Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues

In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coupling of Ca2+ transport to ATP hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum: potential role of the 53-kilodalton glycoprotein

An essential feature of the function of the Ca2+-ATPase of sarcoplasmic reticulum (SR) is the close coupling between the hydrolysis of ATP and the active transport of Ca2+. The purpose of this study is to investigate the role of other components of the SR membrane in regulating the coupling of Ca2+-ATPase in SR isolated from rabbit skeletal muscle, reconstituted SR, and purified Ca2+-ATPase/phospholipid complexes. Our results suggest that (1) it is possible to systematically alter the degree of coupling obtained in reconstituted SR preparations by varying the [KC1] present during cholate solubilization, (2) the variation in coupling is not due to differences in the permeability of the reconstituted SR vesicles to Ca2+, and (3) vesicles reconstituted with purified Ca2+-ATPase are extensively uncoupled under our experimental conditions regardless of the lipid/protein ratio or phospholipid composition. In reconstituted SR preparations prepared by varying the [KC1] present during cholate treatment, we find a direct correlation between the relative degree of coupling between ATP hydrolysis and Ca2+ transport and the level of the 53-kilodalton (53-kDa) glycoprotein of the SR membrane. These results suggest that the 53-kDa glycoprotein may be involved in regulating the coupling between ATP hydrolysis and Ca2+ transport in the SR.     

Functional characterization of reconstituted sarcoplasmic reticulum vesicles

A detailed functional characterization of reconstituted sarcoplasmic reticulum (SR) vesicles with similar lipid content as normal SR was obtained by studies of ATPase activity and calcium transport in transient state, steady state, and equilibrium conditions. For this purpose, enzyme phosphorylation with ATP, hydrolytic activity, calcium transport, phosphorylation with Pi, and ATP synthesis by reversal of the pump were measured, and utilized to demonstrate function and orientation of catalytic sites. The preparations used in these studies displayed the highest activity reported for reconstituted sarcoplasmic reticulum systems. The rates of phosphoenzyme formation from ATP and hydrolysis as well as steady state levels matched the values obtained with normal SR vesicles. Calcium transport and repeated cycles of ATP synthesis by reversal of the pump were also obtained. However, the efficiency of transport and ATP synthesis from a Ca2+ gradient was approximately three times lower than in native vesicles. This deficiency could not be attributed to passive calcium leak from the reconstituted vesicles but, in part, can be explained by the bidirectional alignment of the calcium pump in reconstituted SR. It is suggested that vectorial transport requires a more complex level of protein structure than that for sustaining simple ATPase activity. Time resolution of the phosphorylation reaction by rapid quench methods can be used to estimate the orientation of the calcium pump in the membrane. Such studies indicate that the calcium pump protein is largely bidirectionally oriented in reconstituted SR vesicles.     

Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.     

Functional characteristics of reconstituted sarcoplasmic reticulum membranes as a function of the lipid-to-protein ratio

The ATP-induced Ca2+ accumulation efficiency and rates of Ca2+ uptake of the reconstituted sarcoplasmic reticulum (RSR) model membrane system were measured over an extended range of lipid-to-protein (L/P) molar ratios and were compared to those of isolated light sarcoplasmic reticulum (LSR). Highly purified sarcoplasmic reticulum (SR), dissociated in the presence of deoxycholate, was reconstituted for several L/P ratios, according to the same procedure, forming closed membranes vesicles composed of greater than 95% Ca2+ pump protein and SR lipids which were capable of ATP-induced Ca2+ accumulation in the absence of oxalate or other Ca2+ precipitating agents. This suggests that dissociation of SR and reconstitution to form RSR does not significantly affect the ability of the Ca2+ pump protein incorporated into the SR lipid bilayer to establish Ca2+ gradients. Electron micrographs of fixed and stained dispersions of RSR revealed a structural organization of the membrane that was dependent upon the L/P molar ratio. RSR with L/P greater than 88 were composed of closed vesicles whose membranes stained asymmetrically, similar to that observed for LSR. Closed vesicles of RSR with L/P less than 88 were composed of membrane that stained symmetrically. In addition, reconstituted SR preparations with well-defined L/P molar ratios greater than 88 possess a functional behavior similar to that of LSR (in the absence of oxalate, energy efficiencies are 60-70% and apparent initial uptake rates are 80% that of isolated LSR controls); RSR preparations with L/P less than 88 are characterized by significantly depressed values of the energy efficiencies and apparent initial uptake rates especially at low L/P ratios. Thus, we are the first to report a reconstituted SR model membrane system capable of attaining rates of Ca2+ uptake comparable to isolated LSR controls at comparable L/P ratios in the absence of oxalate or other Ca2+ precipitating agents.     

