The phospholipid headgroup specificity of an ATP-dependent calcium pump.

We have replaced the lipid associated with a purified calcium transport protein with a series of defined synthetic dioleoyl phospholipids in order to determine the effect of phospholipid headgroup structure on the ATPase activity of the protein. At 37 degrees C the zwitterionic phospholipids (dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) support the highest activity, while a phospholipid with two negative charges (dioleoyl phosphatidic acid) supports an activity which is at least twenty times lower. Dioleoyl phospholipids with a single net negative charge support at intermediate ATPase activity which is not affected by the precise chemical structure of the phospholipid headgroup. The protocol used to determine the phospholipid headgroup specificity of calcium transport protein is novel because it establishes the composition of the lipid in contact with the protein without the need to isolate defined lipid-protein complexes. This allows the lipid specificity to be determined using only very small quantities of test lipids. We also determined the ability of the same phospholipids to support calcium accumulation in reconstituted membranes. Two requirements had to be met. The phospholipid had to support the ATPase activity of the pump protein and it had to form sealed vesicles as determined by electron microscopy. Since a number of phospholipids met those requirements it is clear that in vitro the lipid specificity of the calcium-accumulating system is rather broad.     

The hydration of phospholipids and phospholipid-cholesterol complexes

The hydration characteristics of phosphatidylcholines and the effect of cholesterol on these were studied with differential thermal analysis and water vapour adsorption experiments. Also the water adsorption of egg phosphatidylethanolamine and the effect of cholesterol on this was studied and compared with corresponding qualities of phosphatidylcholine.The differential thermal analysis study showed that the monohydrates of egg, dipalmitoyl, and dioleoyl phosphatidylcholine tightly bind ～9 molecules of water per phosphatidylcholine molecule. Cholesterol is proved to somewhat increase the water binding of the phospholipids. Cholesterol is also shown to decrease the heat change of the chain melting transition of dioleoyl phosphatidylcholine, but not to abolish it completely.The water adsorption experiments indicate that the hydration of phosphatidylcholines takes place in two steps; a strong initial water binding and a second phase of weak binding. The adsorption isotherm of egg phosphatidylethanolamine is strikingly different from that of egg phosphatidylcholine. Cholesterol is shown, also by this method, to increase the hydration of phospholipids especially that of dipalmitoylphosphatidylcholine.The results in this study are in good agreement with those presented by many other authors. Starting with the accumulated information of the hydration characteristics of phosphatidylcholines the organization of the bound water around the polar group is discussed and the most probable model is evaluated.     

Lipid phase states influence glycophorin reconstitution

Reconstitution of glycophorin into dimyristoyl phosphatidylcholine and sphingomyelin vesicles was sub-maximal below the phase transition temperatures of these lipids. Reconstitution of glycophorin into diisostearoyl phosphatidylcholine and dioleoyl phosphatidylcholine liposomes was maximal within a range of temperatures below the phase transition temperatures of dimyristoyl phosphatidylcholine and sphingomyelin but above the phase transition temperatures of diisostearoyl phosphatidylcholine and dioleoyl phosphatidylcholine. These findings indicate a greater tendency for reconstitution of glycophorin into fluid as opposed to solid lipid phases.     

Exchange and interactions between lipid layers at the surface of a liposome solution

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. p] Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.     

Oxidative inactivation of lactonase activity of purified human paraoxonase 1 (PON1)

