Mixture Of Phosphatidylserine Research Articles

Phosphorylation of Dynamin I on Ser-795 by Protein Kinase C Blocks Its Association with Phospholipids

Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 +/- 51 nM. Optimal binding occurred with mixtures of phosphatidylserine and phosphatidylcholine of 1:3 with little binding to phosphatidylcholine or phosphatidylserine alone. Phospholipid binding was abolished after dynamin I phosphorylation by PKC and was restored after dephosphorylation by calcineurin. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry revealed the phosphorylation site in PKCalpha-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus, phosphorylation of dynamin I on Ser-795 prevents its association with phospholipid, providing a basis for the cytosolic localization of the minor pool of phospho-dynamin I that mediates synaptic vesicle retrieval in nerve terminals.

Separation of phosphatidylinositol 4-phosphate from a mixture with phosphatidylserine and phosphatidylinositol by Sep-Pak C18 cartridge.

A simple and efficient method for the separation of phosphatidylinositol 4-phosphate (PI 4-P) from phosphatidylinositol (PI) and phosphatidylserine (PS) is described. A mixture of PI, PI 4-P and PS was injected onto a Sep-Pak C18 cartridge. PI and PS were flushed through the cartridge with solvent 1 [methanol-chloroform (3: 1)] while PI 4-P remained in it. Then the cartridge was inverted, and PI 4-P was eluted backward with solvent 2 [chloroform-methanol-0.5 M aqueous ammonium hydroxide (9:7:2)].

The chirality of phosphatidylserine and the activation of protein kinase C.

The properties of phosphatidyl-L-serine (L-PS) and phosphatidyl-D-serine (D-PS) were compared. The two forms of PS have similar but nonidentical L to L phase transition temperatures. Mixtures of phosphatidylserine with phosphatidylethanolamine and cholesterol (molar ratio 1:1:2) show polymorphic behavior at higher temperatures and in the presence of Ca2+. Mixtures with L-PS undergo conversion to nonlamellar phases at lower temperatures than do similar mixtures with D-PS. The aggregation of vesicles upon addition of histones is greater for L-PS than for D-PS. With fluorescence digital imaging microscopy we could show differences in the extent of formation of histone-induced domains enriched in PS or in diacylglycerol. The most enriched domains were induced with histone in membranes containing L-PS. The MARCKS peptide showed no differences in domain formation between L-PS and D-PS. The maximal activity of protein kinase C was greater in the presence of L-PS when histone, which could form more enriched domains, was the substrate. However, with a MARCKS peptide substrate, which formed domains of equal enrichment with L-PS and D-PS, the maximal activity of protein kinase C was the same with D-PS and with L-PS. These observations demonstrate that L-PS and D-PS have different physical properties. These differences likely contribute to the greater ability of L-PS to activate protein kinase C.

Influence of phospholipids on the assessment of factor VIII activity.

In view of reported discrepancies between different factor VIII assays, the influence of phospholipids on the performance of one-stage clotting (OS) and chromogenic substrate (CS) assays was evaluated. The B domain deleted recombinant factor VIII, rVIII SQ, two full-length recombinant products and a plasma derived factor VIII concentrate were each diluted into severe haemophilia A plasma and assayed against a plasma standard. The one-stage activity was 50, 80, 75 and 106%, respectively, of the chromogenic result. Variations in the phospholipid concentration did not affect the chromogenic assay, except at very low levels where the apparent activity increased. In contrast, dilution of the phospholipid reagent had a substantial influence on the activity measured by OS assays, especially in the case of rVIII SQ. At low levels of phospholipid, the one-stage activity of rVIII SQ exceeded the chromogenic result. When mixtures of phosphatidylserine (PS) and phosphatidyl-choline (PC) were used as a source of phospholipid, the OS results for rVIII SQ agreed well with the CS activity as long as the content of PS was below 10%, i.e., closer to the physiological level. At higher levels of PS, as in most commercial APTT reagents, the OS activity decreased. When the APTT reagent was replaced by platelets in the OS assay, the results compared well with those obtained by the CS assay for both t-VIII SQ and full-length factor VIII products.

