Spin-label studies on the origin of the specificity of lipid-protein interactions in Na+,K+-ATPase membranes from Squalus acanthias

The pH dependence and salt dependence of the lipid-protein interactions of phosphatidic acid, phosphatidylserine, and stearic acid with Na+,K+-ATPase membranes from Squalus acanthias have been studied with spin-label electron spin resonance spectroscopy, using lipids with nitroxide labels on the 14-position C atom of the sn-2 chain. For phosphatidic acid and stearic acid, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 6.6 and 8.0, respectively. In contrast, the pKa of stearic acid in the bulk lipid environment of the membrane is estimated from spin-label spectroscopy to be approximately equal to 6.6. The fraction of motionally restricted phosphatidylserine spin-label remains constant over the pH range 4.7-9.2. In the fully dissociated state the fractions of motionally restricted spin-labeled phosphatidic and stearic acids decrease with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M. For stearic acid the net decrease is comparable to that obtained on protonation, but for phosphatidic acid the decrease is considerably smaller (by approximately 55%) than that obtained on protonating the lipid. The fraction of motionally restricted phosphatidylserine spin-label varies relatively little with salt concentration up to 1 M NaCl. Direct electrostatic effects alone cannot account for the whole of the observed specificity of interaction of the two phospholipids with Na+,K+-ATPase membranes.     

Influence of lipid headgroup on the specificity and exchange dynamics in lipid-protein interactions. A spin-label study of myelin proteolipid apoprotein-phospholipid complexes

The pH and salt dependences of the interaction of phosphatidic acid, phosphatidylserine, and stearic acid with myelin proteolipid apoprotein (PLP) in dimyristoylphosphatidylcholine (DMPC) recombinants have been studied by electron spin resonance spectroscopy, using spin-labeled lipids. The two-component spin-label spectra have been analyzed both by spectral subtraction and by simulation using the exchange-coupled Bloch equations to give the fraction of lipids motionally restricted by the protein and the rate of lipid exchange between the fluid and motionally restricted lipid populations. For stearic acid, phosphatidic acid, and phosphatidylserine, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 7.7, 7.6, and ca. 9.4, respectively. The corresponding pKa's for the bulk lipid regions of the bilayer are estimated, from changes in the ESR spectra, to be 6.7, 7.4, and 11, respectively. In the dissociated state at pH 9.0, the fraction of motionally restricted component decreases with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M for all three negatively charged lipids. The net decreases for stearic acid and phosphatidic acid are considerably smaller (by ca. 30%) than those obtained on protonating the two lipids, whereas for phosphatidylserine the fraction of motionally restricted lipid in high salt is reduced to that corresponding to phosphatidylcholine. For a fixed lipid/protein ratio, the on-rate for exchange at the lipid-protein interface is independent of the degree of selectivity and has a shallow temperature dependence, as expected for a diffusion-controlled process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid-protein interactions and effect of local anesthetics in acetylcholine receptor-rich membranes from Torpedo marmorata electric organ

The selectivity of lipid-protein interaction for spin-labeled phospholipids and gangliosides in nicotinic acetylcholine receptor-rich membranes from Torpedo marmorata has been studied by ESR spectroscopy. The association constants of the spin-labeled lipids (relative to phosphatidylcholine) at pH 8.0 are in the order cardiolipin (5.1) approximately equal to stearic acid (4.9) approximately equal to phosphatidylinositol (4.7) > phosphatidylserine (2.7) > phosphatidylglycerol (1.7) > G(D1b) approximately equal to G(M1) approximately equal to G(M2) approximately equal to G(M3) approximately equal to phosphatidylcholine (1.0) > phosphatidylethanolamine (0.5). No selectivity for mono- or disialogangliosides is found over that for phosphatidylcholine. Aminated local anesthetics were found to compete with spin-labeled phosphatidylinositol, but to a much lesser extent with spin-labeled stearic acid, for sites on the intramembranous surface of the protein. The relative association constant of phosphatidylinositol was reduced in the presence of the different local anesthetics to the following extents: tetracaine (55%) > procaine (35%) approximately benzocaine (30%). For stearic acid, only tetracaine gave an appreciable reduction (30%) in association constant. These displacements represent an intrinsic difference in affinity of the local anesthetics for the lipid-protein interface because the membrane partition coefficients are in the order benzocaine > tetracaine approximately procaine.     

