Interaction of glycophorin with lipid bilayer studied by calorimetry, densitometry, static light scattering, and electron microscopy.

In the first part of the present work the interaction of glycophorin with dimyristoylphosphatidylcholine (DMPC) is studied by freeze fracture electron microscopy, densitometry, calorimetry, and 90 degree static light scattering. An exothermic lipid/protein interaction energy of WP = 190 kJ.mol-1 was found by application of the well known Van Laar relation for the displacement of the freezing point and the Gibbs-Duhem relationship. Secondly, the effects of Ca2+ on the lipid/protein interaction were studied. Following Ca2+ addition a remarkable decoupling of the interaction of the glycophorin head group with the bilayer surface was revealed by densitometry and gold-labeling electron microscopy. It is estimated that about 80% of lipid once disturbed by the adsorption of glycophorin head groups is decoupled after addition of Ca2+. Thirdly, the selective interaction of glycophorin with binary lipid mixtures was studied, including the mixtures of DMPC with dimyristoylphosphatidylserine (DMPS) and dilauroylphosphatidylcholine (DLPC), and the mixture of dipalmitoylphosphatidylcholine (DPPC) with DLPC.     

Reconstitution of transferrin receptor in mixed lipid vesicles. An example of the role of elastic and electrostatic forces for protein/lipid assembly

We studied the interaction of transferrin receptors (of cell line Molt-4) with mixed model membranes as a function of lipid chain length (phospholipids with C14:0 and C18:1 hydrocarbon chains) and of the surface charge of the membrane using mixtures of C14:0 lecithin (DMPC) with C14:0 phosphatidylglycerol (DMPG) and C14:0 phosphatidylserine (DMPS). Spontaneous self-assembly of receptors and lipids was achieved by freeze-thaw cycles of a codispersion of mixed vesicles and receptors in buffer and subsequent separation of receptor-loaded and receptor-free vesicles by density gradient centrifugation. Information on specific lipid/protein interaction mechanisms was obtained by evaluation of protein-induced shifts of phase boundaries of lipid mixtures by calorimetry and by FTIR spectroscopy of partially deuterated lipid mixtures. The important role (1) of minimizing the elastic forces caused by the mismatch of the lengths of hydrophobic cores of the protein (lp) and the bilayer (lL) and (2) of the electrostatic coupling of protein head groups with the charged membrane/water interface for the lipid/protein self-assembly is established. The electrostatic interaction energy per receptor is about 10(3) kBT (by coupling to about 1000 charged lipids) which is sufficient to overcompensate the elastic energy associated with a mismatch of lp - lL approximately 1.0 nm. The maximum receptor concentration incorporated was measured as a function of membrane surface charge and lipid chain length. The maximum receptor molar fraction varied from xpmax = 5 x 10(-5) for DMPC to xpmax = 4 x 10(-4) for 1:1 DMPC/DMPG; moreover xpmax is higher for DMPS than for DMPG as charged component. For the long-chain lipids, xpmax is higher for a 9:1 DEPE/DEPC mixture [(4.2-9) x 10(-4)] than for pure DEPC (ca. 3.5 x 10(-4)). By decomposition of reconstituted receptors with proteases, we demonstrated the homogeneous orientation of the receptor with its extracellular head group pointing to the convex side of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of the solubilized insulin receptor in phospholipid vesicles.

The insulin receptor was solubilized from turkey erythrocyte membranes by extraction with 1% beta-octylglucopyranoside. Insulin binding was enhanced when the solubilized material was reconstituted in phospholipid vesicles. The affinity of the reconstituted vesicles for various insulins was similar to that of the intact membranes: porcine insulin greater than proinsulin greater than desoctapeptide insulin. A curvilinear Scatchard plot was obtained for insulin binding to the reconstituted system at 15 degrees C. A high affinity association constant of 1.4 x 10(9) M-1 was obtained from the Scatchard plot. This is a four-fold increase over the value for the turkey erythrocyte membrane, which contains more highly saturated phospholipids. This suggests that the insulin receptor may be sensitive to the lipid composition of the membranes in which it is embedded.     

Binding of branched-chain 2-oxo acids to bovine serum albumin.

1. Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. 2. Equilibrium-dialysis data suggest a two-side model for binding in Krebs-Henseleit saline at 37 degrees C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2-oxovalerate, k1 = 8.7 x 10(3) M-1, k2 = 0.09 x 10(3) M-1; 3-methyl-2-oxovalerate, k1 = 9.8 x 10(3) M-1, k2 = 0.08 x 10(3) M-1; 3-methyl-2-oxobutyrate, k1 = 1.27 x 10(3) M-1, k2 = less than 0.05 x 10(3) M-1. 3. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4, gave the following thermodynamic parameters: primary site delta H0(1) = -28.6kJ . mol-1 and delta S0(1) = -15.2J . mol-1 . K-1 (delta G0(1) = -24.0kJ . mol-1 at 37 degrees C) and secondary sites delta H0(2) = -25.4kJ . mol-1 and delta S0(2) = -46.1J . mol-1 . K-1 (delta G0(1) = -11.2kJ . mol-1 at 37 degrees C). Thus binding at both sites is temperature-dependent and increases with decreasing temperature. 4. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. 5. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. 6. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.     

Synthesis and characterization of 1,2-dimyristoylamido-1, 2-deoxyphosphatidylcholine as an artificial boundary lipid

The synthesis and characterization of an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), are described. DDPC has two amide bonds instead of ester bonds of regular lecithins such as 1,2-dimyristoylphosphatidylcholine (DMPC). In differential scanning calorimetry (DSC) measurements, DDPC gave two endothermic peaks: one was at 18.0 degrees C (delta H = 10.74 kJ.mol-1) and the other at 23.0 degrees C (delta H = 12.91 kJ.mol-1). The former peak was sharp and considered to be the phase transition of the hydrocarbon region, while the latter was assigned to the melt of the hydrogen-belt formed by the amide groups of DDPC. Addition of DDPC to DMPC made the DMPC membrane less fluid in the region close to the surface, and significantly increased the reconstitution efficiency of glycophorin into the membrane. This effect of DDPC was much larger than that of naturally occurring lipid, sphingomyelin.     

Interaction of cytidine 3'-monophosphate and uridine 3'-monophosphate with ribonuclease a at the denaturation temperature

Differential scanning calorimetry (DSC) measurements were performed on the thermal denaturation of ribonuclease a and ribonuclease a complexed with an inhibitor, cytidine or uridine 3'-monophosphate, in sodium acetate buffered solutions. Thermal denaturation of the complex results in dissociation of the complex into denatured ribonuclease a and free inhibitor. Binding constants of the inhibitor to ribonuclease a were determined from the increase in the denaturation temperature of ribonuclease a in the complexed form and from the denaturation enthalpy of the complex. Binding enthalpies of the inhibitor to ribonuclease a were determined from the increase in the denaturation enthalpy of ribonuclease a complexed with the inhibitor. For the cytidine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 87 +/- 8 M-1 (pH 7.0) to 1410 +/- 54 M-1 (pH 5.0), while the binding enthalpies increase from 17 +/- 13 kJ mol-1 (pH 4.7) to 79 +/- 15 kJ mol-1 (pH 5.5). For the uridine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 104 +/- 1 M-1 (pH 7.0) to 402 +/- 7 M-1 (pH 5.5), while the binding enthalpies increase from 16 +/- 5 kJ mol-1 (pH 6.0) to 37 +/- 4 kJ mol-1 (pH 7.0). The binding constants and enthalpies of the cytidine inhibitor in 0.05 M sodium acetate buffered solutions increase respectively from 328 +/- 37 M-1 (pH 6.5) to 2200 +/- 364 M-1 (pH 5.5) and from 22 kJ mol-1 (pH 5.5) to 45 +/- 7 kJ mol-1 (pH 6.5). the denaturation transition cooperativities of the uncomplexed and complexed ribonuclease a were close to unity, indicating that the transition is two state with a stoichiometry of 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Octyl-beta-D-glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer.