Ca 2+ uptake in reconstituted sarcoplasmic reticulum vesicles

The reconstitution of functional sarcoplasmic reticulum vesicles capable of Ca²⁺ transport has been achieved. Sarcoplasmic reticulum vesicles are first solubilized with deoxycholate and then reassembled into membranous vesicles by removal of the detergent using dialysis. The Ca²⁺ pump protein can, by itself, be reconstituted to form membranous vesicles capable of energized Ca²⁺ binding and uptake. The lipid content of the reconstituted vesicles is about the same as that of the original sarcoplasmic reticulum vesicles. The reconstituted vesicles have an elevated ATPase activity. Ca²⁺ binding and uptake in the presence of ATP are restored to about 25% and 50%, respectively.     

Modulation of reconstituted pig kidney Na+/K(+)-ATPase activity by cholesterol in endogenous lipid vesicles: role of lipid domains

Diverse experimental and theoretical evidence suggests that plasma membranes contain cholesterol-induced segregated domains that could play a key role in the modulation of membrane functions, including intrinsic enzyme activity. To gain insight into the role of cholesterol, we reconstituted pig kidney Na+/K+-ATPase into unilamellar vesicles of endogenous lipids mimicking the natural membrane and addressed the question of how modification of the cholesterol content could affect the ATPase activity via changes in the membrane lipid phase and in the protein structure and dynamics. We used steady-state and time-resolved fluorescence spectroscopy with the lipid phase probes DPH and Laurdan and the protein probe fluorescein and also used infrared spectroscopy using attenuated total reflectance. Upon modification of membrane cholesterol content, the ATPase activity did not change monotonically but instead exhibited abrupt changes resulting in two peaks at or close to critical cholesterol mole fractions (25 and 33.3 mol %) predicted by the superlattice or regular distribution model. Fluorescence parameters associated with the membrane probes also showed abrupt changes with peaks, coincident with the cholesterol concentrations associated with the peaks in the enzyme activity, while parameters associated with the protein probes also showed slight but abrupt changes resulting in dips at the same cholesterol concentrations. Notably, the IR amide I band maximum also showed spectral shifts, characterized by a frequency variation pattern with peaks at the same cholesterol concentrations. Overall, these results indicate that the lipid phase had slightly lower hydration, at or near the two critical cholesterol concentrations predicted by the superlattice theory. However, in the protein domains monitored there was a slight but significant hydration increase along with increased peptide backbone flexibility at these cholesterol concentrations. We propose that in the vicinity of the critical mole fractions, where superlattice formation can occur, minute changes in cholesterol concentration produce abrupt changes in the membrane organization, increasing interdomain surfaces. These changes, in turn, induce small changes in the protein's structure and dynamics, therefore acting to fine-tune the enzyme.     