Paraoxonase1 (PON1), one of HDL-associated antioxidant proteins, is known to lose its activity in vivo systems under oxidative stress. Here, we examined the effect of various oxidants on lactonase activity of PON1, and tried to protect the lactonase activity from oxidative inactivation. Among the oxidative systems tested, the ascorbate/Cu²⁺ system was the most potent in inactivating the lactonase activity of purified PON1; in contrast to a limited role of Fe²⁺, Cu²⁺ (0.05–1.0 µM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.02–0.1 mM). Moreover, Cu²⁺ alone inhibited the lactonase activity at concentrations as low as 1 µM. The ascorbate/Cu²⁺-mediated inactivation of PON1 lactonase activity was prevented by catalase, but not general hydroxyl radical scavengers, suggesting the implication of Cu²⁺-bound hydroxyl radicals in the oxidative inactivation. Compared to arylesterase activity, lactonase activity appears to be more sensitive to Cu²⁺-catalyzed oxidation. Separately, ascorbate/Cu²⁺-mediated inactivation of lactonase activity was prevented by oleic acid as well as phoshatidylcholine. Taken together, our data demonstrate that Cu²⁺-catalyzed oxidation may be a primary factor to cause the decrease of PON1 lactonase activity under oxidative stress and that lactonase activity of PON1 is most susceptible to ascorbate/Cu²⁺ among PON1 activities. In addition, we have showed that radical-induced inactivation of lactonase activity is prevented by some lipids.     

Efficient synthesis of the cholinephosphate phospholipid headgroup

In search of an efficient method to prepare cholinephosphate headgroups in phospholipids under mild conditions (where the diacylglycerol moiety is not subject to oxidation), a method was developed for phosphorylation using a trialkyl phosphite and I2. The active intermediate is a phosphoryl iodide formed by oxidation of the phosphite with I2. 2-Bromoethanol, dimethyl chlorophosphite, and an alcohol (diglyceride) are converted to a phosphate triester in a one-pot reaction with high yield. In the second reaction, the phosphate triester is demethylated, and the ethyl bromide group is converted to choline by treatment with aqueous trimethylamine. This procedure is applied to the synthesis of hexadecylphosphocholine, and 1,2-didecanoyl-1-deoxy-1-thio-sn-glyceryo-3-phosphocholine.     

Mapping of the Lipid-Binding and Stability Properties of the Central Rod Domain of Human Dystrophin

Dystrophin is a cytoskeletal protein that confers resistance to the sarcolemma against the stress of contraction-relaxation cycles by interacting with cytoskeletal and membrane partners. Apart from several proteins, membrane phospholipids are a partner of the central rod domain made up of 24 spectrin-like repeats, separated into sub-domains by four hinges. We previously showed that repeats 1 to 3 bind to membrane anionic phospholipids, while repeats 20 to 24 are not able to do so. We focus here on the phospholipid-binding properties of the major part of the central rod domain, namely, the sub-domain delineated by hinges 2 and 3 comprising 16 repeats ranging from repeat 4 to 19 (R4-19). We designed and produced multirepeat proteins comprising three to five repeats and report their lipid-binding properties as well as their thermal stabilities. When these proteins are mixed with liposomes including the anionic lipid phosphatidylserine, they form stable protein-vesicle complexes as determined by gel-filtration chromatography. The absence of an anionic lipid precludes the formation of such complexes. Spectroscopic analyses by circular dichroism and tryptophan fluorescence show that, while the α-helical secondary structures are not modified by the binding, protein trans conformation leads to the movement of tryptophan residues into more hydrophobic environments. In addition, the decrease in the molar ellipticity ratio at 222/208 nm as observed by circular dichroism indicates that lipid binding reduces the inter-helical interactions of multirepeat proteins, thus suggesting partly “opened” coiled-coil structures. Combining these results with data from our previous studies, we propose a new model of the dystrophin molecule lying along the membrane bilayer, in which the two sub-domains R1-3 and R4-19 interact with lipids and F-actin, while the distal sub-domain R20-24 does not exhibit any interaction. These lipid-binding domains should thus maintain a structural link between cytoskeletal actin and sarcolemma via the membrane phospholipids.     

Effects of uranyl ions on lipid bilayer membranes

Uranyl ions (UO₂²⁺) stabilize black lipid membranes (BLM's) as inferred from the doubling of the breakdown voltage and from a considerable increase in the lifetime of the BLM's. These effects are observed also in BLM's made of mono-olein and of oxidized cholesterol. The lytic effect of lysolecithin is significantly reduced in the presence of UO₂²⁺. Uranyl ions adsorb to the interface of BLM's made of phosphatidylcholine (PC) with a dissociation constant of about 3 : 10^?6 M and thereby charge the interface of the membrane and attain almost stoichiometric binding of one molecule of uranyl ion per one molecule of PC at 1 M ionic strength and 20 μM of UO₂²⁺. The membrane conductance induced by ionophores is considerably reduced by UO₂²⁺ and it is inferred by various tests that this is due to the charging of the interface and not to changes in membrane fluidity.     