Interaction of M1 and M2 isozymes pyruvate kinase from human tissues with phosholipids

The effect of pH and the presence of FBP on the interaction of skeletal muscle (PK-M1) and kidney or tumor meningioma (PK-M2) pyruvate kinase with the phospholipids liposomes were investigated by ultracentrifugation and steady-state kinetics and were compared with those results obtained using the bovine heart (PK-M1) isoenzyme which we previously studied. Pyruvate kinase specific activity increases upon the interaction with liposomes.The activation is specifically sensitive to presence of phosphatidylserine (PS) in liposomes. Liposomes made of phosphatidylcholine+phosphatidylserine mixture are good adsorptive systems for both human and bovine of M-type isozymes at low ionic strength. Interaction of PK-M1 with PS liposomes results in the change of V max and K m values for PEP without marked effect on Hill coefficients. Addition of PS liposomes to PK-M2 induces hyperbolic saturation curves for PEP.

Surfactant Proteins Induce Channel-Mediated Transport in Artificial Membranes• 1558

Aim: We have confirmed that natural surfactant, Survanta® (SUR) - containing small, hydrophobic proteins - induces channel-mediated transport of Na+ and K+ in artificial membranes (planar lipid bilayers). Synthetic surfactant, Exosurf® - lacking proteins - does not. The possible participation of SUR proteins in observed channel activities is also suggested by their a-helical structures and mode of insertion in lipid bilayers. We hypothesize that SUR proteins contribute to channel structure as oligomeric complexes. To investigate, SUR proteins were extracted and subsequently tested for the insertion of channels in artificial membranes.Methods: Pooled SUR was extracted with CHCl3: MeOH:H2O(2:2:1.8) by modified Folch method. SUR proteins were separated from SUR lipids by elution through Sephadex LH-20 with CHCl3:MeOH (2:1). Eluents were analyzed by SDS-PAGE confirming one protein band at ≈5kD. Planar lipid bilayers employing phospholipids (1:1 phosphatidylethanolamine(PE):phosphatidylserine (PS)) were painted across an aperture (250mm) connecting solutions of buffered (pH 7.4) NaCl (50-500 mM) with 1mM Ca++. Extracted SUR proteins (EXTR)were introduced to PE:PS bilayers by either: a) direct addition of EXTR to PE:PS mixture (1:1000 protein:lipid) prior to painting the aperture; or b) reconstitution of EXTR with 7:2:1 dipalmitoylphosphatidylcholine (DPPC):phosphatidylglycerol (PG):palmitic acid(PA) (5:1000 protein:lipid) for suspension in one of the NaCl solutions bathing the PE:PS lipid bilayer. Holding potentials (-90 to +90 mV) were applied across the bilayer and currents indicative of salt transport measured.Results: Currents were not observed in the absence of EXTR (DPPC:PG:PA - EXTR). By contrast, both direct addition of EXTR to PE:PS bilayer and suspension of DPPC:PG:PA + EXTR in NaCl solution independently induced cationic current flow characterized by: 1) gating between open and closed channel states; 2) slope conductances = 46±7 pS; 3) sensitivity of open state probability to applied voltage; and 4) Ca++ dependence.Conclusions: SUR proteins extracted from SUR lipids induce channel-mediated transport in lipid bilayers. We speculate that surfactant proteins SP-B and SP-C play key roles in the clearance of lung fluids.Funded by Children's Health Foundation

Three-dimensional structure of brush border myosin-I at ∼20 å resolution by electron microscopy and image analysis

Brush Border Myosin-I (BBMI) is a single-headed, unconventional myosin found in the microvilli of intestinal epithelial cells where it forms lateral bridges between the core bundle of actin filaments and the plasma membrane of the microvillus. A three-dimensional (3D) reconstruction of BBMI was made from images of negatively stained, two-dimensional (2D) crystals grown on lipid monolayers formed from mixtures of phosphatidylserine and phosphatidylcholine. The resolution of the 3D map extends to ∼20 Å and allows identification of all of the major structural domains of BBMI. The BBMI molecule is composed of three domains: a globular motor domain, a light-chain-binding domain and a lipid-binding domain. In our map, the putative motor domain is connected to an extended density, which we believe to be the light-chain-binding domain. This long, narrow region has three distinct bends, which may delineate the bound calmodulin light chains. Following the last calmodulin there is density which extends for a short distance across the lipid surface and is presumably the carboxy-terminal lipid-binding domain.

