the cubic phase.
- Eriksson, Per-Olof,Lindblom, Goeran,Arvidson, Goesta
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p. 846 - 853
(2007/10/02)
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