On the substrate specificity of the red cell calcium pump

ATP-dependent active calcium transport in inside-out human red cell membrane vesicles is stimulated by magnesium essentially parallel with an increase in MgATP concentration. At a constant, low (1 μM) calcium concentration, increasing ATP and magnesium increase the maximum calcium transport rate irrespective of the constant or decreasing concentrations of CaATP present. K_Ca for calcium pumping is practically unchanged at variable ATP and magnesium concentrations. Free magnesium above 1–2 mM inhibits active calcium transport, probably through a direct interaction with the transport enzyme. Based on the experimental findings reported we suggest that the true, physiological substrate of the red cell calcium pump is MgATP.     

Effect of headgroup on the dipole potential of phospholipid vesicles

The dipole potentials, ψ _d, of phospholipid vesicles composed of pure dimyristoylphosphatidylcholine (DMPC) or vesicles in which 50 mol% of the DMPC was substituted by dimyristoylphosphatidylserine (DMPS), dimyristoylphosphatidylglycerol (DMPG), dimyristoylethanolamine (DMPE), dimyristoylphosphatidic acid (DMPA) or monomyristoylphosphatidylcholine (MMPC) were measured via a fluorescent ratiometric method utilizing the probe di-8-ANEPPS. The PS and PG headgroups were found to cause only minor changes in ψ _d. PE caused an increase in ψ _d of 51 mV. This could be explained by a decrease in the dielectric constant of the glycerol backbone region as well as a movement of the P⁻–N⁺ dipole of the less bulky PE headgroup to a position more parallel to the membrane surface than in PC. The negatively charged PA headgroup increases ψ _d by 215 mV relative to PC alone. This indicates that the positive pole of the dipole predominantly responsible for the dipole potential is located at a position closer to the interior of the membrane than the phosphate group. The increase in the charge of the negative pole of the dipole by the phosphate group of PA increases the electrical potential drop across the lipid headgroup region. The incorporation of the single chain lipid MMPC into the membrane causes a decrease in ψ _d of 142 mV. This can be explained by a decrease in packing density within the membrane of carbonyl dipoles from the sn-2 chain of DMPC. The results presented should contribute to a better understanding of the electrical effect of lipid headgroups on the functioning of membrane proteins.     

The determination of the separate Ca2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47–72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.     

Quantitation of cholesterol incorporation into extruded lipid bilayers

Cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. To this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures 33-75 mol% cholesterol have been measured and compared with the original mixture before lipid hydration. There is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the extruded bilayers is much lower than predicted. A quantitative analysis of the vesicles is thus required before any experimental study is undertaken.     

The influence of headgroup structure and fatty acyl chain saturation of phospholipids on monolayer behavior: a comparative rheological study

This paper compares six phospholipidic monolayers at the water/chloroform interface by performing dilational rheological measurements with a drop tensiometer apparatus. The chosen lipids differ both in their headgroup structure and fatty acyl chain saturation or symmetry. The study concentrated on monolayers formed with DPPC, DPPE, DOPC, DOPE, POPC and POPE. Using a generalized Maxwell rheological model, transposed at the interface, the intimate intermolecular interactions between amphiphilic molecules are studied on and off the monolayer plane. The equilibrium and nonequilibrium phenomena are analyzed and, respectively, correlated with monolayer cohesion and with monolayer/sub-surface interactions. The purpose of this work is to gain further insights into the influences (as slight as they are) of the weak changes in phospholipid structure and on the behavior of the monolayers. The results, widely described, provide further details on nuances existing between very similar molecules, and likewise, on the synergies created between the different effects.     