Lipid-protein interactions in ADP-ATP carrier/egg phosphatidylcholine recombinants studied by spin-label ESR spectroscopy

The stoichiometry and specificity of lipid-protein interaction, as well as the lipid exchange rates at the protein interface, have been determined from the electron spin resonance spectra of spin-labeled lipids in reconstituted complexes of the mitochondrial ADP-ATP carrier with egg phosphatidylcholine. With the exception of cardiolipin and phosphatidic acid, the lipids studied are found to compete for approximately 50 sites at the intramembranous surface of the protein dimer. This number of first-shell lipid sites is unusually large for a protein of this size. The specificity for the protein is in the order stearic acid approximately phosphatidic acid approximately cardiolipin greater than phosphatidylserine greater than phosphatidylglycerol approximately phosphatidylcholine, with the maximum association constant relative to phosphatidylcholine being approximately 4. The selectivity for anionic lipids was partially screened with increasing ionic strength, but to a lesser extent for cardiolipin and phosphatidic acid than for stearic acid. Only in the case of phosphatidylserine was the selectivity reduced at high ionic strength to a level close to that for phosphatidylcholine. The off rates for lipid exchange at the protein surface were independent of lipid/protein ratio and correlated in a reciprocal fashion with the different lipid selectivities, varying from 5 x 10(6) s-1 for stearic acid at low ionic strength to 2 x 10(7) s-1 for phosphatidylcholine and phosphatidylglycerol. The off rates for cardiolipin were unusually low in comparison with the observed selectivity, and indicated the existence of a special population of sites (ca. 30% of the total) for cardiolipin, at which the exchange rate was very low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutral Phospholipids Stimulate Na,K-ATPase Activity: A SPECIFIC LIPID-PROTEIN INTERACTION

Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein. Most recently the phosphatidylserine binding site has been located between transmembrane segments of αTM8–10 and the FXYD protein. This paper describes stimulation of Na,K-ATPase activity of the purified human α1β1 or α1β1FXYD1 complexes by neutral phospholipids, phosphatidylcholine, or phosphatidylethanolamine. In the presence of phosphatidylserine, soy phosphatidylcholine increases the Na,K-ATPase turnover rate from 5483 ± 144 to 7552 ± 105 (p < 0.0001). Analysis of α1β1FXYD1 complexes prepared with native or synthetic phospholipids shows that the stimulatory effect is structurally selective for neutral phospholipids with polyunsaturated fatty acyl chains, especially dilinoleoyl phosphatidylcholine or phosphatidylethanolamine. By contrast to phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine destabilizes the Na,K-ATPase. Structural selectivity for stimulation of Na,K-ATPase activity and destabilization by neutral phospholipids distinguish these effects from the stabilizing effects of phosphatidylserine and imply that the phospholipids bind at distinct sites. A re-examination of electron densities of shark Na,K-ATPase is consistent with two bound phospholipids located between transmembrane segments αTM8–10 and TMFXYD (site A) and between TM2, -4, -6, -and 9 (site B). Comparison of the phospholipid binding pockets in E2 and E1 conformations suggests a possible mechanism of stimulation of Na,K-ATPase activity by the neutral phospholipid.     

Pulmonary surfactant protein SP-B is significantly more immunoreactive in anionic than in zwitterionic bilayers

Binding of polyclonal and monoclonal antibodies, quantitated by enzyme-linked immunosorbent assay, to porcine SP-B reconstituted in different phospholipid bilayers has been used to assess differences in protein structure due to lipid-protein interactions. SP-B bound significantly more antibodies when it was reconstituted in bilayers made of anionic phospholipids (phosphatidic acid, cardiolipin, phosphatidylglycerol, phosphatidylinositol or phosphatidylserine) than in zwitterionic bilayers (phosphatidylcholine, phosphatidylcholine/cholesterol, or phosphatidylethanolamine) or in fatty acid micelles (made of salts of palmitic or stearic acids). These differences in immunoreactivity can be important in the development of quantitation methods for SP-B in clinical samples based on immunological techniques.     

A single-residue deletion alters the lipid selectivity of a K+ channel-associated peptide in the beta-conformation: spin label electron spin resonance studies.

