Boundary Lipid Research Articles

Effect of Artificial Boundary Lipid on the Membrane Dynamics of Human Glycophorin-Containing Liposome

Abstract Membrane dynamics of egg yolk phosphatidylcholine (egg PC) lipid bilayer which contains human glycophorin with an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), were investigated by ESR spin probe technique. 2-Tridecyl-2-(4-hydroxy-4-oxoundecyl)-4,4-dimethyl-3-oxazolidinyloxy (5-NS), 2-octyl-2-(7-ethoxy-7-oxoheptyl)-4,4-dimethyl-3-oxazolidinyloxy (8-ENP), and 2-hexyl-2-(11-methoxy-11-oxoundecyl)-4,4-dimethyl-3-oxazolidinyloxy (12-MNS) were used as the spin probe to measure the fluidity in different regions and depths of the lipid bilayer membrane. Studies on lipid bilayer containing 5-NS revealed that the reconstitution of glycophorin to egg PC bilayer slightly decreased the membrane fluidity in the vicinity of membrane surface. Further addition of DDPC to this liposome brought about further decrease in the fluidity of this region. Liposomes containing 12-MNS revealed, however, that the addition of glycophorin had an opposite effect on the deep hydrophobic domain of the membrane by increasing the fluidity and the addition of DDPC to the liposome makes this domain less fluid. Corresponding changes were observed in the energy of activation for the rotation of 12-MNS during each case.

Effects of melittin on lipid-protein interactions in sarcoplasmic reticulum membranes

To investigate the physical mechanism by which melittin inhibits Ca-adenosine triphosphatase (ATPase) activity in sarcoplasmic reticulum (SR) membranes, we have used electron paramagnetic resonance spectroscopy to probe the effect of melittin on lipid-protein interactions in SR. Previous studies have shown that melittin substantially restricts the rotational mobility of the Ca-ATPase but only slightly decreases the average lipid hydrocarbon chain fluidity in SR. Therefore, in the present study, we ask whether melittin has a preferential effect on Ca-ATPase boundary lipids, i.e., the annular shell of motionally restricted lipid that surrounds the protein. Paramagnetic derivatives of stearic acid and phosphatidylcholine, spin-labeled at C-14, were incorporated into SR membranes. The electronic paramagnetic resonance spectra of these probes contained two components, corresponding to motionally restricted and motionally fluid lipids, that were analyzed by spectral subtraction. The addition of increasing amounts of melittin, to the level of 10 mol melittin/mol Ca-ATPase, progressively increased the fraction of restricted lipids and increased the hyperfine splitting of both components in the composite spectra, indicating that melittin decreases the hydrocarbon chain rotational mobility for both the fluid and restricted populations of lipids. No further effects were observed above a level of 10 mol melittin/mol Ca-ATPase. In the spectra from control and melittin-containing samples, the fraction of restricted lipids decreased significantly with increasing temperature. The effect of melittin was similar to that of decreased temperature, i.e., each spectrum obtained in the presence of melittin (10:1) was nearly identical to the spectrum obtained without melittin at a temperature approximately 5 degrees C lower. The results suggest that the principal effect of melittin on SR membranes is to induce protein aggregation and this in turn, augmented by direct binding of melittin to the lipid, is responsible for the observed decreases in lipid mobility. Protein aggregation is concluded to be the main cause of inactivation of the Ca-ATPase by melittin, with possible modulation also by the decrease in mobility of the boundary layer lipids.

Direct transfer of tumor surface antigenic protein (TSAP) from tumor cell to liposome for making liposomal vaccine

Abstract For the production of an effective liposomal vaccine, tumor surface antigenic proteins (TSAP) were directly transferred from BALB RVD leukemia cells to a lecithin liposome containing an artificial boundary lipid (DDPC, 1,2-dimyristoylamido-1,2-deoxyphospbatidylcholine). When DMPC (20 mol%)-DDPC (80 mol%) liposomes were employed the highest efficiency of membrane protein transfer was achieved. From studies of specific lectin-induced aggregation of the membrane protein-transferred liposome, saccharide moieties of proteins transferred were found to contain at least sialic acid, galactose, and mannose moieties. This membrane protein-transferred liposome showed significantly higher efficiency of macrophage uptake compared with the other two liposomes, namely no membrane protein-reconstituted liposome and liposome with proteins which were first extracted by butanol from turner cell and then reconstituted to liposome. Judging from results obtained, TSAP was considered to be certainly involved in membrane proteins transferred from the tumor cell to liposome. When mice were immunized by the TSAP-transferred liposome (liposomal vaccine) and subsequently challenged by BALB RVD, high CTL induction and excellent prevention of tumor growth were observed, as expected.

