Natural Phosphatidylcholine Research Articles

Natural Phosphatidylcholine Is Actively Translocated across the Plasma Membrane to the Surface of Mammalian Cells

The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established.

Separation and purification of phosphatidylcholine and phosphatidylethanolamine from soybean degummed oil residues by using solvent extraction and column chromatography

Natural phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were separated and purified from soybean degummed oil residues in this work. Crude PC and PE were first separated from degummed oil residues by extraction with 95% ethanol, and then the crude PC and PE were used as raw materials to prepare high purity PC and PE by using column chromatography of silica gel (100–200 mesh) with different eluents and elution modes. The high purity PC (content >90%) was obtained from the crude PC by using isocratic elution with methanol as eluent. Compared with the methods reported by using isocratic elution with mixed solvents as eluent or gradient elution, the procedure proposed exhibits low cost and industry potentialities because of some advantages, such as operation simplicity, cheap equipment and solvent to be recovered easily. The purity of the PE product prepared from the crude PE was more than 75%. The gradient elution was preferable to isocratic elution for reducing the elution time and eluent consumption when to prepare PE from the crude PE. The effects of loading amount and the flow-rate on separation efficiency were also investigated. For obtaining high separation efficiency, the loading amount should be less than 2.0 g crude PC or PE/100 g silica gel, and the flow-rate should be controlled under 4 ml/min for crude PC and 3 ml/min for crude PE, respectively.

Effects of an amphiphilic peptide on membrane order of phosphatidylcholine

Amphiphilic sequences located at N terminal of secretory proteins and integral membrane proteins, the so called signal sequences or topogenic sequences, lead insertion of the following polypeptide segments into biomembranes. We have a lot of knowledge about the sequential structure and the steric conformation, whereas poor knowledge about interaction between the signal sequences and membranes. We studied effects of a synthetic amphiphilic peptide on chain orientational order of lipid membranes by the optical birefringence method at high peptide concentrations. The synthetic peptide is modeled on the signal sequences. The peptide with 20 amino acid residues has a hydrophobic region (Ala(7), Leu(7) and Trp) with hydrophilic amino acids (Lys(6)) at the end. The hydrophobic region forms α-helix structure. Multilamellar vesicles of synthetic dimyristoyl- (DMPC) and dipalmitoyl-phosphatidylcholines (DPPC), and of natural egg yolk phosphatidylcholine (Egg-PC) were prepared as lipid model membranes. It was observed that interference light due to the membrane birefringence considerably decreased by addition of the peptide for both DMPC and DPPC multilamellar bilayers, but did not change for Egg-PC multilamellar bilayers at high peptide concentrations. In addition, the peptide induced a strong aggregation of Egg-PC unilamellar small vesicles. These results indicate that the peptide penetrates into both the synthetic and the natural lipid membranes, and the penetration induces a decrease in chain order for the synthetic lipid bilayers whereas no change in chain order of Egg-PC bilayers. This behavior of Egg-PC bilayers seems to be resulted from lower order in the chain packing of the bilayers. The strong aggregation of small unilamellar vesicles may be induced by peptides adsorbed onto the vesicle surfaces. The result suggests that signal sequences of bio-synthetic proteins mediate initiation of the protein insertion into biomembranes without a significant disturbance in the chain order.

Liposomes with phosphatidylethanol as a carrier for oral delivery of insulin: studies in the rat