Lipid-peptide interactions with the 27-residue peptide of sequence KLEALYILMVLGFFGFFTLGIMLSYIR reconstituted as beta-sheet assemblies in dimyristoylphosphatidylcholine bilayers have been studied by electron spin resonance (ESR) spectroscopy with spin-labeled lipids. The peptide corresponds to residues 42-68 of the IsK voltage-gated K+ channel protein and contains the single putative transmembrane span of this protein. Lipid-peptide interactions give rise to a second component in the ESR spectra of lipids spin-labeled on the 14C atom of the chain that corresponds to restriction of the lipid mobility by direct interaction with the peptide assemblies. From the dependence on the lipid/peptide ratio, the stoichiometry of lipid interaction is found to be about two phospholipids/peptide monomer. The sequence of selectivity for lipid association with the peptide assemblies is in the order phosphatidic acid > stearic acid = phosphatidylserine > phosphatidylglycerol = phosphatidylcholine. Comparison with previous data for a corresponding 26-residue mutant peptide with a single deletion of the apolar residue Leu2 (Horvath et al., 1995. Biochemistry 34:3893-3898), indicates a very similar mode of membrane incorporation for native and mutant peptides, but a strongly modified pattern and degree of specificity for the interaction with negatively charged lipids. The latter is interpreted in terms of the relative orientations of the charged amino acid side chains in the beta-sheet assemblies of the native and deletion-mutant peptides.     

Mechanism for the Activation of Plasma Membrane H-ATPase from Rice (Oryza sativa L.) Culture Cells by Molecular Species of a Phospholipid

The activation of H⁺-ATPase solubilized from plasma membrane of rice (Oryza sativa L. var Nipponbare) culture cells was examined by the exogenous addition of various phospholipids, free fatty acids, glycerides, polar head groups of phospholipids and molecular species of phosphatidylcholine (PC). H⁺-ATPase activity appeared to be stimulated by phospholipids in the following order: asolectin > phosphatidylserine > PC > lysophosphatidylcholine > phosphatidylglycerol, and maximal ATPase activation was noted at around 0.05 to 0.03% (w/v) of asolectin or molecular species of PC. Polar head groups such as glycerol, inositol, and serine only slightly activated ATPase activity or not at all, while ethanolamine and choline had no effect. Activation was dependent on the degree of saturation or unsaturation of the fatty acyl chain and its length. The activity decreased with increase in the length of fatty acyl chain from dimyristoryl(14:0)-PC to distearoyl(18:0)-PC and the degree of unsaturation from dioleoyl(18:1)-PC to dilinolenoyl(18:3)-PC. Maximum activation was observed when PC possessing 1-myristoyl(14:0)-2-oleoyl(18:1) or 1-oleoyl-2-myristoyl was added to the reaction mixture. These data show that the activation of plasma membrane H⁺-ATPase by PC depends on a combination of saturated (myristic acid 14:0, palmitic acid 16:0, and stearic acid 18:0) and unsaturated (oleic acid 18:1, linoleic acid 18:2, and arachidonic acid 20:4) fatty acids at the sn-1 and sn-2 positions of the triglycerides.     

Spin-label electron spin resonance study of bacteriophage M13 coat protein incorporation into mixed lipid bilayers

The major coat protein of bacteriophage M13 was incorporated in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (80/20 w/w) vesicles probed with different spin-labeled phospholipids, labeled on the C-14 atom of the sn-2 chain. The specificity for a series of phospholipids was determined from a motionally restricted component seen in the electron spin resonance (ESR) spectra of vesicles with the coat protein incorporated. At 30 degrees C and pH 8, the fraction of motionally restricted phosphatidic acid spin-label is 0.36, 0.52, and 0.72 for lipid/protein ratios of 18, 14, and 9 mol/mol, respectively. The ESR spectra, analyzed by digital subtraction, resulted in a phospholipid preference following the pattern cardiolipin = phosphatidic acid greater than stearic acid = phosphatidylserine = phosphatidylglycerol greater than phosphatidylcholine = phosphatidylethanolamine. The specificities found are related to the composition of the target Escherichia coli cytoplasmic membrane.     

Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes.

Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate. With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI). Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4. When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed. The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0. PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L. monocytogenes and by the homologous enzyme from B. cereus. The water-soluble hydrolysis product was identified as inositol-1-phosphate. For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed. 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC. The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE. I-, Cl- and F- stimulated activity on either PC- Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B. cereus which is strongly inhibited by halides. Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L. monocytogenes and B. cereus.     