Analysis of cell membrane micro-heterogeneity using the fluorescence lifetime of DPH-type fluorophores

Heterogeneity in the lipid organization in lipid bilayers and cell membranes was probed by using the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 position of phosphatidylcholine (DPH-PC). In the presence of protein, it is proposed that the bulk lipids and boundary lipids can potentially provide distinct enough fluorophore environments for two different lifetime centers to be recovered from the analysis of the fluorescence decay. To test this model experiments were performed with cytochrome b 5 in 1-palmitoyl-2-oleoylphosphatidylcholine bilayers. The number of boundary lipids of cytochrome b 2 is known from the literature or can be calculated from known dimensions, so that for a known protein:lipid ratio the fraction of lipids in the bulk and boundary lipid regions is known. These values were found to closely correspond to the fractions associated with the lifetime centers recovered from an analysis of the fluorescence decay assuming two major fluorophore populations. This indicated that the DPH distributed in a similar manner to the lipids and that its boundary lipid residency time was greater than the excited state lifetime, showing the validity of the approach. An important requirement was that the protein should influence the fluorephore decay sufficiently enough to enable separate lifetime centers for the bulk and boundary lipid fluorophores to be recovered by the analysis. Attempts were made to analyze the fluorescence decay of DPH in liver plasma membranes and microsomes as arising from two distinct fluorophore populations, however, the basic condition was not satisfied. By contrast, using DPH-PC it was possible to extract two separate lifetime centers. The limitations and potential of this approach are critically assessed and it is concluded that in certain circumstances information pertaining to the protein-lipid interfacial region of membranes can be extracted from fluorescence decay heterogeneity properties.

Direct Transfer of Membrane Proteins from B16 Melanoma Cell to Artificial Cell Liposome.

Direct transfer of membrane proteins of B16 melanoma cell to lecithin liposome has been tried in two different cell culturing methods; namely, monolayer and suspension culture systems. For this purpose an appropriate amount of an artificial boundary lipid, 1, 2-dimyristoylamido-1, 2-deoxyphosphatidylcholine (DDPC), was added to 1, 2-dimyristoylphosphatidylcholine (DMPC) liposome. An increase in the amount of DDPC to DMPC led to an increase in both the total amount and the kind of proteins transferred from the cell to the liposome. In addition, the amount and kind of proteins were differred also by altering the method of cell culture. These results indicate that the location and the mobility of the proteins in cytoplasm membrane are largely affected by the situation of the cells whether the cell adheres on a substrate or suspends in a medium.

Structural Stability of Lecithin Liposomes as Improved by Adding an Artificial Boundary Lipid

Abstract Enzymatic lyses of phosphatidylcholine liposomes by phospholipase A2 and D are significantly depressed by adding an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC). Most interestingly, DDPC was not the substrate even for phospholipase D and phospholipase A2 as well.

Effective Transfer of Membrane Proteins from Human Erythrocytes to Artificial Boundary Lipid-Containing Liposomes

Abstract Several proteins were directly and effectively transferred from human erythrocyte membranes to dimyristoylphosphatidylcholine (DMPC) liposomes which contain an artificial boundary lipid (1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine, DDPC). Of the membrane proteins, the activity of acetylcholine esterase (AchE) was monitored. The activity of AchE and the amount of total protein transferred were highest when 40 mol% of DDPC was added to DMPC.

