Enzymatic deacylation of l,2-diacyl-sn-glycero-3-phosphocholines to sn-glycerol-3-phosphocholine

This research was undertaken to study the enzymatic deacylation of l,2-diacyl-sn-glycero-3-phosphocholines (sn-1,2-PC) to sn-glycerol-3-phosphocholine (GPC); this compound could be an useful intermediate in the synthesis of “structured” sn-1,2-PC, after re-acylations of the two sn-positions of the glycerol backbone. The enzymatic reactions represent a valid alternative to the chemical deacylation that can be simply obtained in alkaline conditions. High conversion were achieved using a lipase selective for the sn-1-position of sn-1,2-PC (Lipozyme IM, from Mucor miehei) together with a Phospholipase A₂ from hog pancreas, enzyme selective for the sn-2-position; the best results were obtained carrying out the enzymatic reaction in a microemulsion system.     

In vitro stereoselective hydrolysis of diacylglycerols by hormone-sensitive lipase

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, racemic 1,2(2,3)-sn-diolein was used as a substrate and the enantiomeric excess (ee%) of residual 1,2-sn-diolein over 2,3-sn-diolein was measured as a function of DAG hydrolysis. Enantiomeric DAGs were separated by performing chiral-stationary-phase HPLC after direct derivatization from lipolysis product extracts. The fact that the ee% of 1,2-sn-diolein over 2,3-sn-diolein increased with the level of hydrolysis indicated that HSL has a preference for 2,3-sn-diolein as a substrate and therefore a stereopreference for the sn-3 position of dioleoylglycerol. The ee% of 1,2-sn-diolein reached a maximum value of 36% at 42% hydrolysis. Among the various mammalian lipases tested so far, HSL is the only lipolytic carboxylester hydrolase found to have a pronounced stereospecificity for the sn-3 position of dioleoylglycerol.     

Conversion of monogalactosyldiacylglycerols to triacylglycerols in ozone-fumigated spinach leaves

Molecular species and fatty acid distribution of triacylglycerol (TG) accumulated in spinach (Spinacia oleracea L.) leaves fumigated with ozone (0.5 microliter per liter) were compared with those of monogalactosyldiacylglycerol (MGDG). Analysis of positional distribution of the fatty acids in MGDG and the accumulated TG by the enzymatic digestion method showed that hexadecatrienoate (16:3) was restricted to sn-2 position of the glycerol backbone in both MGDG and TG, whereas α-linolenate (18:3) was preferentially located at sn-1 position in MGDG, and sn-1 and/or sn-3 positions in TG, suggesting that 1,2-diacylglycerol moieties of MGDG are the direct precursor of TG in ozonefumigated leaves. Further analysis of TG molecular species by argentation chromatography and mass spectrometry showed that TG increased with ozone fumigation consisted of approximately an equal molar ratio of sn-1,3-18:3-2-16:3 and sn-1,2,3-18:3. Because the molecular species of MGDG in spinach leaves is composed of a similar molar ratio of sn-1-18:3-2-16:3 and sn-1,2-18:3, we concluded that MGDG was converted to 1,2-diacylglycerol and acylated with 18:3 to TG in ozone-fumigated spinach leaves.     

Chemical and physicochemical studies of lysylphosphatidylglycerol derivatives. Occurrence of a2' yields 3' lysyl migration

Syntheses of 1,2-didodecanoyl-sn-glycero-3-phosphoryl-1′-(3′-O-L-lysyl)-sn-glycerol (IV) and 1,2-didodecanoyl-sn-glycero-3-phosphoryl-1′-(2′-O-L-lysyl)-sn-glycerol (VIII) as well as 1,2-didodecanoyl-sn-glycerol-3-phosphoryl-1′-sn-glycerol (XII) are described. 2′- and 3′-lysylphosphatidylglycerol are obtained as pure isomers and can be distinguished spectroscopically (infrared, 100 and 300 MHZ NMR). By these criteria a migration of the lysyl group from the 2′ to the 3′ position of the glycerol occurs in the presence of a strong acid catalyst such as HCl. On the other hand, a weak acid such as acetic acid appears ineffective in inducing lysyl migration, even at very high concentrations.Spectroscopic analysis furthermore demonstrated that lysylphosphatidylglycerol extracted from the Staphylococcus aureus membrane, is a 3′-isomer.     

Infrared and raman spectra of specifically deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholines

Deuterated methylene groups have been introduced synthetically in selected positions of the sn-2 palmitoyl chain of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and deuterated methyl groups in the sn-1 and sn-2 palmitoyl chains as well as in the sn-3 phosphocholine group.The vibrational spectra of seven such deuterium labelled derivatives of the title compound have been studied as the assignment of the C–D stretching vibrations is discussed.     