The interaction of atebrin with phospholipid vesicles

The interaction of atebrin with phosphatidylcholine and phosphatidylcholine-phosphatidic acid vesicles has been followed by equilibrium dialysis, and by photometric, fluorimetric and NMR techniques. The presence of negative charges in the phospholipids enhances the binding of atebrin. The absorbance and NMR spectral changes and fluorescence quenching occurring with phosphatidic acid are attributed to dimerization of the dye interacting electrostatically with negative groups.The dissociation constant of the binding of the dye to phosphatidylcholine vesicles was 1.4 mM; those of binding to the negative sites of phosphatidic acid were approx. 150 and 3 μM.The dye is probably located at the interphase with the acridine ring interacting with the anionic groups of phosphatidic acid and the tail freely floating in the aqueous phase. The results are discussed also in view of the use of atebrin as a probe of the energized state in natural membranes and of the suggestion that atebrin may be used as a transmembrane pH indicator in liposomes or natural membranes.     

Lipid-protein interactions in Escherichia coli membranes over-expressing the sugar-H(+) symporter,GalP EPR of spin-labelled lipids

The D-galactose-H(+) symport protein (GalP) of Escherichia coli is a homologue of the human glucose transport protein, GLUT1. After amplified expression of the GalP transporter in E. coli, lipid-protein interactions were studied in gradient-purified inner membranes by using spin-label electron paramagnetic resonance (EPR) spectroscopy. Phosphatidylethanolamine, -glycerol, -choline and -serine, in addition to phosphatidic and stearic acids, were spin-labelled at the 14 C-atom of the sn-2 chain. EPR spectra of these spin labels at probe amounts in GalP membranes consist of two components. One component corresponds to a lipid population whose motion is restricted by direct interaction with the transmembrane sections of the integral protein. The other component corresponds to a lipid population with greater chain mobility, and is similar to the single-component EPR spectrum of the spin-labelled lipids in membranes of E. coli lipid extract. Quantitation of the protein-interacting spin-label component allows determination of the stoichiometry and selectivity of lipid-protein interactions. On average, approximately 20 mol of lipid are motionally restricted per 52 kDa of protein in GalP membranes. At the pH of the transport assay, there is relatively little selectivity between the different phospholipids tested. Only stearic acid displays a stronger preferential interaction with this protein.     

Specific interaction of cytosolic and mitochondrial glyoxalase II with acidic phospholipids in form of liposomes results in the inhibition of the cytosolic enzyme only

Kinetics of cytosolic recombinant human glyoxalase II and bovine liver mitochondrial glyoxalase II were studied in the presence of liposomes made of different phospholipids (PLs). Neutral PLs such as egg phosphatidylcholine or dipalmitoylphosphatidylcholine did not affect the enzymatic activity of either enzymatic form. Liposomes made of dioleoyl phosphatidic acid or cardiolipin or phosphatidylserine also did not affect the enzymatic activity of mitochondrial glyoxalase II. Conversely, these negatively charged PLs exerted noncompetitive inhibition on cytosolic glyoxalase II only, dioleoyl phosphatidic acid and bovine brain phosphatidylserine exerting the highest and lowest inhibition, respectively. Binding studies, carried out by using a resonant mirror biosensor, revealed that liposomes made of negatively charged PLs interact specifically with both enzymatic forms of glyoxalase II, whereas interactions were not detected with neutral PLs. Once bound on glyoxalase II, negatively charged liposomes could not be removed by 3 M NaCl, suggesting that interactions between glyoxalase II and negatively charged PLs, besides ionic, may be also hydrophobic. These data suggest a possible role of negatively charged phospholipids in the regulation of level of lactoylglutathione in the cell. The data are also discussed in terms of a possible regulation of reduced glutathione supply to mitochondria.     

Correlation of phospholipid structure with functional effects on the nicotinic acetylcholine receptor. A modulatory role for phosphatidic acid.