Organic pesticides modify lipid-lipid and lipid-protein domains in model membranes. A laser Raman study

The effect of hexachlorocyclohexane (all isomers) on the thermal transition properties of phospholipid liposomes was determined by Raman spectroscopy. Raman spectra of liposomes with and without the presence of hexachlorocyclohexanes were recorded in the C-H stretching region which shows three major bands around 2850, 2880 and 2930 cm −1. Thermal transition properties were estimated from plots of I 2880 I 2850 and or I 2930 I 2850 vs. temperature, where I represents the intensity of the respective band. Our data on phospholipid liposomes reveal that δ- and γ-hexachlorocyclohexanes drastically reduce and broaden the main thermal transitions of phospholipids at toxic level concentrations. These effects are more pronounced in liposomes containing 18 or more carbon atom long acyl chains. α- and β-isomers at similar concentrations show a minimum effect on the thermal transition properties of phospholipids. Raman analysis of phospholipid liposomes containing melittin, interestingly, reveal that the δ-isomer unlike the γ-isomer strongly alters the transition properties of boundary lipids. These data suggest that the effect of hexachlorocyclohexanes on the thermal transition properties of membranes is stereo specific and that the δ-isomer preferably disrupts the lipid-protein domains. Results are explained on the basis of the dynamic flexibility owing to the equatorial and axial chlorine atoms of various hexachlorocyclohexane isomers.

Deuterium nuclear magnetic resonance studies on the interaction of glycophorin with 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine

Membrane dynamics of dimyristoylphosphatidylcholine (DMPC) lipid bilayer which contains glycophorin with an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), was investigated by 2H-NMR technique. For this purpose, both DMPC and DDPC were deuterated at the position of the 8th carbon atom of their acyl chains. Comparing, with DMPC bilayers, DDPC bilayers showed larger deuterium quadrupole splitting ( Δν Q ) by approx. 2 kHz. This was explicable in terms of the stabilization of the membrane due to the formation of a strong hydrogen bonding in bilayers. Addition of glycophorin to the DMPC or DDPC single bilayers caused an increase in the Δν Q value. The Δν Q value of DMPC/DDPC mixed lipid bilayer was smaller than that of each single lipid bilayer. DDPC in the DDPC/DMPC mixed bilayer was not phase-separated but homogeneously distributed. In glycophorin-reconstituted DMPC- d 4/DDPC mixed bilayers, the Δν Q of DMPC- d 4 was almost identical to that of the simple DMPC- d 4 bilayer. On the other hand, the Δν Q of DDPC- d 4 in the DMPC/DDPC- d 4 mixed bilayer increased significantly upon the reconstitution of glycophorin. Judging from these data, we concluded that, in the DDPC/DMPC mixed bilayer which contains glycophorin, DMPC simply behaves as the matrix lipid, while DDPC surrounds glycophorin and certainly plays a role of the boundary lipid.

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°C ( ΔH = 10.74 kJ·mol −1) and the other at 23.0°C ( ΔH = 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.

Identification of trapped and boundary lipid binding sites in M13 coat protein/lipid complexes by deuterium NMR spectroscopy

The major coat protein of M13 bacteriophage has been incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the trimethyl segments of the choline headgroup (DMPC-d9). Two-component deuterium and phosphorus-31 NMR spectra have been observed from bilayer complexes containing the coat protein, indicating slow exchange (on the deuterium quadrupole anisotropy and phosphorus-31 chemical shift averaging time scales) of lipid molecules of less than 10(3) Hz between two motionally distinct environments in the complexes. The fraction of the isotropic spectral component increases with increasing M13 protein concentration, and this component is attributed to lipid headgroups, which are disordered relative to their order in protein-free bilayers. The activation energy of the fast local motions of the trimethyl groups of the choline residue in the headgroup decreases from 23 kJ mol-1 in the pure lipid bilayers to 20 kJ mol-1 for the protein-associated lipid headgroups. The chemical exchange rate of lipid molecules between the two motionally distinct environments has been estimated to be 20-50 Hz by steady-state line-shape simulations of the deuterium spectra of DMPC-d9/M13 coat protein complexes using exchange-coupled modified Bloch equations. The off-rate was, as expected from one-to-one exchange, independent of the L/P ratio; tau off -1 = 0.23 kHz. It is suggested that the protein-associated lipid may be trapped between closely packed parallel aggregates of M13 coat protein and that the high local concentration of protein in a one-dimensional arrangement in lipid bilayers may be required for the fast reassembly of phage particles before release from an infected cell.