In this study, phosphatidylethanol formed by phospholipase D catalysed transphosphatidylation of phosphatidylcholine was employed as a component for preparation of liposomal carrier for oral delivery of insulin. Thermotropic behaviour of liposomes from mixtures of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanol, and their resistance to pancreatic phospholipase A 2 catalysed hydrolysis were studied. Three kinds of liposomes with insulin were prepared to examine the pharmacological availability of liposomes with phosphatidylethanol: (i) dipalmitoyl phosphatidylcholine/dipalmitoyl phosphatidylethanol (1:1 w/w) liposomes; (ii) dipalmitoyl phosphatidylcholine/dipalmitoyl phosphatidylethanol/palmitoyl–stearoyl sucrose (1:1:0.2) liposomes; and (iii) liposomes composed of natural phosphatidylcholine and phosphatidylinositol (1:1). The resultant liposomes were orally administrated to rats with blood glucose concentration of 270 mg/100 ml in a dose of 12 IU/kg body weight. Blood samples were collected 0.5, 1.5, 3, 5, and 24 h after treatment. Oral administration of all liposomal species resulted in hyperinsulinemia. Hyperinsulinemia induced by liposomes containing dipalmitoyl phosphatidylethanol was attended by a decrease of blood glucose concentration. No correlation between insulin level and glucose concentration in the rat blood after oral administration of phosphatidylinositol-containing liposomes was observed.

The substrate requirements of phospholipase D

The hydrolysis rates of different diphosphates, compared with the one observed with natural phosphatidylcholine, are used to identify the molecular basis for phospholipase D (PLD) catalysis. Experimental data strongly support the idea that PLD is a rather generic phosphodiesterase with very wide substrate specificity and a net preference for lipophilic substrates. The presence of choline in the polar head is not required for activity although it improves hydrolysis efficiency. Choline esters are found to be substrates for PLD hydrolysis, but only with long chain fatty acids.

Synthesis of the diacetylenic phospholipids 1,2-(10',12'-heptadecadiynoyl)-sn-glycero-3-phophatidylcholine and 1,2-(4',6'-tricosadiynoyl)-sn-glycero-3-phophatidylcholine

Diacetylenic phosphatidylcholines are synthetic compounds analogous to natural phospholipids. These molecules can be hydrated to form bilayered aggregates known as liposomes in a manner similar to natural phosphatidylcholines. A particular feature of these liposomes is their capability of undergoing polymerization when submitted to ultraviolet (UV) light irradiation. This process allows the construction of polymerized liposomes possessing interesting physical properties making these vesicles appropriate for slow-release drug delivering systems. In this article we describe the total syntheses of diacetylenic phosphatidylcholines 1,2-bis(10',12'-heptadecadiynoyl)-sn-glycero-3-phosphatidylcholine (1a), 1,2-bis(10',12'-tricosadiynoyl)-sn-glycero-3-phosphatidylcholine (1b) and 1,2-bis(4',6'-tricosadiynoyl)-sn-glycero-3-phosphatidylcholine (1c). Phospholipids 1a and 1c, displaying the butadiinyl chromophores closer to the chain terminus, or closer to the polar heads respectively, have not been reported before. Noteworthy was the observation that liofilization of the diacetylenic alcohols and acids intermediates significantly reduced undesired polymerization, allowing to stock these compounds for a short period of time (~24 h).

New ultradeformable drug carriers for potential transdermal application of interleukin-2 and interferon-alpha: theoretic and practical aspects.

Transfersomes (TFs) are highly deformable hydrophilic lipid vesicles that are able to penetrate the skin barrier spontaneously because of their characteristics. Transfersomes are able to transport noninvasively low- and high-molecular-weight molecules into the body. We describe the formulation and several biologic characteristics of interleukin-2 (IL-2)- and interferon-alpha (IFNalpha )-containing TFs. TFs contain natural phosphatidylcholine and sodium cholate. Recombinant human IL-2 and human hybrid IFNalpha were added to TFs and incubated for 24 hours at 4 degrees C. Immunotransfersomes were isolated from free IL-2 and IFNalpha by filtration (Centrisart, Sartorius). The biologic activity of immunotransfersomes was measured by a cytotoxic lymphoid line assay for IL-2 and by an A549-encephalomyocarditis virus assay for IFN; concentrations of proteins were determined by the enzyme-linked immunosorbent assay (ELISA). It was possible to incorporate a large amount of IL-2 and IFN in TFs (75-80%), and the incorporated IL-2, and IFN were biologically active. The increased lipid/protein ratio (90.9/1.0) led to a growing probability of association. We were thus able to show that IL-2 and IFN are trapped by transfersomes in a biologically active form and in sufficient concentrations for immunotherapy. In upcoming experiments these IL-2- and IFN-containing TFs will be used for a transdermal approach in the murine RENCA cell line model.