Nucleophilic substitution in glycerol derivatives. Part V. Formation of 1,3-diacylglycerol-2-phosphates in the reaction of 1,2-diacyl-3-iodoeoxy-rac-glycerol with silver dibenzyl phosphate

The reaction of 1,2-dipalmitoyl-3-iododeoxy-rac-glycerol with silver dibenzyl phosphate gave 1,2-dipalmitoyl-rac-glycerol-3-(dibenzyl phosphate) as the major product, contamined with ca. 2.0% of 1,3-dipalmitoyl-rac-glycerol-2-(dibenzyl phosphate). This contamination is acceptable in most preparations; in particular it cannot account for the low optical rotation values which have been recorded in the literature for sn-glycerol-3-phosphates prepared from sn-3-iododeoxy derivatives. The reaction thus remains a valuable key step in the total synthesis of rac- and sn-glycero-3-phosphoric acid esters. Factors controlling regioselectivity in such reactions are discussed.     

Chemical structures of mono-, di-, tri- and tetraglycosyl glycerides in rice bran

• 1.1. Monoglycosyl monoglyceride, mono-, di-, tri- and tetraglycosyl diglycerides were isolated from rice bran and characterized for their chemical structures.
• 2.2. Monoglycosyl monoglycerides were characterized as Gal(β1' → 3)-1- or 2-monoacyl-sn-glycerol and Glc(β1' → 3)-1- or 2-monoacyl-sn-glycerol.
• 3.3. The structures ofmonoglycosyl diglyceride were Gal(β1' → 3)-1,2-diacyl-sn-glycerol and Glc(β1' → 3)-1,2-diacyl-sn-glycerol. Epimeric separation of the galactosyl and glucosyl glycerides was for the first time achieved by thin-layer chromatography.
• 4.4. The main diglycosyl diglyceride was shown to be Gal(α1' → 6')-Gal(β1' → 3)-1,2-diacyl-sn-glycerol.
• 5.5. The major structure of triglycosyl diglyceride was characterized as Gal(α1″' → 6″)-Gal(α1″ → 6')-Gal(β1' → 3)-1,2-diacyl-sn-glycerol.
• 6.6. The representative structure of tetraglycosyl diglyceride was for the first time established as Gal(α1″ → 6″')-Gal(α1″' α 6″)-Gal(α1″ → 6')-Gal(βl' → 3)-1, 2-diacyl-sn-glycerol.

     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252–6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 μmol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

The active site and the phospholipid activation of rat liver lysosomal lipase are not stereospecific

The stereochemical specificity of lysosomal lipase of rat liver was investigated using enantiomeric triacylglycerol analogs, sn-1-alkyl-2,3-diacylglycerol and sn-3-alkyl-1,2-diacylglycerol as substrates. Lysosomal lipase utilized both substrates with equal rates. The dependence of the activity of lysosomal lipase on the stereoconfiguration of activating acidic phospholipid was also studied. Our results showed that both sn-3-phospholipids (diphosphatidylglycerol, phosphatidylserine) and sn-1-phospholipids (bis(monoacylglycero)phosphate (BMP) were efficient activators of this enzyme and thus the stereochemical configuration of the activating phospholipid is not important. Accordingly, the rat liver lysosomal lipase lacks stereospecificity with respect to both the triacylglycerol substrate and the acidic phospholipid activator.     

Structure and Dynamics of the Myristoyl Lipid Modification of Src Peptides Determined by H Solid-State NMR Spectroscopy

Lipid modifications of proteins are widespread in nature and play an important role in numerous biological processes. The nonreceptor tyrosine kinase Src is equipped with an N-terminal myristoyl chain and a cluster of basic amino acids for the stable membrane association of the protein. We used ²H NMR spectroscopy to investigate the structure and dynamics of the myristoyl chain of myr-Src(2-19), and compare them with the hydrocarbon chains of the surrounding phospholipids in bilayers of varying surface potentials and chain lengths. The myristoyl chain of Src was well inserted in all bilayers investigated. In zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes, the myristoyl chain of Src was significantly longer and appears “stiffer” than the phospholipid chains. This can be explained by an equilibrium between the attraction attributable to the insertion of the myristoyl chain and the Born repulsion. In a 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] membrane, where attractive electrostatic interactions come into play, the differences between the peptide and the phospholipid chain lengths were attenuated, and the molecular dynamics of all lipid chains were similar. In a much thicker 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine]/cholesterol membrane, the length of the myristoyl chain of Src was elongated nearly to its maximum, and the order parameters of the Src chain were comparable to those of the surrounding membrane.     