Lipid-Protein Interaction in Mixed Monolayers from Phospholipids and Proteins

Abstract The thermotropic behavior of different proteins with phospholipids in monolayers was investigated by measuring the surface pressure and the surface viscosity. When phospholipids, such as dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC), were mixed with the hydrophobic proteins, such as hemoglobin and β-casein, below their phase transition temperatures (Tc), a considerable expansion of monolayers was observed. On the other hand, when phospholipids were mixed with the hydrophilic proteins, such as bovine serum albumin (BSA) and lysozyme below Tc, no expansion was observed. When DMPC was mixed with different proteins at Tc or above, all mixtures formed slightly deviated monolayers from ideal mixing, independent of a variety of proteins. This type of behavior suggests that the hydrophobic proteins perturb the structure of boundary lipids near the protein molecule below Tc, while the hydrophobic proteins do not perturb the phospholipid structure below Tc because of their low hydrophobicities. At Tc or above the membrane properties are independent of a variety of proteins, because all lipid hydrocarbon chains cause a disordering, even without proteins. In addition, when DPPC was mixed with a hydrophobic protein, such as hemoglobin, the fluidity of monolayer increased significantly.

Structure-activity relationship of gramicidin S analogues on membrane permeability

The previous study of the action of gramicidin S on bacteria (Katsu, T., Kobayashi, H. and Fujita, Y. (1986) Biochim. Biophys. Acta 860, 608–619) prompted us to investigate further the structure-activity relationship of the gramicidin S analogues on membrane permeability. Two types of the gramicidin S analogues were used in the present study: (1) cyclo(- X- d-Leu- d-Lys- d-Leu- l-Pro -) 2 , where X = Gly, d-Leu and d-cyclohexylalanine ( d -c HxAla ); (2) N, N′-diacetyl derivative of gramicidin S (diacetyl-gramicidin S) which lacks a cationic moiety of gramicidin S. All the analogues have a β-sheet conformation as gramicidin S. The following cellular systems were used: Staphylococcus aureus as Gram-positive bacteria, Escherichia coli as Gram-negative bacteria, human erythrocytes, rat liver mitochondria and artificial liposomal membranes. It was found that gramicidin S and one of the type 1 analogues having X = d -c HxAla induced the efflux of K + through the cytoplasmic membrane of all types of the cells. In addition, these two peptides had the ability to lower the phase transition temperature of dipalmitoylphosphatidylcholine. Accordingly, it was concluded that, if peptides can expand greatly the membrane structure of neutral lipids which constitute main parts of the biological membrane, they can stimulate the permeability of cells without any selectivity. The action of the type 2 peptide, diacetyl-gramicidin S, was strongly cell dependent. Although this peptide stimulated the efflux of K + from mitochondria, it did not do so efficiently, if at all, from S. aureus, E. coli and erythrocytes. In experiments using liposomes, diacetyl-gramicidin S increased markedly the permeability of liposomes composed of egg phosphatidylcholine. The presence of egg phosphatidylethanolamine or cholesterol reduced its activity. These results on liposomes explained well the low sensitivity of diacetyl-gramicidin S against E. coli and erythrocytes in terms of lipid constituents of the membranes. The mechanism of action of diacetyl-gramicidin S was discussed from the formation of a boundary lipid induced by this peptide.

Circular dichroism study of bacteriorhodopsin-lipid interaction

Delipidated bacteriorhodopsin purified from purple membrane of H. halobium was reconstituted with the circular dichroism active phospholipid. The observed circular dichroism spectra in the 450-700 nm region characteristic of bacteriorhodopsin showed the temperature dependence characterized by a midpoint at ca. 45 degrees C and this spectral change showed the disaggregation of bacteriorhodopsin trimer to monomer. The circular dichroism spectra in the 250-400 nm region characteristic of the azo chromophore of phospholipid exhibited a remarkable temperature dependence synchronized with the disaggregation of bacteriorhodopsin, suggesting that a large proportion of the phospholipid is present as boundary lipid.

Lipid bilayer polypeptide interactions studied by molecular dynamics simulation.