Mechanism of liposome destabilization by polycationic amino acids.

Polycationic amino acids induce the leakage and fusion of liposomes containing anionic lipids. We have investigated the nature and extent of the changes in membrane physical properties caused by these polypeptides which could result in the observed membrane destabilization. We found that in the range of pH 5 to pH 7 both poly-L-histidine and poly-L-lysine were ineffective in shifting the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine, either in the presence of absence of 1-palmitoyl-2-oleoylphosphatidylserine. We also studied the gel to liquid crystalline phase transition properties of 1:1 mixtures of phosphatidylserine and phosphatidylethanolamine, both in dimyristoyl forms as well as the 1-palmitoyl-2-oleoyl forms, as a function of pH and in the presence and absence of polycationic amino acids. We observed that these two lipids were largely miscible at all pH values and in the presence and absence of the polypeptides. However, there was some increased tendency for phase separation at higher pH and in the absence of polypeptide. Thus neither changes in curvature strain nor lateral phase separation induced by the polycationic amino acids could account for their marked ability to induce leakage and fusion. Phosphatidylethanolamine labelled with pyrene on one of the acyl chains gives rise to fluorescent emission from both monomer and excimer forms. The ratio of emission intensity from these two forms is indicative of lateral phase separation and the degree of lateral mobility of this probe. In equimolar mixtures of the 1-palmitoyl-2-oleoyl forms of phosphatidylserine and phosphatidylethanolamine in the liquid crystalline phase at 30 degrees C we find little effect of pH on the ratio of excimer to monomer emission intensity. However poly-L-lysine markedly lowers the fraction of excimer emission from these liposomes through the pH range from 5 to 7. Poly-L-histidine lowers the excimer to monomer emission ratio at pH 5 but not at pH 7. This is opposite to what one would expect for lateral phase separation and is interpreted at being the consequence of the polypeptide lowering the rate of lateral diffusion of the lipids. This effect of poly-L-histidine is observed over a range of temperatures from 0 to 40 degrees C in both gel and liquid crystalline phases. There is no evidence from the behaviour of the pyrene fluorescent probe for lipid interdigitation.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of human factor V by meizothrombin.

A recombinant human prothrombin was prepared in which Arg155 was replaced by Ala. The recombinant prothrombin was converted into a meizothrombin derivative (R155A meizothrombin) that was resistant to autocatalytic removal of the fragment 1 domain. R155A meizothrombin appeared to be a potent factor V activator in reaction mixtures that contained negatively charged phospholipid vesicles. Factor V activation by R155A meizothrombin was characterized by second-order rate constants of 0.06 x 10(6) M-1 S-1 in the absence of phospholipid and 18 x 10(6) M-1 S-1 in the presence of 60 microM phospholipid vesicles composed of a 10:90 mol/mol mixture of phosphatidylserine (PS) and phosphatidylcholine (PC). The rate constant for thrombin-catalyzed activation of factor V was hardly affected by the presence of phospholipid vesicles and was 4.0 x 10(6) M-1 S-1. The initial rate of activation of 3 nM factor V by R155A meizothrombin was a function of the concentration of PS/PC vesicles present in the reaction mixture, and the calculated rate constant reached a plateau value at > or = 50 microM PS/PC. Gel electrophoretic analysis of factor V activation showed that R155A meizothrombin and thrombin cleaved the susceptible peptide bonds in factor V at different rates. However, both activators finally generated a factor Va molecule composed of a heavy chain with an M(r) of 104,000 and a light chain doublet with M(r) values of 74,000 and 71,000. Since meizothrombin is one of the major reaction products formed during the initial phase of prothrombin activation, these findings are indicative of a significant contribution of meizothrombin to in vivo factor V activation.

Comparison of the membrane binding kinetics of bovine prothrombin and its fragment 1.