Specific proteins are required to translocate phosphatidylcholine bidirectionally across the endoplasmic reticulum.

A long-standing problem in understanding the mechanism by which the phospholipid bilayer of biological membranes is assembled concerns how phospholipids flip back and forth between the two leaflets of the bilayer. This question is important because phospholipid biosynthetic enzymes typically face the cytosol and deposit newly synthesized phospholipids in the cytosolic leaflet of biogenic membranes such as the endoplasmic reticulum (ER). These lipids must be transported across the bilayer to populate the exoplasmic leaflet for membrane growth. Transport does not occur spontaneously and it is presumed that specific membrane proteins, flippases, are responsible for phospholipid flip-flop. No biogenic membrane flippases have been identified and there is controversy as to whether proteins are involved at all, whether any membrane protein is sufficient, or whether non-bilayer arrangements of lipids support flip-flop. To test the hypothesis that specific proteins facilitate phospholipid flip-flop in the ER, we reconstituted transport-active proteoliposomes from detergent-solubilized ER vesicles under conditions in which protein-free liposomes containing ER lipids were inactive. Transport was measured using a synthetic, water-soluble phosphatidylcholine and was found to be sensitive to proteolysis and associated with proteins or protein-containing complexes that sedimented operationally at 3.8S. Chromatographic analyses indicated the feasibility of identifying the transporter(s) by protein purification approaches, and raised the possibility that at least two different proteins are able to facilitate transport. Calculations based on a simple reconstitution scenario suggested that the transporters represent approximately 0.2% of ER membrane proteins. Our results clearly show that specific proteins are required to translocate a phosphatidylcholine analogue across the ER membrane. These proteins are likely to be the flippases, which are required to translocate natural phosphatidylcholine and other phospholipids across the ER membrane. The methodology that we describe paves the way for identification of a flippase.

Degradation of pyrene-labelled phospholipids by lysosomal phospholipases in vitro. Dependence of degradation on the length and position of the labelled and unlabelled acyl chains

The hydrolysis of pyrenylacyl phosphatidylcholines (PyrnPCs)(n indicates the number of aliphatic carbons in the pyrene-chain) by crude lysosomal phospholipases in vitro was investigated. PyrnPCs consist of several sets in which the length of the pyrene-labelled or the unlabelled acyl chain, linked to the sn-1 or sn-2 position, was systematically varied. Lysophosphatidylcholine and fatty acid were the only fluorescent breakdown products detected, thus indicating that PyrnPCs were degraded by A-type phospholipases and lysophospholipases. Of these, mainly A1-type phospholipases appear to be involved, as determined from the relative amounts of labelled fatty acid and lysolipid released from the positional isomers. Based on the effects of the length and position of the pyrene-labelled and unlabelled chains it is suggested that (1) the lysosomal A-type phospholipases acting on PyrnPCs recognize the carboxy-terminal part of the lipid acyl chains and (2) the relevant part of the binding site is relatively narrow. Thus phospholipids with added bulk in the corresponding region, such as those that are peroxidized and polymerized, may not be good substrates for the lysosomal phospholipases mentioned. The impaired hydrolysis of the most hydrophobic PyrnPCs indicates that lysosomal phospholipases may not be able to penetrate significantly into the substrate interphase, but upward movement of the lipid may be required for efficient hydrolysis. Finally, the rate of hydrolysis of many pyrenyl derivatives was found to be comparable to that of a natural phosphatidylcholine species, both in micelles and in lipoprotein particles, indicating that these derivatives can be used as faithful reporters of lysosomal degradation of natural lipids in vivo and in vitro.