A model membrane with a polypeptide alpha-helix inserted has been simulated by molecular dynamics at a temperature well above the gel/liquid crystalline phase transition temperature. Order parameters of the lipids and other equilibrium and dynamic quantities have been calculated. Three systems, polyglycine constrained into an alpha-helical configuration, glycophorin with similarly conformationally constrained backbone and finally glycophorin free to change its backbone conformation, have been studied. In all cases there was an ordering of the chains close to the helix. This effect was, however, much smaller for glycophorin with its rather bulky side chains than for polyglycine. The dynamics of the lipids were affected by the neighbouring helix, not drastically however. Lateral diffusion and reorientational time correlations of lipids close to the helix were slower than for the bulk ones, but not more than two or three times. Thus, we did not find any evidence of bound or frozen boundary lipids.

Effect of dietary cholesterol on microsomal membrane composition, dynamics and kinetic properties of UDPglucuronyl transferase

The effect of cholesterol administration in vivo on the lipid composition, dynamic properties of the microsomal membrane of guinea pig livers and the kinetic properties of UDPglucuronyl transferase were studied. Cholesterol administration in the diet evoked an increase of microsomal cholesterol, but no significant changes in the fatty-acid composition of total lipids or of each phospholipid class. Instead, the phosphatidylethanolamine, phosphatidylcholine molar ratio of the membrane was markedly decreased from 0.57 to 0.38. This decline was not enough to counterbalance the overall ‘ordering’ effect of cholesterol and consequently, the fluorescence anisotropy of the membranes labeled with 1,6-diphenylhexatriene was increased. The lateral diffusion evaluated by measuring the pyrene excimer formation was decreased by the cholesterol incorporation. These physical changes were associated with changes in the kinetic properties of UDPglucuronyl transferase: V max increased, while the K m of the different steps of the reaction decreased in the modified microsomes. Furthermore, a shift of the non-michaelian kinetics to michaelian, equivalent to a decrease of a negative homotropic effect and apparent cooperativity of UDPglucuronic acid was observed since the Hill coefficient changed, approaching 1. A non-michaelian kinetics of this enzyme is an indication of boundary lipids in the gel phase and a shift to michaelian, a change of the surrounding lipids to a liquid-crystalline structure. In consequence, our results suggest that cholesterol incorporation in the microsomal membrane while producing a condensing effect of bulk lipids would produce an opposite effect on the UDPglucuronyl transferase boundary lipids.

Effect of the Gel-Liquid Crystal Transition on the Lipid-Preference of Protein in Lipid Bilayer Membranes

A theoretical model of lipid bilayers including a protein is presented to make clear how far from the protein the perturbation of the conformation and packing of lipid molecules due to the protein extends and what factors control the extent. The conformation and packing of lipid molecules under the presence of inter-molecular interactions are described effectively on the basis of the isomeric bond rotation scheme for free polymer chains and of the hard disk models respectively. When the boundary lipid chains have less gauche bonds than the bulk lipids, the perturbation decays smoothly in the bilayer. The perturbation does not propagate into the bilayer, when the boundary lipids are packed more roughly than the bulk lipids. The number of gauche bonds and the number density of bulk lipid molecules are changed drastically by the gell-liquid crystal phase transition. Therefore, the phase transition affects essentially the propagation of the perturbation due to the protein.

Theoretical studies of phospholipid bilayers and monolayers. Perturbing probes, monolayer phase transitions, and computer simulations of lipid–protein bilayers

This paper describes some mathematical models for studying properties of lipid bilayer membranes. It is shown that there is evidence from fluorescent probe and electron spin resonance studies that some integral proteins are "randomly" distributed in the plane of a lipid bilayer for T greater than Tc, so that protein-protein "contacts" can occur. This implies that there is no permanent annulus of lipids around these proteins, so that there is no unique stoichiometric ratio of "boundary lipids" to protein. Computer simulation techniques are reviewed and comments are made about some recently introduced methods. Dynamical models of lipid--integral protein bilayers are outlined and it is shown that much differential scanning calorimetry, freeze-fracture, X-ray, and 2H nuclear magnetic resonance data can be understood. It is predicted that specific heat curves should show a rise at a temperature, TK less than Tc, at which a protein-rich phase starts to "melt"; that dipalmitoyl phosphatidylcholine (DPPC) hydrocarbon chains in such a protein-rich phase should exhibit some static disorder down to approximately -10 degrees C, around which they should freeze noncooperatively; and that the crossing of phase boundaries, as protein concentration expressed as mole fraction X changes just below Tc, should be reflected in a complicated dependence of protein lateral motion upon X. Models to study the liquid condensed-liquid expanded (LC-LE) transition of phosphatidylcholine (PC) monolayers at the air-water interface are described. The rounding in the LC phase of pressure-area isotherms are understood as the melting of microscopic gel-phase "domains" into macroscopic regions of fluid-phase lipids. There is some experimental support for this. Finally, to model the difference between a monolayer and the corresponding bilayer, an interaction is introduced between the two halves of a bilayer. It is shown to be very weak in the case of PC bilayers and it is outlined in the paper how monolayer and bilayer thermodynamics can be related. It was found that the internal lateral pressure of a DPPC bilayer is approximately 30 dyn/cm (1 dyn = 10 microN).