The interaction of the nonionic detergent octyl-beta-D-glucopyranoside (OG) with lipid bilayers was studied with high-sensitivity isothermal titration calorimetry (ITC) and solid-state 2H-NMR spectroscopy. The transfer of OG from the aqueous phase to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be investigated by employing detergent at concentrations below the critical micellar concentration; it can be defined by a surface partition equilibrium with a partition coefficient of K = 120 +/- 10 M-1, a molar binding enthalpy of delta H degrees D = 1.3 +/- 0.15 kcal/mol, and a free energy of binding of delta G degrees D = -5.2 kcal/mol. The heat of transfer is temperature dependent, with a molar heat capacity of delta CP = -75 cal K-1 mol-1. The large heat capacity and the near-zero delta H are typical for a hydrophobic binding equilibrium. The partition constant K decreased to approximately 100 M-1 for POPC membranes mixed with either negatively charged lipids or cholesterol, but was independent of membrane curvature. In contrast, a much larger variation was observed in the partition enthalpy. delta H degrees D increased by about 50% for large vesicles and by 75% for membranes containing 50 mol% cholesterol. Structural changes in the lipid bilayer were investigated with solid-state 2H-NMR. POPC was selectively deuterated at the headgroup segments and at different positions of the fatty acyl chains, and the measurement of the quadrupolar splittings provided information on the conformation and the order of the bilayer membrane. Addition of OG had almost no influence on the lipid headgroup region, even at concentrations close to bilayer disruption. In contrast, the fluctuations of fatty acyl chain segments located in the inner part of the bilayer increased strongly with increasing OG concentration. The 2H-NMR results demonstrate that the headgroup region is the most stable structural element of the lipid membrane, remaining intact until the disordering of the chains reaches a critical limit. The perturbing effect of OG is thus different from that of another nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), which produces a general disordering at all levels of the lipid bilayer. The OG-POPC interaction was also investigated with POPC monolayers, using a Langmuir trough. In the absence of lipid, the measurement of the Gibbs adsorption isotherm for pure OG solutions yielded an OG surface area of AS = 51 +/- 3 A2. On the other hand, the insertion area AI of OG in a POPC monolayer was determined by a monolayer expansion technique as AI = 58 +/- 10 A2. The similar area requirements with AS approximately AI indicate an almost complete insertion of OG into the lipid monolayer. The OG partition constant for a POPC monolayer at 32 mN/m was Kp approximately 320 M-1 and thus was larger than that for a POPC bilayer.     

Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.     

Flexible charged macromolecules on mixed fluid lipid membranes: theory and Monte Carlo simulations

Fluid membranes containing charged lipids enhance binding of oppositely charged proteins by mobilizing these lipids into the interaction zone, overcoming the concomitant entropic losses due to lipid segregation and lower conformational freedom upon macromolecule adsorption. We study this energetic-entropic interplay using Monte Carlo simulations and theory. Our model system consists of a flexible cationic polyelectrolyte, interacting, via Debye-Hückel and short-ranged repulsive potentials, with membranes containing neutral lipids, 1% tetravalent, and 10% (or 1%) monovalent anionic lipids. Adsorption onto a fluid membrane is invariably stronger than to an equally charged frozen or uniform membrane. Although monovalent lipids may suffice for binding rigid macromolecules, polyvalent counter-lipids (e.g., phosphatidylinositol 4,5 bisphosphate), whose entropy loss upon localization is negligible, are crucial for binding flexible macromolecules, which lose conformational entropy upon adsorption. Extending Rosenbluth's Monte Carlo scheme we directly simulate polymer adsorption on fluid membranes. Yet, we argue that similar information could be derived from a biased superposition of quenched membrane simulations. Using a simple cell model we account for surface concentration effects, and show that the average adsorption probabilities on annealed and quenched membranes coincide at vanishing surface concentrations. We discuss the relevance of our model to the electrostatic-switch mechanism of, e.g., the myristoylated alanine-rich C kinase substrate protein.     

Adsorption of proteins on a lipid bilayer

In our analysis of protein adsorption on a lipid bilayer, the protein surface is considered to contain one or a few charged spots, and the bilayer contains a significant amount of lipids with oppositely charged head groups. After adsorption, a folded protein is assumed to change its shape slightly due to the electrostatic attraction, so that one of the spots forms a flat contact with the oppositely charged lipid heads of the lipid bilayer. With realistic parameters, this model predicts that the contribution of electrostatic interactions to the protein adsorption energy per charged amino acid–lipid pair is 16–25 kJ/mol. Thus, a few (four or five) pairs is sufficient for irreversible adsorption.     