Total internal reflection fluorescence microscopy has been used to compare the membrane binding characteristics of fluorescein-labeled bovine prothrombin and fluorescein-labeled bovine prothrombin fragment 1. The Ca(2+)-dependent association of these proteins with quartz-supported planar membranes composed of mixtures of phosphatidylserine (2-10 mol%) and phosphatidylcholine was examined. Equilibrium binding measurements showed that the apparent equilibrium dissociation constants increased with decreasing molar fractions of phosphatidylserine and that the dissociation constants were somewhat lower for intact prothrombin. Kinetic measurements, using fluorescence photobleaching recovery, showed that the measured dissociation rates were approximately equivalent for prothrombin and fragment 1 and did not change with the protein solution concentration or the molar fraction of phosphatidylserine. The kinetic data also implied that the surface binding mechanism for both proteins is more complex than a simple reversible reaction between monovalent proteins and monovalent surface sites. Measured equilibrium and kinetic constants are reported and compared for prothrombin and fragment 1 on planar membranes.

Lysogenic activity of enhancer caltrin and the influence of phospholipids on its expression.

Enhancer caltrin permeabilizes the plasma membrane of bovine epididymal spermatozoa as indicated by the release of hyaluronidase from the acrosome and lactate dehydrogenase (LDH) from the sperm cytosol. A previously reported increased calcium uptake by the sperm in the presence of enhancer caltrin was apparently due, in part, to calcium entry into the mitochondria, which had become accessible to external calcium. At 37 microM (200 micrograms/ml), enhancer caltrin released about 30% of the total hyaluronidase in the acrosome and 50% of the cytosolic LDH from epididymal sperm (4 x 10(7)/ml). This event was prevented by phosphatidylserine (PS), presumably through caltrin-phospholipid complex formation, whereas phosphatidylcholine (PC) was ineffective. Cardiolipin induced the release of LDH and this too was prevented by enhancer caltrin. Lysophosphatidylserine (LPS), on the other hand, potentiated the lysogenic activity of enhancer caltrin, promoting the release of the full complement of hyaluronidase and LDH even at a molar ratio of only 1:1 with caltrin. The effect of mixtures of LPS and PS on the lysogenic property of enhancer caltrin was investigated, and it was found that PS suppressed the potentiating effect of LPS. Release of hyaluronidase and LDH took place only when the LPS/PS molar ratio was greater than 2. The implications of these findings for the role of caltrin in mammalian fertilization are discussed.

Expression of Modified Human Cytochrome P450 3A4 in Escherichia coli and Purification and Reconstitution of the Enzyme

A full-length human cytochrome P450 (P450) 3A4 cDNA clone and four derivatives in which the N-terminus was modified were inserted into a pCW vector and used to transform Escherichia coli DH5α cells. Little expression was seen with the native sequence; the highest level of expression (range of 40-110 membrane-bound nmol P450 liter −1) was achieved with a construct (NF14) in which residues 3-12 were deleted. In all of the constructs P450 was found primarily in the membranes. The modified P450 3A4 (construct NF14) showed typical P450 hemoprotein spectra. The protein was purified to electrophoretic homogeneity in a five-step procedure [nominally 23 nmol P450 (mg protein) −1]. For most purposes it was found to be more practical to purify the modified P450 3A4 to ∼70% homogeneity [nominally 15 nmol P450 (mg protein) −1] in a simple two-step process. The modified P450 3A4 (NF14) or P450 3A4 purified from human liver could be mixed with rabbit liver NADPH-P450 reductase to achieve catalytic activities nearly as high as those found in human liver microsomes (on a nmol P450 basis), but the optimal reconstitution conditions included not only a mixture of phosphatidylserine, L-α-dilauroyl- and L-α-dioleoyl- sn-glycero-3-phosphocholines, cholate, and cytochrome b 5 suggested by others but also glutathione during the preincubation. Several other thiols were found not to substitute in this role. Good catalytic activity was seen for nifedipine oxidation, testosterone 6β-hydroxylation, and the 8,9-epoxidation and 3α-hydroxylation of aflatoxin B 1, reactions previously ascribed to the enzyme. These procedures provide a relatively convenient and reliable means of producing, purifying, and reconstituting a catalytically active and useful derivative of P450 3A4, a human P450 enzyme that has many roles in the oxidation of drugs and other xenobiotic chemicals.

Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate.

We studied the binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta) to vesicles containing the negatively charged phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). PLC-delta did not bind significantly to large unilamellar vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but bound strongly to vesicles formed from mixtures of PC and PIP2. The apparent association constant for the putative 1:1 complex formed between PLC-delta and PIP2 was Ka congruent to 10(5) M-1. The binding strength increased further (Ka congruent to 10(6) M-1) when the vesicles also contained 30% PS. High-affinity binding of PLC-delta to PIP2 did not require Ca2+. PLC-delta bound only weakly to vesicles formed from mixtures of PC and either PS or phosphatidylinositol (PI); binding increased as the mole fraction of acidic lipid in the vesicles increased. We also studied the membrane binding of a small basic peptide that corresponds to a conserved region of PLC. Like PLC-delta, the peptide bound weakly to vesicles containing monovalent negatively charged lipids; unlike PLC-delta, it did not bind strongly to vesicles containing PIP2. Our data suggest that a significant fraction of the PLC-delta in a cell could be bound to PIP2 on the cytoplasmic surface of the plasma membrane.

Characterization and Subcellular Localization of Calcium-Dependent Phospholipid Binding Proteins (Annexins) in Normal Human Skin and Reconstituted Epidermis

Annexins represent a widespread family of Ca(++)-dependent phospholipid binding proteins. Although their precise functions are still unknown, they probably play an important role in cell regulation because they are major substrates for various growth factor receptor kinases. We characterized annexins in human skin using three different antisera raised against annexin II, annexin V, and a synthetic peptide that resembles the consensus sequence of all annexins. In normal human skin, using SDS-PAGE, two-dimensional gel electrophoresis and immunoblot analysis, we identified two major 34-kDa proteins and one 36-kDa protein, with respective isoelectric points of 6.5, 5.2, and 7.2-7.9. According to these criteria they were identified as annexins, I, V, and II, respectively. Minor 45-51 kDa and 68-kDa proteins with 6.1-6.7 and 6.8-7.1 isoelectric points were also present, and likely corresponded to annexins VII and VI, respectively. We investigated the ability of these proteins to bind phospholipids in the presence of calcium using liposomes formed from a mixture of phosphatidylserine and phosphatidylcholine. The cellular distribution of annexins in normal human skin was determined by immunofluorescence with antiannexin II and anti-annexin V antibodies. Labeling with both antibodies was observed predominantly at the cell membrane with some cytoplasmic staining also being apparent. Specificity was confirmed by the absence of staining using pre-immune sera or after the absorption of the antibodies with their corresponding antigens. These proteins were also characterized in vitro in a reconstituted human skin model. All were present in this system except annexin VI and VII, which were lost after phospholipid purification. Further experiments should now be carried out using this system to clarify the role and regulation of these proteins within the epidermis.

Bilayer Membrane Bending Stiffness by Tether Formation From Mixed PC-PS Lipid Vesicles

Recently, a new approach to measure the bending stiffness (curvature elastic modulus) of lipid bilayer membrane was developed (Biophys. J., Vol. 55; pp. 509-517, 1989). The method involves the formation of cylindrical membrane strands (tethers) from bilayer vesicles. The bending stiffness (B) can be calculated from measurements of the tether radius (Rt) as a function of the axial force (f) on the tether: B = f.Rt/2 pi. In the present report, we apply this method to determine the bending stiffness of bilayer membranes composed of mixtures of SOPC (1-stearoyl-2-oleoyl phosphatidyl choline) and POPS (1-palmitoyl-2-oleoyl phosphatidyl serine). Three different mixtures were tested: pure SOPC, SOPC plus 2 percent (mol/mol) POPS, and SOPC plus 16 percent POPS. The bending stiffness determined for these three different lipid mixtures were not significantly different (1.6-1.8 x 10(-12) ergs). Because POPS carries a net negative charge, these results indicate that changes in the density of the membrane surface charge have no effect on the intrinsic rigidity of the membrane. The values we obtain are consistent with published values for the bending stiffness of other membranes determined by different methods. Measurements of the aspiration pressure, tether radius and the tether force were used to verify a theoretical relationship among these quantities at equilibrium. The ratio of the theoretical force to the measured force was 1.12 +/- 0.17.