A Comparison of the Membrane Binding Properties of C1B Domains of PKCγ, PKCδ, and PKC?

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical γ, and the novel δ and ?), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bγ and C1B? exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1B? being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1B? domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.     

Digestion and absorption of a sulphoxide analogue of triacylglycerol in the rat

The stereochemistry of fat digestion and absorption was studied by feeding a triacylglycerol analogue to rats with a thoracic duct cannula. The analogue, rac-1,2-dioleoyl-3-S-tetradecyl-3-thioglycerol-S-oxide was chosen since its enantiomers exhibited high rotation in optical rotatory dispersion (ORD) and circular dichroism (CD). In the chyle, triacylglycerol was the major lipid but X-1,2-diacyl-3-S-tetradecyl-3-thioglycerol-S-oxide constituted 8% of lipid weight. It was resolved by thin-layer chromatography (TLC) into two diastereomers. Each of the diastereomers were analyzed for the proportions of 1-thio-sn-glycerol/3-thio-sn-glycerol isomers by ORD and CD. The 1-thio-sn-glycerol isomers dominated for both compounds indicating that they were enriched during the absorption processes, since a racemic compound was fed. The stereospecificities are probably exerted by acyltransferase(s) during chyle lipid synthesis. The methods used will be valuable tools in studies on the metabolism of enantiomeric glycerides, and also for characterization of naturally occurring sulphur-containing lipids.     

Synthesis of 1,2-diacyloxypropyl-3-(1′,2′-diacyl-sn-glycero)-phosphonate

The total synthesis of 1,2-diacyloxypropyl-3-(1′,2′-diacyl-sn-glycero)phosphonate is described. The 1,2-dipalmitoyloxypropyl phosphonic acid was prepared by an Arbusov reaction of 1,2-diacylglycerol bromohydrin with trimethyl phosphite; the final product was obtained by a coupling reaction involving the diacyloxypropyl-3-phosphonic acid and 1,2-dipalmitoyl-sn-glycerol, catalysed by tri-isopropylbenzene sulfonyl chloride. The resulting synthetic product was characterised by elemental analysis, phosphono-phosphorus determinations and IR spectroscopy.     

Glycerolipid biosynthesis in rat adipose tissue. Effect of polyamines on triglyceride synthesis.

Triacylglycerol formation from sn-glycerol 3-phosphate and 1,2-diacyl-sn-glycerol was markedly elevated in the presence of spermine and spermidine. This was attributed to the activation of microsomal sn-glycerol 3-phosphate acyltransferase and 1,2-diacyl-sn-glycerol acyltransferase and to the inhibition of palmitoyl-CoA hydrolase. Spermine was more effective than spermidine, and putrescine did not stimulate triacylglycerol formation. The stimulatory effect of spermine on triacylglycerol-forming enzymes was observed in the presence of Mg²⁺ and was apparent in the presence or absence of bovine serum albumin. The activation of 1,2-diacyl-sn-glycerol acyltransferase by spermine was specific, and other diacylglycerol-utilizing enzymes were not affected under these conditions. These studies demonstrate that polyamines may be important regulators of triacylglycerol formation in adipose tissue.     

Synthesis of 3-O-(2-acetamido-2-deoxy-3-O-β-d-galactopyranosyl-β-d-galactopyranosyl)-1,2-di-O-tetradecyl-sn-glycerol

Stereo- and regio-selective synthesis of 3-O-(2-acetamido-2-deoxy-3-O-β-d- galactopyranosyl-β-d-galactopyranosyl)-1,2-di-O-tetradecyl-sn-glycerol by use of 1,2-di-O-tetradecyl-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-galactopyranosyl)-sn-glycerol as a key intermediate is described.     