Effect of Lipid Head Groups on Double-Layered Two-Dimensional Crystals Formed by Aquaporin-0

Aquaporin-0 (AQP0) is a lens-specific water channel that also forms membrane junctions. Reconstitution of AQP0 with dimyristoyl phosphatidylcholine (DMPC) and E. coli polar lipids (EPL) yielded well-ordered, double-layered two-dimensional (2D) crystals that allowed electron crystallographic structure determination of the AQP0-mediated membrane junction. The interacting tetramers in the two crystalline layers are exactly in register, resulting in crystals with p422 symmetry. The high-resolution density maps also allowed modeling of the annular lipids surrounding the tetramers. Comparison of the DMPC and EPL bilayers suggested that the lipid head groups do not play an important role in the interaction of annular lipids with AQP0. We now reconstituted AQP0 with the anionic lipid dimyristoyl phosphatidylglycerol (DMPG), which yielded a mixture of 2D crystals with different symmetries. The different crystal symmetries result from shifts between the two crystalline layers, suggesting that the negatively charged PG head group destabilizes the interaction between the extracellular AQP0 surfaces. Reconstitution of AQP0 with dimyristoyl phosphatidylserine (DMPS), another anionic lipid, yielded crystals that had the usual p422 symmetry, but the crystals showed a pH-dependent tendency to stack through their cytoplasmic surfaces. Finally, AQP0 failed to reconstitute into membranes that were composed of more than 40% dimyristoyl phosphatidic acid (DMPA). Hence, although DMPG, DMPS, and DMPA are all negatively charged lipids, they have very different effects on AQP0 2D crystals, illustrating the importance of the specific lipid head group chemistry beyond its mere charge.     

Pentachlorophenol-induced change of zeta-potential and gel-to-fluid transition temperature in model lecithin membranes

We have determined zeta-potentials for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) membranes by measuring the electrophoretic mobility of multilayered vesicles and the temperatures of the gel-to-ripple-to-fluid phase transitions of sonicated vesicles by a photometric method. Some conclusions are: (1) The zeta-potentials of DMPC and DPPC vesicles become negative due to adsorption of ionized pentachlorophenol (PCP), (2) their magnitude changes, step-like, on gel-to-fluid transition and (3) the temperature of the step-like change in zeta-potential decreases with an increase in PCP concentration. (4) PCP exhibits a large effect on membrane structure: It induces an isothermal phase change from the ordered to disordered state, which is enhanced by monovalent salt in the aqueous phase. (5) Both ionized and unionized PCP decrease the melting phase transition temperature and abolish the pretransition, (6) the unionized species increases the melting transition width and (7) the ionized species is more potent in abolishing the pretransition. (8) The shorter chain lipid (DMPC) is more sensitive to the presence of PCP; the maximum decrease in delta Tt is 13 K (DMPC) and 7 K (DPPC) in the presence of ionized PCP. We have shown experimentally, by comparing the delta Tt from photometric studies with the density of adsorbed PCP derived from zeta-potential isotherms, that (9) the shift of the melting phase transition temperature increases linearly with the density of adsorbed PCP. (10) In contrast to membranes made of negatively charged lipids, the transition temperature of DMPC and DPPC membranes in the presence of PCP further decreases in the presence of monovalent salt. The salt effect is due to screening of the membrane surface leading to enhanced adsorption of ionized PCP and a depression in transition temperature. (11) It is shown that both the adsorption and the changes of gel-to-fluid phase transition temperature can be described in terms of the Langmuir-Stern-Grahame model and (12) proposed that future studies of membrane toxicity of PCP should be focused on its pH dependence.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance

Gramicidin A was incorporated at a peptide/lipid ratio of 1:10 mol/mol in aligned bilayers of dimyristoyl phosphatidylcholine (DMPC), phosphatidylserine (DMPS), phosphatidylglycerol (DMPG), and phosphatidylethanolamine (DMPE), from trifluoroethanol. Orientations of the peptide and lipid chains were determined by polarized attenuated total reflection infrared spectroscopy. Lipid-peptide interactions with gramicidin A in DMPC bilayers were studied with different spin-labeled lipid species by using electron spin resonance spectroscopy. In DMPC membranes, the orientation of the lipid chains is comparable to that in the absence of peptide, in both gel and fluid phases. In gel-phase DMPC, the effective tilt of the peptide exceeds that of the lipid chains, but in the fluid phase both are similar. For gramicidin A in DMPS, DMPG, and DMPE, the degree of orientation of the peptide and lipid chains is less than in DMPC. In the fluid phase of DMPS, DMPG, and DMPE, gramicidin A is also less well oriented than are the lipid chains. In DMPE especially, gramicidin A is largely disordered. In DMPC membranes, three to four lipids per monomer experience direct motional restriction on interaction with gramicidin A. This is approximately half the number of lipids expected to contact the intramembranous perimeter of the gramicidin A monomer. A selectivity for certain negatively charged lipids is found in the interaction with gramicidin A in DMPC. These results are discussed in terms of the integration of gramicidin A channels in lipid bilayers, and of the interactions of lipids with integral membrane proteins.     

Deuterium and phosphorus nuclear magnetic resonance studies on the binding of polymyxin B to lipid bilayer-water interfaces

Deuterium and phosphorus NMR methods have been used to study the binding of polymyxin B to the surface of bilayers containing lipids that were deuterated at specific positions in the polar head-group region. The binding of polymyxin B to acidic dimyristoylphosphatidylglycerol (DMPG) membranes induces only small structural distortions of the glycerol head group. The deuterium spin-lattice relaxation times for the different carbon-deuterium bonds in the head group of the same phospholipid are greatly reduced on binding of polymyxin B, indicating a restriction of the motional rate of the glycerol head group. Only very weak interactions were detected between polymyxin B and bilayers of zwitterionic dimyristoylphosphatidylcholine (DMPC). In mixed bilayers of the two phospholipid types, in which either of the two phospholipids was deuterated, the presence of polymyxin B caused a lateral phase separation into DMPG-enriched phospholipid-peptide clusters and a DMPG-depleted phase. Complete phase separation did not occur: peptide-containing complexes with charged phosphatidylglycerol contained substantial amounts of zwitterionic phosphatidylcholine. Exchange of both phospholipid types between complexes and the bulk lipid matrix was shown to be fast on the NMR time scale, with a lifetime for phospholipid-peptide association of less than 1 ms.     

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.     

The interaction of water with the phospholipid head group and its relationship to the lipid electrical conductivity

We have studied the interaction of water with the lipid head group by gravimetrically measuring the lipid water adsorption and the lateral dc electrical conductivity increase resulting from this hydration. We have done this for dimyristoyl phosphatidylcholine (DMPC) having protonated or deuterated hydrocarbon chains. These studies were also done for two cationic lipids having rather different polar head groups. All three lipids behave as strong water adsorbers and all three display a steep, logarithmic increase in the conductivity as the first 1-3 waters per lipid are adsorbed. This increase is usually 5-6 orders of magnitude. After the initial 1-3 waters are adsorbed, the conductivity increases much more gradually, upon additional water adsorption. This electrical behavior is also found for weak water adsorbers and appears to be independent of the head group composition. The conductivity behavior suggests two types of water interacting with the head group. Our studies also indicate that a choline-like component is responsible for the strong water binding nature of the lipids, although, both phosphate and choline make significant contributions to the total amount of adsorbed water. The conductivity behavior, however, does not depend on the presence of both these head group components.     

Binding of apolipoprotein A-I and A-II after recombination with phospholipid vesicles to the high density lipoprotein receptor of luteinized rat ovary