Electrical potential accelerates the E1P(Na)----E2P conformational transition of (Na,K)-ATPase in reconstituted vesicles

We have used renal (Na,K)-ATPase, covalently labeled with fluorescein, and phospholipid vesicles reconstituted with labeled enzyme, to detect conformational transitions induced by acetyl phosphate in the presence of Mg2+ and Na+ ions. Equilibrium fluorescence measurements show quenching of the fluorescein fluorescence, which is thought to reflect conversion of the initial E1 form to the phosphorylated E2P form. These fluorescence changes occur on inside-out-oriented pumps. The rates of acetyl phosphate-induced fluorescence changes have been measured using a stopped-flow fluorimeter. The rate of fluorescence quenching (1.5-3 s-1) is a measure of the rate of the E1P(Na)----E2P transition. The quenching is preceded by a fast fluorescence increase (12.3 +/- 4 s-1) associated with phosphorylation of E1 to E1P(Na), shown clearly in experiments with enzyme treated with oligomycin. Oligomycin greatly reduces the rate of the fluorescence quenching (0.044 +/- 0.01 s-1). Using potassium-loaded vesicles treated with valinomycin or lithium-loaded vesicles treated with Li+ ionophore N,N'-diheptyl-N,N'-didiethyl ether, 5,5-dimethyl-3,7-dioxanonanediamide in order to induce electrical diffusion potentials, negative inside, the rates of the fluorescence quenching are accelerated by up to 4-fold. The experiments demonstrate that the conformational transition E1P(Na)----E2P, associated with transport of 3 Na+ ions, is a voltage-sensitive reaction, carrying a net positive charge. This confirms a prediction based on transport experiments. In experiments with fluorescein-labeled (Na,K)-ATPase, the use of acetyl phosphate rather than ATP, which does not bind, provides a valuable tool to detect fluorescence signals accompanying steps in the turnover cycle.     

The nature of the modulation of Ca2+ transport as studied by reconstitution of cardiac sarcoplasmic reticulum

Membrane vesicles capable of energized Ca2+ pumping have been reconstituted from cardiac sarcoplasmic reticulum (SR). Cardiac SR was solubilized with Triton X-100 in a detergent to protein weight ratio of 0.8, and membranous vesicles were reconstituted by removal of detergent with Bio-Beads SM-2 (a neutral porous styrene-divinylbenzene copolymer). The reconstituted vesicles exhibited ATP-dependent oxalate-facilitated Ca2+ accumulation with rates and efficiency comparable to the best reconstituted skeletal muscle preparation (Ca2+-loading rate = 1.65 +/- 0.31 mumol mg-1 min-1, Ca2+-activated ATPase activity = 2.39 +/- 0.25 mumol mg-1 min-1, efficiency (Ca2+/ATP) = 0.69 +/- 0.09). Phospholamban in the reconstituted vesicles was phosphorylated with added catalytic subunit of cAMP-dependent protein kinase to almost the same extent as that in original vesicles. However, phosphorylation of phospholamban had no effect on the Ca2+ accumulation of the reconstituted vesicles. This is to be contrasted with a decrease in the half-maximal concentration of Ca2+ for Ca2+ accumulation (KCa) in the original vesicles from 1.35 +/- 0.08 microM to 0.75 +/- 0.12 microM by cAMP-dependent phosphorylation of phospholamban. On the other hand KCa for the reconstituted vesicles was about 0.5 microM and remained unchanged by phosphorylation, indicating that the Ca2+ pump in the reconstituted vesicles is already fully activated. These results suggest that in normal cardiac SR, phospholamban in the dephosphorylated state acts as a suppressor of the Ca2+ pump and that phosphorylation of phospholamban serves to reverse the suppression.     

Prolonged exercise induces structural changes in SR Ca(2+)-ATPase of rat muscle.