HIV Fusion Peptide Penetrates, Disorders, and Softens T-Cell Membrane Mimics

This work investigates the interaction of N-terminal gp41 fusion peptide (FP) of human immunodeficiency virus type 1 (HIV-1) with model membranes in order to elucidate how FP leads to fusion of HIV and T-cell membranes. FP constructs were (i) wild-type FP23 (23 N-terminal amino acids of gp41), (ii) water-soluble monomeric FP that adds six lysines on the C-terminus of FP23 (FPwsm), and (iii) the C-terminus covalently linked trimeric version (FPtri) of FPwsm. Model membranes were (i) LM3 (a T-cell mimic), (ii) 1,2-dioleoyl-sn-glycero-3-phosphocholine, (iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol, (iv) 1,2-dierucoyl-sn-glycero-3-phosphocholine, and (v) 1,2-dierucoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol. Diffuse synchrotron low-angle x-ray scattering from fully hydrated samples, supplemented by volumetric data, showed that FP23 and FPtri penetrate into the hydrocarbon region and cause membranes to thin. Depth of penetration appears to depend upon a complex combination of factors including bilayer thickness, presence of cholesterol, and electrostatics. X-ray data showed an increase in curvature in hexagonal phase 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, which further indicates that FP23 penetrates into the hydrocarbon region rather than residing in the interfacial headgroup region. Low-angle x-ray scattering data also yielded the bending modulus K_C, a measure of membrane stiffness, and wide-angle x-ray scattering yielded the S_xray orientational order parameter. Both FP23 and FPtri decreased K_C and S_xray considerably, while the weak effect of FPwsm suggests that it did not partition strongly into LM3 model membranes. Our results are consistent with the HIV FP disordering and softening the T-cell membrane, thereby lowering the activation energy for viral membrane fusion.     

Assessing the size, stability, and utility of isotropically tumbling bicelle systems for structural biology

Aqueous phospholipid mixtures that form bilayered micelles (bicelles) have gained wide use by molecular biophysicists during the past 20 years for spectroscopic studies of membrane-bound peptides and structural refinement of soluble protein structures. Nonetheless, the utility of bicelle systems may be compromised by considerations of cost, chemical stability, and preservation of the bicelle aggregate organization under a broad range of temperature, concentration, pH, and ionic strength conditions. In the current work, ³¹P nuclear magnetic resonance (NMR) and atomic force microscopy (AFM) have been used to monitor the size and morphology of isotropically tumbling small bicelles formed by mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DIOMPC) with either 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) or 1,2-di-O-hexyl-sn-glycero-3-phosphocholine (DIOHPC), testing their tolerance of variations in commonly used experimental conditions. ¹H-¹⁵N 2D NMR has been used to demonstrate the usefulness of the robust DMPC-DIOHPC system for conformational studies of a fatty acid-binding protein that shuttles small ligands to and from biological membranes.     

Phosphatidylserine Inhibits and Calcium Promotes Model Membrane Fusion

PEG-mediated fusion of SUVs composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, sphingomyelin, cholesterol, and dioleoylphosphatidylserine was examined to investigate the effects of PS on the fusion mechanism. Lipid mixing, content mixing, and content leakage measurements were carried out with vesicles containing from 0 to 8 mol % PS and similar amounts of phosphatidylglycerol. Fitting these time courses globally to a 3-state (aggregate, intermediate, pore) sequential model established rate constants for each step and probabilities of lipid mixing, content mixing, and leakage in each state. Charged lipids inhibited both the rates of intermediate and pore formation as well as the extents of lipid and contents mixing, although electrostatics were not solely responsible for inhibition. Ca²⁺ counteracted this inhibition and increased the extent of fusion in the presence of PS to well beyond that seen in the absence of charged lipids. The effects of both PS and Ca²⁺ could be interpreted in terms of a previous proposal for the nature of lipid fluctuations that account for transition states for the two steps of the fusion process examined. The results suggest a more significant role for Ca²⁺-lipid interactions than is acknowledged in current thinking about cell membrane fusion.Abbreviations used: SUVs, small unilamellar vesicles; DOPC, 1,2-dioleoyl-3-sn-phosphatidylcholine; DOPE, 1,2-dioleoyl-3-sn-phosphatidylethanolamine; SM, sphingomyelin (bovine brain); CH, Cholesterol; DOPS, 1,2-dioleoyl-3-sn-phosphatidylserine; PS, phosphatidylserine; DOPG, 1,2-dioleoyl-3-sn-phosphatidylglycerol; PG, phosphatidylglycerol; TES, N- tris(hydroxymethyl)methyl}2-2-aminoethane sulfonic acid; PEG, poly(ethylene glycol); CM, contents mixing; LM, lipid mixing     

An improved method for the preparation of 1,2-isopropylidene-SN-glycerol

A simple and fast route for the preparation of 1,2-isopropylidene-sn-glycerol from D-mannitol in 45% yield is described. The value of optical rotation, [α]_D²⁰ + 15.2°, is higher than usual indicating considerable racemization for other procedures. Since 1,2-isopropylidene-sn-glycerol serves as general intermediate for the synthesis of glycerides and of phosphoglycerides these lipids contain substantial amounts of the isomer, for instance 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine may consist of up to 15% of 2,3-dipalmitoyl-sn-glycerol-1-phosphocholine in earlier preparations.