Effects of phospholipase A2 on gastric microsomal H+, K+-ATPase system: role of "boundary lipids" and the endogenous activator protein.

Pig gastric microsomal vesicles enriched in gastric H+,K+-ATPase and K+-pNPPase were digested with bee venom phospholipase A2 at 21 or 37 degrees C. The unattacked phospholipids were then related to the remaining enzyme activities, followed by reconstitution with microsomal phospholipids and the endogenous activator protein. Gastric K+-stimulated ATPase was nearly abolished within 10 min of phospholipase A2 treatment. A substantial amount of pNPPase activity remained unaffected under identical conditions. About 80% of the microsomal phosphatidylethanolamine was attacked by phospholipase A2 at both temperatures while 60 and 79% of the phosphatidylcholine was hydrolyzed at 21 and 37 degrees C, respectively. Analysis of the phospholipids revealed that phospholipase A2 attacked only the phosphatidylcholine and phosphatidylethanolamine molecules enriched in polyunsaturated fatty acids. Microsomal H+,K+-ATPase system inactivated by phospholipase A2 at 21 degrees C could be largely restored by the endogenous activator alone. On the other hand, those inactivated at 37 degrees C needed pretreatment with phosphatidylcholine before assaying with the activator protein for maximal reconstitution; phosphatidylethanolamine was totally ineffective in restoration of the enzyme activity. Analysis of the fatty acid composition of the lysophosphatidylcholine following phospholipase A2 treatment at 21 and 37 degrees C suggested involvement of some phosphatidylcholine molecules relatively enriched in saturated fatty acids and extremely poor in polyunsaturated fatty acids in gastric ATPase function. The data not only pointed out the importance of phosphatidylcholine and the endogenous activator in gastric microsomal H+,K+-ATPase reaction but also demonstrated considerable heterogeneity within the same species of microsomal phospholipids.

A spin-label and hydrogen-deuterium exchange reaction kinetics study of protein-lipid interactions in lipid-replaced Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum.

The Ca2+-ATPase of sarcoplasmic reticulum from rabbit skeletal muscle was incorporated into vesicles made from dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The Ca2+-ATPase activity of these reconstituted membranes became appreciable above 20 degrees C and 30 degrees C, respectively, in accord with the results of previous investigators. Measurement by the spin-labeling technique of the fluidity of the bulk lipid revealed the gel-to-liquid crystalline phase transition at 29 degrees C and 39 degrees C, respectively, while the fluidity of the boundary lipid in both samples was found to be low throughout the temperature range studied. The rotational mobility of the Ca2+-ATPase protein in both samples, measured by saturation transfer electron spin resonance, was also very low throughout the temperature range studied and its temperature-dependence did not show any break or jump corresponding to the phase transition of the bulk lipid. On the other hand, the structural fluctuation of the Ca2+-ATPase protein in dimyristoylphosphatidylcholine-recombinant, measured in terms of hydrogen-deuterium exchange reaction kinetics, showed a jump at about 27 degrees C, apparently in accordance with the phase transition of the bulk lipid. Results obtained in this study suggested that the Ca2+-ATPase protein molecules are in an aggregated state in these reconstituted membranes and that the Ca2+-ATPase activity is neither directly correlated to the fluidity of the boundary lipid nor to the rotational mobility of the Ca2+-ATPase, contrary to the suggestions of previous investigators (Hesketh et al. (1976) Biochemistry 15, 4145-4151; Hidalgo et al. (1978) J. Biol. Chem. 253, 6879-6887).