Sarcoplasmic reticulum (SR) isolated from the deep red portion of the gastrocnemius muscle of Sprague-Dawley rats after a single bout of prolonged exercise was shown to have depressed Ca(2+)-stimulated Mg(2+)-dependent ATPase activity over a temperature range of 15 to 42.5 degrees C when compared to SR obtained from control muscle. Inclusion of the calcium ionophore, A23187, failed to restore the depressed ATPase activity from SR of exercised muscle to control values, but it did normalize the stimulatory effect of temperature on ATPase activity. This depression was also manifested as an increased activation energy when the data were converted to an Arrhenius plot. SR vesicles from both groups showed no differences or discontinuities in plots of steady-state fluorescence anisotropy. When the binding characteristics of the fluorescent probe, fluorescein isothiocyanate (FITC), were analyzed, SR vesicles prepared from exercised muscle displayed a 40% reduction in binding capacity with no apparent change in Kd. These findings support the conclusion that a single bout of exercise induces a structural change in the Ca(2+)-ATPase protein of rat red gastrocnemius muscle that is not a direct result of gross lipid alterations or increased muscle temperature.     

Purification of a K(+)-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system

A K(+)-channel protein of the sarcoplasmic reticulum (SR) was purified by assaying the channel activity in a planar lipid bilayer system. The light fraction of SR vesicles was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and fractionated by an anion-exchange chromatography and followed by gel filtration chromatography and affinity chromatography with concanavalin A. All fractions in each steps were mixed with asolectin solubilized in CHAPS and reconstituted into vesicles by dialysis. The channel activity of each fraction was assayed after the reconstituted vesicles had been fused into a planar lipid bilayer. The final fraction which showed the K(+)-channel activity contained only 100 kDa protein in a silver-stained gel after SDS-PAGE and an anti-Ca(2+)-ATPase antibody did not recognize the protein. The characteristics of the K(+)-channel were identical to those observed in native SR vesicles when using the same method. The channel showed a single-channel conductance of 120 pS in 0.1 M KCl and marked voltage dependence. The channel did not permeate Ca2+ and Cl- and was blocked by neomycin B.     

Chemical modification of the Ca2+ -dependent ATPase of sarcoplasmic reticulum from skeletal muscle. II. Use of 2, 4, 6-trinitrobenzenesulfonate to show functional movements of the ATPase molecule.

1. By changing the concentrations of Mg2+ and Ca2+ ions and ATP, the Ca2+, Mg2+-dependent ATPase [EC 3.6.1.3] of SR was converted to various enzymatic states, E, MgE, MgECa, MgEATP' CaECa, and CaECap at pH 8.0 and 0 degrees (cf. Eq. 1). 2. SR vesicles were allowed to react with 0.5 mM 2,4,6-trinitrobenzenesulfonate (TBS) at pH 8.0 and 0 degrees, keeping the ATPase in one of the enzymatic states listed above. Trinitrophenyl (TNP)-protein and TNP-lipid were separated by gel-filtration, and the amounts of TNP incorporated into protein and lipid were determined. 3. In all the enzymatic states of ATPase tested, the amount of TBS bound with SR protein increased exponentially with time, and reached a maximum level 10 min after starting the reaction. On the other hand, the amount of TBS bound to lipid increased with time, and did not reach a maximum level for at least 20 min. 4. The SR ATPase activity and the amount of EP formed decreased only slightly, even when the amount of TBS bound to SR protein reached the maximum level. 5. The maximum amount of TBS bound to SR protein varied on changing the enzymatic state of SR ATPase. Namely, about 2,3,1,3, or 4, and 3 moles of TNP were incorporated per mole of SR ATPase, when SR was allowed to react with TBS in the enzymatic states MgE, MgECa, MgEATP' CaECa, and CaECap, respectively. 6. When the enzymatic state was changed from MgE to MgECa 10 min after starting the reaction with TBS, about 4 moles of TBS was bound per mole of ATPase within 10 min, while the maximum levels of TBS bound in states MgE and MgECa were about 2 and 3 moles per mole of ATPase, respectively, as mentioned above. 7; When the enzymatic state was changed from MgE to MfEATP 10 min after starting the reaction with TBS, about 3 moles of TBS was bound per mole of ATPase within 10 min, while the maximum levels of TBS bound in states MgE and MgEATP were about 2 and 1 moles per mole of ATPase, respectively, as mentioned above. 8. When the enzymatic state was changed from CaECa to CaECap 10 min after starting the reaction with TBS, about 4 moles of TBS was bound per mole of ATPase, while the maximum levels of TBS bound in states CaECa and CaECap were about 3 or 4 moles per mole of ATPase, respectively. 9. A diagrammatic model of functional movements of the ATPase molecule coupled with elementary steps in the ATPase reaction is proposed on the basis of these results.     