Inhibition of protein kinase C phosphorylation by mono- and divalent cations

The effect of a matrix of concentrations of Ca 2+ (0.01, 0.1, 0.5, 5 mM), Mg 2+ (0.2, 0.5, 1, 2, 5, 10 mM), and Na + (50, 100, 150 mM) on the phosphorylation of histone H-1 by protein kinase C was measured in the presence of 5 mol % diacylglycerol and Mg-ATP in both phosphatidylserine micelles and liposomes formed from a 1:4 mixture of phosphatidylserine and phosphatidylcholine. Monovalent cations (150 mM) reduced activity by 60 and 84% in the micelle and liposome assay systems, respectively. Inhibition was also observed with 5 mM Ca 2+ and 10 mM Mg 2+. The phosphorylating activity was compared with computer calculations of the negative electrostatic potentials (ψ o) of the phospholipid membranes in the presence of the cations.

Identification of calcium-dependent phospholipid-binding proteins in higher plant cells

Calcium-dependent phospholipid-binding proteins of apparent M r 33 000 and 35 000 were isolated from suspension cultures of tomato cells. Two-dimensional gel electrophoresis showed the proteins to have isoelectric points of approx. 5.7 and 5.6, respectively. In the presence of calcium, both proteins bound to liposomes formed from a mixture of phosphatidylserine and phosphatidylcholine, but not to liposomes of phosphatidylcholine alone. Both proteins showed immunological similarities to previously characterized calcium-dependent phospholipid-binding proteins (annexins) from Torpedo marmorata and mammalian species. The protein of M r 33 000 cross-reacted with three separate antisera raised to the annexin Torpedo calelectrin, whereas that of M r 35 000 cross-reacted with antisera to the bovine annexins p68 and p32/34. We suggest that the two proteins may represent the first identification in higher plants of the annexin family of calcium-dependent phospholipid-binding proteins.

Isolation and characterization of the major phospholipase a 2 from the venom of trimeresurus purpureomaculatus (shore pit viper)

1. 1. The major phospholipase A 2 (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity. 2. 2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14.000 as estimated by SDS-polyacrylamide gel electrophoresis. 3. 3. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC > PE > PS = 0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme. 4. 4. The phospholipase A 2 exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 μg/g by i.v. route.

Reconstitution of affinity-purified dopamine D 2 receptor binding activities by specific lipids

The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D 2 receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [ 3H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [ 3H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [ 3H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [ 3H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D 2 receptor and should prove useful in studies on functional reconstitution of the receptor.

Physico-chemical properties and organization of lipids in membranes: their possible role in myocardial injury.

Lipids in biological membranes are organized in a bilayer configuration in order to form a semi-permeable barrier. The lipids are freely mobile in the bilayer, which is denoted as "fluid" or liquid-crystalline. For plasma membranes it is assumed that the lipids are not homogeneously distributed over the two leaflets or monolayers. This so-called lipid asymmetry is established for the erythrocyte membrane. There it was found that phosphatidylserine (PS) and phosphatidylethanolamine (PE) are present exclusively and predominantly in the cytoplasmic leaflet, respectively. It is shown that isolated PE at physiological conditions forms a non-bilayer configuration the so-called hexagonal HII phase. Moreover, isolated PS can undergo a transition from the fluid into the solid state upon addition of calcium. In mixtures of PS and PE, calcium is able to induce fusion events, possibly formation of the HII phase and phase separation of solid PS. The physico-chemical behaviour of these phospholipids will be discussed in the light of the structural changes of the sarcolemma of heart muscle cells observed by freeze-fracturing and thin section electron microscopy after ischaemia, ischaemia and reperfusion and the calcium paradox. The lateral phase separation of intramembranous particle aggregation is explained as isothermic phase separation by H+ and calcium. The disruption of the sarcolemma by the formation of blebs (liposomal structures) is interpreted as a destabilization of the bilayer configuration since PE prefers the HII phase and thus induces uncontrolled fusion events. This all leads to an irreversible disruption of the sarcolemma.