To determine the apolipoprotein specificity of high density lipoprotein (HDL) receptor, apolipoprotein A-I (apo-AI) and apolipoprotein A-II (apo-AII) purified from high density lipoprotein3 (HDL3) were reconstituted into dimyristoyl phosphatidylcholine vesicles (DMPC) and their ability to bind to luteinized rat ovarian membranes was examined. Both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC were shown to bind to ovarian membranes with Kd = 2.87 and 5.70 micrograms of protein/ml, respectively. The binding of both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC was inhibited by unlabeled HDL3, apo-A-I.DMPC, apo-A-II.DMPC, apo-C-I.DMPC, apo-C-II.DMPC, apo-C-III1.DMPC, and apo-C-III2.DMPC, but not by DMPC vesicles, bovine serum albumin.DMPC or low density lipoprotein. Since the binding labeled apo-A-I.DMPC and apo-A-II.DMPC was inhibited by the DMPC complexes of apo-C groups, the direct binding of 125I-apo-C-III1.DMPC was also demonstrated with Kd = 9.6 micrograms of protein/ml. In addition, unlabeled apo-A-I.DMPC, and apo-A-II.DMPC, as well as apo-C.DMPC, inhibited 125I-HDL3 binding. 125I-apo-A-I, 125I-apo-A-II, and 125I-apo-C-III1 in the absence of DMPC also bind to the membranes. These results suggest that HDL receptor recognizes apolipoprotein AI, AII, and the C group and that the binding specificity of the reconstituted lipoproteins is conferred by their apolipoprotein moiety rather than the lipid environment. In vivo pretreatment of rats with human chorionic gonadotropin resulted in an increase of 125I-apo-A-I.DMPC, 125I-apo-A-II.DMPC, and 125I-apo-C-III1.DMPC binding activities. However, no induction of binding activity was observed when the apolipoprotein was not included in DMPC vesicles. An examination of the equilibrium dissociation constant and binding capacity for 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC after human chorionic gonadotropin treatment revealed that the increase in binding activity was due to an increase in the number of binding sites rather than a change in the binding affinity. These results further support our contention that apo-A-I, apo-A-II, and the apo-C group bind to HDL receptor. In conclusion, the HDL receptor of luteinized rat ovary recognizes apolipoproteins A-I, A-II, and the C group but not low density lipoprotein, and the binding is induced by human chorionic gonadotropin in vivo.     

Anion binding to neutral and positively charged lipid membranes

Aqueous anion binding to bilayer membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated by using deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. Only those anions that exhibit chaotropic properties showed significant binding to POPC membranes. A detailed investigation of thiocyanate binding to neutral POPC and to positively charged mixed POPC/dihexadecyldimethylammonium bromide (DHDMAB) (8:2 mol/mol) membranes revealed changes in the 2H NMR quadrupole splittings from POPC specifically deuteriated at either the alpha-segment or the beta-segment of the choline head group which were consistent with a progressive accumulation of excess negative charge at the membrane surface with increasing SCN- concentration. Both the 2H and 31P NMR spectra indicated the presence of fluid lipids in a bilayer configuration up to at least 1.0 M NaSCN with no indication of any phase separation of lipid domains. Calibration of the relationship between the change in the 2H NMR quadrupole splitting and the amount of SCN- binding provided thiocyanate binding isotherms. At a given SCN- concentration the positively charged membranes bound levels of SCN- 3 times that of the neutral membranes. The binding isotherms were analyzed by considering both the electrostatic and the chemical equilibrium contributions to SCN- binding. Electrostatic considerations were accounted for by using the Gouy-Chapman theory. For 100% POPC membranes as well as for mixed POPC/DHDMAB (8:2 mol/mol) membranes the thiocyanate binding up to concentrations of 100 mM was characterized by a partition equilibrium with an association constant of K approximately 1.4 +/- 0.3 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lipid composition on the "membrane response" induced by a fusion peptide

To understand the initial stages of membrane destabilization induced by viral proteins, the factors important for binding of fusion peptides to cell membranes must be identified. In this study, effects of lipid composition on the mode of peptides' binding to membranes are explored via molecular dynamics (MD) simulations of the peptide E5, a water-soluble analogue of influenza hemagglutinin fusion peptide, in two full-atom hydrated lipid bilayers composed of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). The results show that, although the peptide has a common folding motif in both systems, it possesses different modes of binding. The peptide inserts obliquely into the DMPC membrane mainly with its N-terminal alpha helix, while in DPPC, the helix lies on the lipid/water interface, almost parallel to the membrane surface. The peptide seriously affects structural and dynamical parameters of surrounding lipids. Thus, it induces local thinning of both bilayers and disordering of acyl chains of lipids in close proximity to the binding site. The "membrane response" significantly depends upon lipid composition: distortions of DMPC bilayer are more pronounced than those in DPPC. Implications of the observed effects to molecular events on initial stages of membrane destabilization induced by fusion peptides are discussed.