Purification, characterization, and reconstitution of the Ca2+-transport system (high-affinity Ca2+, Mg2+-ATPase) of the human erythrocyte membrane

The Ca2+-transport system of human erythrocyte membranes was solubilized by deoxycholate in the presence of the nonionic detergent Tween 20 and was purified by calmodulin affinity chromatography. The method yields a functional enzyme, which as compared with the erythrocyte membrane was purified 207-fold based on specific activity, and about 330-fold based on protein content. The activity of the isolated enzyme can be increased about 9-fold by the addition of calmodulin, resulting in a specific activity of 10.1 mumoles/mg . min at 37 degrees C. Triton X-100 and deoxycholate stimulate the calmodulin-deficient Ca2+-ATPase in a concentration dependent manner, which results in a loss of the calmodulin-sensitivity. The Ca2+-transport ATPase could be reconstituted after solubilization of the ATPase by deoxycholate and controlled dialysis near room temperature. The system was reconstituted to form membraneous vesicles capable of energized Ca2+ accumulation. The membrane vesicles showed a protein to lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original erythrocyte membrane. The stimulation by calmodulin of the calmodulin-depleted membrane-bound and partially purified Ca2+-ATPase is strongly time dependent. At a Ca2+-concentration of 40 microM and low calmodulin concentrations, approx. 120 min are required to regain full activity. This time period is decreased to about 15 min in the presence of a high excess of calmodulin. Vice versa, at fixed concentrations of calmodulin, the time necessary for regain of full activity is decreased as the Ca2+ concentrations is increased. The dependence of the Ca2+-ATPase activity on the calmodulin concentration shows strong deviation from Michaelis-Menten kinetics at Ca2+ concentrations below (4--10 microM) and above (200 microM) the optimum concentration of 40 microM. Mathematical analysis of the results at 200 microM Ca2+ leads to the assumption that 4 calmodulin molecules interact with one oligomer of Ca2+-ATPase consisting of 4 identical subunits.     

The blocking effect of aliphatic hydrocarbons on Ca2+ leakage channels formed by aggregates of Ca2+-ATPase of the sarcoplasmic reticulum

The effects of aliphatic hydrocarbons within the liposomes on the Ca2+ transport function of isolated sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle, vesiculate preparation of Ca2+ dependent ATPase and proteoliposomes reconstituted from Ca2+-ATPase and egg phosphatidylcholine, were studied. It was shown that liposomes prepared from dipalmitoyl phosphatidylcholine containing aliphatic hydrocarbons increase 2 to 3 times Ca2+ accumulation by Ca2+-dependent ATPase from rabbit skeletal muscle SR. Ca2+ transport by SR vesicles increases in the presence of hydrocarbons by 15--20%. The activating effect of hydrocarbons on Ca2+ transport by proteoliposomes depends on the lipid/protein ratio. The proteoliposomes with a high lipid/protein ratio are practically insensitive to the effects of hydrocarbons. It was suggested that activation of Ca2+ transport by hydrocarbons is due to blocking of Ca2+ leakage channels formed during the aggregation of Ca2+-ATPase molecules. Treatment of membranes by formaldehyde results in the oligomerization of Ca2+-ATPase and decreases 2--4-fold the ATP-dependent accumulation of Ca2+. Subsequent addition of decane restores Ca2+ transport practically completely.     

Reconstitution of the Ca2+-transport system of human erythrocytes.