Fourier transform infrared spectroscopy is used to characterize specific interactions between negatively charged lipids, such as phosphatidic acid, and the purified nicotinic acetylcholine receptor from Torpedo californica. The specific interaction of phosphatidic acid with acetylcholine receptor is demonstrated by the receptor-induced perturbation of the lipid ionization state, which is monitored using Fourier transform infrared bands arising from the phosphate head group. The acetylcholine receptor shifts the pKa of phosphatidic acid molecules adjacent to the receptor to a lower value by almost 2 pH units from 8.5 to 6.6. Decreased pH also leads to changes in ion channel function and to changes in the secondary structure of the acetylcholine receptor in membranes containing ionizable phospholipids. Phospholipase D restores functional activity of acetylcholine receptor reconstituted in an unfavorable environment containing phosphatidylcholine by generating phosphatidic acid. Lipids such as phosphatidic acid may serve as allosteric effectors for membrane protein function and the lipid-protein interface could be a site for activity-dependent changes that lead to modulation of synaptic efficacy.     

Neutral lipids, phospholipids and higher fatty acids in bull's testes

The lipid fractions were studied in the testicular tissue of mature bulls, of the lowland black-and-white breed. It was found that the main component of neutral lipids was cholesterol (48%) followed by triglycerides (24%), cholesterol esters (16%) and free fatty acids (12%). In cholesterol esters the main component was palmitic acid (41%) followed by oleic acid (22%), stearic acid (14%) and linoleic acid (14%). In phospholipids the main fraction was composed of lecithins (48%) followed by phosphatidylethanolamine (20%) and phosphatidic acids and phosphatidylglycerol (13%). Palmitic acid was found mainly in the fractions of lecithins and sphingomyelins, stearic acid in fractions of phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. Linoleic acid was found in the fractions of phosphatidylethanolamine, phosphatidylcholine and sphingomyelin. Arachidonic, docosatetraenoic and docosapentaenoic acids in the fractions of phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylcholine.     

The interfacial lipid binding site on the potassium channel KcsA is specific for anionic phospholipids

Lipid binding to the potassium channel KcsA from Streptomyces lividans has been studied using quenching of the fluorescence of Trp residues by brominated phospholipids. It is shown that binding of phospholipids to nonannular lipid binding sites on KcsA, located one each at the four protein-protein interfaces in the tetrameric structure, is specific for anionic phospholipids, zwitterionic phosphatidylcholine being unable to bind at the sites. The binding constant for phosphatidylglycerol of 3.0 ± 0.7 mol fraction⁻¹ means that in a membrane containing ~20 mol% phosphatidylglycerol, as in the Escherichia coli inner membrane, the nonannular sites will be ~37% occupied by phosphatidylglycerol. The binding constant for phosphatidic acid is similar to that for phosphatidylglycerol but binding constants for phosphatidylserine and cardiolipin are about double those for phosphatidylglycerol. Binding to annular sites around the circumference of the KcsA tetramer are different on the extracellular and intracellular faces of the membrane. On the extracellular face of the membrane the binding constants for anionic lipids are similar to those for phosphatidylcholine, the lack of specificity being consistent with the lack of any marked clusters of charged residues on KcsA close to the membrane on the extracellular side. In contrast, binding to annular sites on the intracellular side of the membrane shows a distinct structural specificity, with binding of phosphatidic acid and phosphatidylglycerol being stronger than binding of phosphatidylcholine, whereas binding constants for phosphatidylserine and cardiolipin are similar to that for phosphatidylcholine. It is suggested that this pattern of binding follows from the pattern of charge distribution on KcsA on the intracellular side of the membrane.     

The interaction of insulin with phospholipids

1. A simple two-phase chloroform–aqueous buffer system was used to investigate the interaction of insulin with phospholipids and other amphipathic substances. 2. The distribution of ¹²⁵I-labelled insulin in this system was determined after incubation at 37°C. Phosphatidic acid, dicetylphosphoric acid and, to a lesser extent, phosphatidylcholine and cetyltrimethylammonium bromide solubilized ¹²⁵I-labelled insulin in the chloroform phase, indicating the formation of chloroform-soluble insulin–phospholipid or insulin–amphipath complexes. Phosphatidylethanolamine, sphingomyelin, cholesterol, stearylamine and Triton X-100 were without effect. 3. Formation of insulin–phospholipid complex was confirmed by paper chromatography. 4. The two-phase system was adapted to act as a simple functional system with which to investigate possible effects of insulin on the structural and functional properties of phospholipid micelles in chloroform, by using the distribution of [¹⁴C]glucose between the two phases as a monitor of phospholipid–insulin interactions. The ability of phospholipids to solubilize [¹⁴C]glucose in chloroform increased in the order phosphatidylcholine<sphingomyelin<phosphatidylethanolamine<phosphatidic acid. Insulin decreased the [¹⁴C]glucose solubilized by phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid, but not by sphingomyelin. 5. The significance of these results and the molecular requirements for the formation of insulin–phospholipid complexes in chloroform are discussed.     