Manipulation of phospholipid polar headgroup composition in primary cultures of rat hepatocytes

Isolated hepatocytes in suspension or in primary culture were incubated with different phospholipid bases and the effects on the synthesis and composition of phospholipids were studied. After incubation in the presence of 1 mM diethylethanolamine for three days, an unnatural phospholipid, phosphatidyl-diethylethanolamine, constituted more than 20% of total phospholipids. Its fatty acid composition differed from that of other phospholipids. Incubation of hepatocytes with ethanolamine gave smaller effects and in this case the increased synthesis of phosphatidylethanolamine was compensated for by methylation to phosphatidylcholine. This system can be used for studies on the functional significance of phospholipid polar headgroups in a specialized type of cell.     

The effect of calcium on phospholipid peroxidation

The effect of calcium ions on the peroxidation of ox-brain phospholipid liposomes in different free-radical catalysing systems has been assessed. Using thiobarbituric acid-reactivity (TBA) as a measure of lipid peroxidation, calcium ions both inhibited and enhanced peroxidation in the different systems.Changing the composition of the ox-brain phospholipid liposome with synthetic non TBA-reactive phosphatidylcholine, significantly altered its susceptibility to peroxidation both in the presence and absence of calcium ions.The results are discussed with reference to the possibility that calcium ions induce conformational changes in membrane phospholipids. Susceptibility to peroxidation is then influenced by a complex interrelationship between the qualitative lipid composition of the membrane, the pro-oxidant catalyst and the presence of calcium or other active ions.     

Mode of binding of cytochrome b5 to phospholipid bilayers in lamellar structure

X-ray diffraction studies were made on the multilamellar systems produced by incubation of phospholipid bilayers and the membrane protein, cytochrome b₅, or non-membrane proteins (albumin, ovalbumin and β-lactoglobulin A) at pH 8.1 in aqueous 5 mM CaCl₂ solutions.Detergent-extracted cytochrome b₅ (soluble aggregate) forms two types of lamellar phase with dipalmitoyl phosphatidylcholine bilayers, depending upon the incubation temperature. One type, which has a repeat distance of 114Å, is formed above 34°C, where the binding of cytochrome b₅ to the bilayers is hydrophobic. The other type, with a repeat distance of 153 Å, is formed below 34°C, where the binding is electrostatic. It is also suggested that cytochrome b₅ is monomeric in the former phase but remains aggregated in the latter phase.When dimyristoyl phosphatidylcholine is used, the boundary temperature for the two types shifts to 12°C. These boundary temperatures coincide with the thermal pretransition points of hydrated dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, respectively.Trypsin-treated cytochrome b₅ (monomeric) and the three non-membrane proteins exhibit only binding of the electrostatic type to the bilayers, independently of the incubation temperature. The observed repeat distances suggest that in these cases two layers of protein molecules are incorporated between the bilayers.     

Cell signalling through phospholipid breakdown

There is much evidence that G-proteins transduce the signal from receptors for Ca²⁺-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein -subunits. It is proposed that this protein is the a-subunit of the G-protein that regulates the phospholipase in this tissue.Ca²⁺-mobilizing agonists and certain growth factors also promote the hydrolysis of phosphatidylcholine through the activation of phospholipases C and D in many cell types. This yields a larger amount of diacylglycerol for a longer time than does the hydrolysis of inositol phospholipids. Consequently phosphatidylcholine breakdown is probably a major factor in long-term regulation of protein kinase C. The functions of phosphatidic acid produced by phospholipase D are speculative, but there is evidence that it is a major source of diacylglycerol in many cell types. The regulation of phosphatidylcholine phospholipases is multiple and involves direct activation by G-proteins, and regulation by Ca²⁺ protein kinase C and perhaps growth factor receptor tyrosine kinases.     

The effect of indoleacetic acid on phospholipid metabolism in pea stems

Indole-3-acetic acid (IAA) was found to stimulate stem elongation but inhibit the incorporation of [¹⁴C]choline into phosphatidylcholine within 1 h