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte 'ghosts' was solubilized and reconstituted to form membranous vesicles capable of energized Ca2+ accumulation. The erythrocyte 'ghosts' for this purpose were prepared by using isoosmotic freeze-haemolysis in the presence of Tween 20 and proteinase inhibitors to stabilize the preparation. The reconstitution procedure is similar to that developed by Meissner & Fleischer [(1974) J. Biol. Chem. 249, 302-309] for skeletal-muscle sarcoplasmic-reticulum in that: (1) deoxycholate is used for the solubilization of the membrane; (2) controlled dialysis at near room temperature, rather than 0 degree C, is required in order to obtain a functional preparation capable of Ca2+ accumulation; and (3) membrane vesicles can be reassembled with protein/lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original membrane.     

Uptake of Ca2+ mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles

The (Ca2+ + Mg2+)-ATPase was purified from skeletal muscle sarcoplasmic reticulum and reconstituted into sealed phospholipid vesicles by solution in cholate and deoxycholate followed by detergent removal on a column of Sephadex G-50. The level of Ca2+ accumulated by these vesicles, either in the presence or absence of phosphate within the vesicles, increased with increasing content of phosphatidylethanolamine in the phospholipid mixture used for the reconstitution. The levels of Ca2+ accumulated in the absence of phosphate were very low for vesicles reconstituted with egg yolk phosphatidylcholine alone at pH 7.4, but increased markedly with decreasing pH to 6.0. Uptake was also relatively low for vesicles reconstituted with dimyristoleoyl- or dinervonylphosphatidylcholine, and addition of cholesterol had little effect. The level of Ca2+ accumulated increased with increasing external K+ concentration, and was also increased by the ionophores FCCP and valinomycin. Vesicle sizes changed little with changing phosphatidylethanolamine content, and the sidedness of insertion of the ATPase was close to random at all phosphatidylethanolamine contents. It is suggested that the effect of phosphatidylethanolamine on the level of Ca2+ accumulation follows from an effect on the rate of Ca2+ efflux mediated by the ATPase.     

Reconstitution of vesicles capable of energy transformation from phospholipids and adenosine triphosphatase of a thermophilic bacterium.

1. A stable ATPase [EC 3.6.1.3] complex (TF0-F1) from the thermophilic bacterium PS3 was reconstituted into vesicles capable of energy transformation,measured as ATP-dependent enhancement of fluorescence of 8-anilinonoaphthalene-1-sulfonate. 2. The factors necessary for obtaining highly active vesicles were investigated. Cholate and deoxycholate were both required for solubilization of TF0-F1 and P-lipids, and removal of the detergents by dialysis resulted in vesicle formation. Medium of around pH 8 and low ionic strength containing 2.5 mM MgSO4 was found suitable for dialysis. The optimal temperature for reconstitution was 30 degrees with soybean P-lipids and 45 degree with PS3 P-lipids. The optimal ratio of protein to lipid was about 1/50. 3. The vesicles obtained under these conditions were mainly 100-200 nm in diameter, covered with 9.5 nm spheres, and had a bouyant density of 1.06 in sucrose andan internal volume of about 0.5 mul per mg of P-lipids.     

Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation

In some cases, lipids in one leaflet of an asymmetric artificial lipid vesicle suppress the formation of ordered lipid domains (rafts) in the opposing leaflet. Whether this occurs in natural membranes is unknown. Here, we investigated this issue using plasma membrane vesicles (PMVs) from rat leukemia RBL-2H3 cells. Membrane domain formation and order was assessed by fluorescence resonance energy transfer and fluorescence anisotropy. We found that ordered domains in PMVs prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ～37°C, whereas ordered domains in symmetric vesicles formed from the extracted PMV lipids were stable up to 55°C, indicating the stability of ordered domains was substantially decreased in intact PMVs. This behavior paralleled lesser ordered domain stability in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles, suggesting intact PMVs exhibit some degree of lipid asymmetry. This was supported by phosphatidylserine mislocalization on PMV outer leaflets as judged by annexin binding, which indicated NEM-induced PMVs are much more asymmetric than PMVs formed by dithiothreitol/paraformaldehyde treatment. Destroying asymmetry by reconstitution of PMVs using detergent dilution also showed stabilization of domain formation, even though membrane proteins remained associated with reconstituted vesicles. Similar domain stabilization was observed in artificial asymmetric lipid vesicles after destroying asymmetry via detergent reconstitution. Proteinase K digestion of proteins had little effect on domain stability in NEM PMVs. We conclude that loss of PMV lipid asymmetry can induce ordered domain formation. The dynamic control of lipid asymmetry in cells may regulate domain formation in plasma membranes.     