Phase transitions and phase separations in phospholipid membranes induced by changes in temperature, pH, and concentration of bivalent cations.

Differential scanning calorimetry (DSC) and fluorescence polarization of embedded probe molecules were used to detect phase behavior of various phospholipids. The techniques were directly compared for detecting the transition of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA) dispersed in aqueous salt solutions. Excellent agreement occurred in the case of phosphatidylcholine; however, in the case of phosphatidic acid, at pH 6.5, transitions detected by fluorescence polarization using the disc-like perylene molecule occurred about 10 degrees lower than those detected by DSC. Discrepancy between fluorescence and DSC methods is eliminated by using a rod-like molecule, diphenylhexatriene (DPH). Both techniques show that doubly ionizing the phosphate group reduces the Tc by about 9 degrees. Direct pH titration of fluidity can be accomplished and this effect is most dramatic when membranes are in their transition temperature range (ca. 50 degrees). Phosphatidic acid transitions occur at higher temperatures, and have appreciably lower transition enthalpies and entropies than phosphatidylcholine. These effect could not be explained simply on the basis of double layer electrostatics and several other factors were discussed in an attempt to rationalize the results. Addition of monovalent cations (0.01-0.5 M) is shown to increase the Tc of dipalmitoylphosphatidylglycerol by less than 3 degrees. However, addition of (1 x 10-3 M) Ca2+ abolishes the phase transition of both phosphatidyglycerol and phosphatidylserine in the range 0-70 degrees. Preliminary X-ray evidence indicates the phosphatidylserine-Ca2+ bilayers are in a crystalline state at 24 degrees. In contrast, 5 x 10-3 M Mg2+ only broadens the transition and increases the Tc indicating a considerable difference between the effects of Ca2+ and Mg2+. Neutralization of PS increases the Tc from 6 degrees (at pH 7.4) to 20-26 degrees (at pH 2.5-3.0) but does not abolish the transition, suggesting the Ca2+ effect involves more than charge neutralization. Addition of Ca2+ to mixed phosphatidylserine-phosphatidylcholine dispersions, induces a phase separation of the dipalmitoyl- (and also distearoyl-) phosphatidylcholine as seen by the appearance of a new endothermic peak at 41 degrees (58 degrees). Similarly, in mixed (dipalmitoyl) phosphatidic acid-phosphatidylcholine (2:1) dispersions, Ca2+ again can separate the phosphatidylcholine component.     

Hydrolysis of lipid mixtures by rat hepatic lipase

The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in conformation of spin-labeled calmodulin by phospholipids

Spin-labeled calmodulin was synthesized and the effects of phospholipids on its conformation were examined by ESR spectroscopy. Phosphatidylserine (0.1-1.0 mM) increased the signal intensity of the ESR spectrum of spin-labeled calmodulin and decreased the apparent rotational correlation time in the presence of 0.1 mM CaCl2. This change was reversed by addition of excess calcium, and in the absence of calcium phosphatidylserine did not change the spectrum, suggesting that the change in spin-labeled calmodulin brought about by phosphatidylserine was not induced by a hydrophobic interaction of the two, but by inhibition of the binding of calcium to calmodulin. L-Serine and O-phospho-L-serine had no effect on the ESR signals of spin-labeled calmodulin. The effects of various other phospholipids were also examined. Their inhibitory activities were in the order phosphatidic acid greater than phosphatidylserine greater than phosphatidylglycerol = phosphatidylinositol; phosphatidylethanolamine and phosphatidylcholine had no effect on the spectra. The effects of these phospholipids were dependent on their binding activities toward calcium. Furthermore, phosphatidic acid and phosphatidylserine at 1 mM reduced the activity of calmodulin-dependent phosphodiesterase by 16.4 and 8.7%, respectively. These findings indicate that spin-labeled calmodulin did not interact with the phospholipids by a hydrophobic interaction, but that calcium binding to spin-labeled calmodulin interfered with phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol, and some of these phospholipids inactivated calmodulin. Thus the activity of calmodulin may be regulated in part by some phospholipids.