Preparative transformation of natural phospholipids catalysed by phospholipase D from Streptomyces

Phospholipase D from Stretomyces sp. catalyses the transphosphatidylation reaction of various primary and secondary alcohols with natural phosphatidylcholine in an emulsion system in a preparatively useful manner.

Enzyme-mediated synthesis of two diastereoisomeric forms of phosphatidylglycerol and of diphosphatidylglycerol (cardiolipin)

3-sn-Phosphatidyl-1′-sn-glycerol and its stereoisomer 3-sn-phosphatidyl-3′-sn-glycerol are prepared through phospholipase D-catalysed transphosphatidylation of natural phosphatidylcholine with the two enantiomeric (R)- and (S)-isopropylideneglycerols and subsequent hydrolysis of the phospholipids obtained. If glycerol is the alcohol donor in the same reaction, a mixture of stereoisomers is obtained. When the enzyme from savoy cabbage is used, the two compounds are obtained as the only products. With PLD from Streptomyces as catalyst, the initially formed phosphatidylglycerols are quantitatively transformed into diphosphatidylglycerol (cardiolipin).

Polysiloxane-based biomembranes

Natural phosphatidylcholine was attached to a polysiloxane backbone. The Langmuir film, Langmuir-Blodgett deposition and Montal-Mueller properties were studied. The material forms exceptionally stable Langmuir films. Montal-Mueller bilayers have a resistance of about 2 × 10 4 Ω cm −2 and a breakdown voltage of about 350 mV. Langmuir-Blodgett bilayer films have a low-voltage resistance of about 2 × 10 5 Ω cm −2, a transition to a square-law dependence at around 0.1 V, and a breakdown voltage greater than 1 V. There is evidence that bacteriorhodopsin in readily incorporated into such films.

Hyperinsulinemia in rats with alloxan diabetes after oral administration of insulin-containing negatively charged liposomes

The effect of oral administration of insulin-containing negatively charged liposomes on the immunoreactive insulin level and glucose concentration in the blood of rats with alloxan diabetes was studied. Liposomes were formed from equimolar mixtures of either natural phosphatidylcholine and phosphatidylinositol or semisynthetic dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanol. More than 70% of insulin was encapsulated in liposomes at initial molar lipid to insulin ratio 100:1. Oral administration of both liposomal species results in hyperinsulinemia. Hyperinsulinemia induced by liposomes from semisynthetic phospholipids is attended by a decrease of blood glucose concentration. No correlation between insulin level and glucose concentration in the rat blood after oral intake of phosphatidylinositol-containing liposomes is observed.

Indirect enzymatic phosphorylation: preparation of dihydroxyacetone phosphate

Starting from natural phosphatidyl choline, the substitution of the choline polar head for an alcohol donor is catalysed by phospholipase D; subsequent hydrolysis of the new phospholipid obtained in the presence of phospholipase C allows the obtainment of the phosphate of the alcohol donor; the overall process is the net transfer of the phosphate moiety from a phospholipid to a primary alcohol and the procedure is verified in the preparation of dihydroxyacetone phosphate

Phospholipase D from Streptomyces catalyses the transfer of secondary alcohols

The first preparatively useful transphosphatidylation reaction of secondary alcohols with natural phosphatidyl choline in an emulsion system, catalysed by phospholipase D of commercial origin is reported.

Mixed monolayers of natural and polymeric phospholipids: structural characterization by physical and enzymatic methods

This study has focused on physical characterization and enzymatic hydrolysis of mixed monolayers of a natural phospholipid substrate and a polymerizable phospholipid analogue. Such a mixed system presents the possibility to stabilize model biomembranes, vary the molecular environment within the layer through polymerization and simultaneously examine these influences on monolayer structure. Phospholipase A 2 was used here as a sensitive probe of the molecular environment within these mixed, polymerizable monolayers to complement information obtained from isotherm and isobar data. The results clearly show a strong influence of molecular environment on phospholipase A 2 activity, even if differences in the physical state of mixed monolayers are not detectable with isotherm and isobar measurements. Physical characterization indicated that both monomeric and polymeric mixed monolayers were phase-mixed. Enzyme hydrolysis, however, showed large differences in the ability of the enzyme to selectively hydrolyze the natural phosphatidylcholine component from the monomeric as opposed to the polymeric mixtures. This demonstrates a high sensitivity of phospholipase A 2 to distinguish subtle differences in molecular arrangement within mixed monolayers on a molecular level.