Fourier-transform infrared studies of CaATPase/phospholipid interaction: survey of lipid classes

CaATPase from rabbit skeletal muscle has been isolated, purified, delipidated, and reconstituted with retention of ATPase activity into lipid vesicles consisting respectively of 1,2-dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), and egg sphingomyelin. The effect of the enzyme on phospholipid order and melting characteristics were determined with Fourier-transform infrared spectroscopy. Taken together with prior data from this laboratory for 1,2-dipalmitoylphosphatidylcholine and 1,2-dioleoylphosphatidylcholine (DOPC), as well as for native sarcoplasmic reticulum (SR), three types of lipid response to protein incorporation have been observed: (1) Phospholipids with high levels of acyl chain unsaturation (DOPC or native SR) have their lipid acyl chains slightly ordered by CaATPase incorporation. The effect of protein on the gel-liquid crystal phase transition cannot be easily determined, since the cooperative melting even in these systems occurs at temperature well below 0 degrees C. (2) Phospholipids with saturated acyl chains show slightly lowered melting temperatures and reduced cooperativity of melting upon CaATPase insertion. In addition, protein induces (at most) slight disorder into the acyl chains at temperatures removed from the lipid melting point. (3) The strongest response is observed for phospholipids containing one saturated and one unsaturated chain (POPE or SOPC) or heterogeneous systems with low levels of unsaturation (egg sphingomyelin). In these cases, relatively low protein levels diminish the magnitude of or completely abolish the phospholipid phase transition. In addition, substantial disorder is introduced into the acyl chain compared with the pure lipid both above and below its transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the membrane association of two antimicrobial peptides, magainin 2 and indolicidin

Interactions of two antimicrobial peptides, magainin 2 and indolicidin, with three different model biomembranes, namely, monolayers, large unilamellar vesicles (LUVs), and giant liposomes, were studied. Insertion of both peptides into lipid monolayers was progressively enhanced when the content of an acidic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in a film of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) was increased. Indolicidin and magainin 2 penetrated also into lipid monolayers containing cholesterol (mole fraction, X = 0.1). Membrane association of magainin 2 attenuated lipid lateral diffusion in POPG-containing LUVs as revealed by the decrease in the excimer/monomer fluorescence ratio I(e)/I(m) for the pyrene fatty-acid-containing phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl) decanoyl]-sn-glycero-3-phospho-rac-glycerol (PPDPG). Likewise, an increase in steady-state fluorescence anisotropy of the membrane-incorporated diphenylhexatriene (DPH) was observed, revealing magainin 2 to increase acyl chain order and induce segregation of acidic phospholipids. Similar effects were observed for indolicidin. The topological effects of magainin 2 and indolicidin on phospholipid membranes were investigated using optical microscopy of giant vesicles. Magainin 2 had essentially no influence on either SOPC or SOPC:cholesterol (X = 0.1) giant liposomes. However, effective vesiculation was observed when acidic phospholipid (X(PG) = 0.1) was included in the giant vesicles. Indolicidin caused only a minor shrinkage of giant SOPC vesicles whereas the formation of endocytotic vesicles was observed when the giant liposome contained POPG (X(PG) = 0.1). Interestingly, for indolicidin, vesiculation was also observed for giant vesicles composed of SOPC/cholesterol (X(chol) = 0.1). Possible mechanisms of membrane transformation induced by these two peptides are discussed.