Partial characterization of testosterone 5 alpha-reductase solubilized from rat testicular microsomes.

Testosterone 5 alpha-reductase was successfully solubilized by the use of digitonin from rat testicular microsomes and then partially purified by polyethylene glycol fractionation and DEAE-Sephacel column chromatography. The 5 alpha-reductase activity of the partially purified preparation was significantly stimulated by addition of phosphatidylserine (bovine brain). Synthetic dilauroylphosphatidylcholine also increased the reductase activity to a somewhat lesser extent than did phosphatidylserine, whereas natural phosphatidylcholine from bovine liver did not exhibit any stimulation. When synthetic phosphatidylcholines with varying acyl chain lengths were tested for their stimulatory effects on the reductase activity, dilauroylphosphatidylcholine was most active; dimyristoylphosphatidylcholine was less active; dioleoylphosphatidylcholine was almost inactive.

13C nuclear magnetic resonance study of some phosphinolipids: Assignments and conformational studies

Abstract13C chemical shifts and 31P, 13C spin‐spin coupling constants are reported for six model phosphinates and eight synthetic phosphinolipids. The complex spectra of the synthetic phosphinolipids could be assigned from increments and couplings derived from the model compounds. Based on these investigations the 3J(PC) couplings of 7–15 Hz indicate a preferential trans orientation of the respective head‐group carbon relative to the phosphorus. This behaviour is similar to that of natural phosphatidylcholine lipids.

A phospholipid spin label used as a liposome‐associated MRI contrast agent

Given current clinical use of phospholipid bilayer structures (liposomes/vesicles) as nontoxic drug delivery vehicles, we have addressed the possibility of employing the phospholipids themselves as MRI contrast agents. To this end we have synthesized phosphatidylcholine with a nitroxide spin label replacing one methyl residue of the choline headgroup. This material was mixed with natural phosphatidylcholine in mole ratios from 1:50 to 1:1 and used to prepare sonicated unilamellar vesicles in saline. Expected structural features of these vesicles were verified by freeze-fracture electron microscopy. Proton T1 values of saline were readily decreased to less than 0.3 s by such preparations, yielding a net relaxivity of 0.6 M-1 s-1. The approach seems to be a realistic way of firmly associating a contrast agent of minimal toxicity with ordinary liposomes/vesicles in a manner that is not subject to leakage.

Function of the delipidated beta-adrenergic receptor appears to require a fatty acid or a neutral lipid in addition to phospholipids.

Detergent-solubilized preparations of the beta-adrenergic receptor (R) and of the guanyl nucleotide binding proteins (Gs) were extensively treated to remove phospholipids and cholesterol. Reconstitution of an R-Gs system was subsequently performed in the presence of a mixture of natural phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine or the synthetic dioleoyl derivatives of the same phospholipids. In both cases, an additional lipid was required for the agonist-dependent activation of Gs. The requirement could be fulfilled by alpha-tocopherol, or by unsaturated fatty acids such as oleic acid. Inclusion of this non-phosphorylated lipid in the reconstituted system enhanced the isoproterenol-dependent activation of Gs by guanosine 5'-O-[gamma-thio]triphosphate 16-33-fold. The rate of activation was largely dependent on the addition of the agonist. Efficient functional reconstitution of R-Gs was thus achieved in a totally defined lipid system. Additional studies of the reconstituted system and of the native membrane led to the notion that the non-phosphorylated lipid plays a role in the function of the hormone-R complex.