Calorimetric and Fourier transform infrared spectroscopic studies on the interaction of glycophorin with phosphatidylserine/dipalmitoylphosphatidylcholine-d62 mixtures

Glycophorin has been isolated in pure form from human erythrocyte membranes and reconstituted into lipid vesicles composed of binary mixtures of bovine brain phosphatidylserine (PS) and acyl-chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62). The effect of protein on lipid melting behavior and order has been monitored with differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR). The phase diagram for PS/DPPC-d62 is consistent with that previously reported for PS/DPPC (Stewart et al. (1979) Biochim. Biophys. Acta 556, 1-16) and indicates that acyl chain perdeuteration does not greatly alter the lipid mixing characteristics. The use of deuterated lipid allows the examination of lipid order by FT-IR of each lipid component in the binary mixtures as well as in the ternary (lipid/lipid/protein) systems. Addition of glycophorin to a 30:70 PS/DPPC-d62 binary lipid mixture results in a preferential glycophorin/PS interaction leading to bulk lipid enriched in DPPC-d62. This is revealed in two ways: first, through cooperative calorimetric transitions increased in temperature from the binary lipid system and second, through FT-IR melting curves of the DPPC-d62 component which shows transitions increased in both onset and completion temperatures in the presence of protein. In addition, non-cooperative melting events are observed at temperatures below the onset of phase separation. The FT-IR data are used to assign these non-cooperative events to the melting of the PS component. For the 50:50 lipid mixture with protein, two transitions are observed in the DSC experiments. The IR results indicate that both lipid components are involved with the lower temperature event.     

Comparison of DPPC and DPPG environments in pulmonary surfactant models

Deuterium nuclear magnetic resonance was used to monitor lipid acyl-chain orientational order in suspensions of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) containing Ca(2+) and the lung surfactant proteins SP-A and SP-B separately and together. To distinguish between protein-lipid interactions involving the PC and PG lipid headgroups and to examine whether such interactions might influence spatial distribution of lipids within the bilayer, acyl chains on either the DPPC or the DPPG component of the mixture were deuterated. The lipid components of the resulting mixtures were thus either DPPC-d(62)/DPPG (7:3) or DPPC/DPPG-d(62) (7:3), respectively. SP-A had little effect on DPPC-d(62) chain order but did narrow the temperature range over which DPPG-d(62) ordered at the liquid-crystal-to-gel transition. No segregation of lipid components was seen for temperatures above or below the transition. Near the transition, though, there was evidence that SP-A promoted preferential depletion of DPPG from liquid crystalline domains in the temperature range over which gel and liquid crystal domains coexist. SP-B lowered average chain order of both lipids both above and below the main transition. The perturbations of chain order by SP-A and SP-B together were smaller than by SP-B alone. This reduction in perturbation of the lipids by the additional presence of SP-A likely indicated a strong interaction between SP-A and SP-B. The competitive lipid-lipid, lipid-protein, and protein-protein interactions suggested by these observations presumably facilitate the reorganization of surfactant material inherent in the transformation from lamellar bodies to a functional surfactant layer.     

Effect of hydrophobic surfactant proteins SP-B and SP-C on binary phospholipid monolayers: II. Infrared external reflectance-absorption spectroscopy

In situ external reflection infrared spectroscopy at the air-water interface was used to study the influence on phospholipid structure of an endogenous mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, which are thought to play pivotal roles in the adsorption and function of pulmonary surfactant. Mixtures studied were 1:1, 2:1, and 7:1 (mol:mol) DPPC-d(62):DPPG, and 7:1 DPPC-d(62):DOPG, alone and in the presence of 0.5-10 wt % mixed SP-B/C purified chromatographically from calf lung surfactant extract. Perdeuteration of DPPC produced a shift in vibrational frequencies so that it could be differentiated spectroscopically from the phosphoglycerol component in the surface monolayer. CH(2) antisymmetric and symmetric stretching bands ( approximately 2920 and 2852 cm(-1)) along with the analogous CD(2) stretching bands ( approximately 2194 and 2089 cm(-1)) were analyzed, and band heights and peak wavenumber positions were assessed as a function of monolayer surface pressure. Small, near-physiological contents of 1-2 wt % SP-B/C typically produced the maximum observed spectroscopic effects, which were abolished at high protein contents of 10 wt %. Analysis of CH(2) and CD(2) stretching bands and C-H/C-D band height ratios indicated that SP-B/C affected PC and PG lipids differently within the surface monolayer. SP-B/C had preferential interactions with DPPG in 1:1, 2:1, and 7:1 DPPC-d(62):DPPG films that increased its acyl chain order. SP-B/C also interacted specifically with DOPG in 7:1 DPPC-d(62):DOPG monolayers, but in this case an increase in CH(2) band heights and peak wavenumber positions indicated a further disordering of the already fluid DOPG acyl chains. CD(2) band height and peak wavenumber analysis indicated that SP-B/C had no significant effect on the structure of DPPC-d(62) chains in 7:1 films with DPPG or DOPG, and had only a slight tendency to increase the acyl chain order in 1:1 films of DPPC-d(62):DPPG. SP-B/C had no significant effect on DPPC-d(62) structure in films with DOPG. Infrared results also indicated that interactions involving SP-B/C and lipids led to exclusion of PC and PG lipids from the compressed interfacial monolayer, in agreement with our previous report on the phase morphology of lipid monolayers containing 1 wt % SP-B/C.     

Fourier-transform infrared studies of CaATPase partitioning in phospholipid mixtures of 1,2-dipalmitoylphosphatidylcholine-d62 with 1-palmitoyl-2-oleoylphosphatidylethanolamine and 1-stearoyl-2-oleoylphosphatidylcholine

CaATPase from rabbit sarcoplasmic reticulum has been reconstituted into binary lipid mixtures of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/1,2-dipalmitoylphosphatidylcholine-d62 (DPPC-d62) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC)/DPPC-d62. Fourier-transform infrared (FT-IR) spectroscopy has been used to monitor temperature-induced structural alterations in the individual lipid components in the presence and absence of protein. A simple two-state model is used to construct a phase diagram that is in good agreement with one constructed from differential scanning calorimetry data, for the POPE/DPPC-d62 (protein-free) system. Although these two lipids are miscible over at least most of the composition range, substantial deviations from ideal behavior are observed. An estimate of the nonideality of mixing in both the gel and liquid-crystalline phases is obtained from regular solution theory. The phase diagram for SOPC/DPPC-d62 shows gel-phase immiscibility. FT-IR studies of ternary (POPE/DPPC-d62/CaATPase) complexes indicate that both lipid components are disordered by protein at all temperatures studied. In addition, their melting events are broadened and shifted to lower temperatures compared with the appropriate binary lipid mixture. Semiquantitative estimates for the fraction of each lipid melted are obtained from the model. The effect of protein on SOPC/DPPC-d62 mixtures depends on that total lipid to protein ratio. At low protein levels, SOPC is preferentially selected by CaATPase, so that bulk lipid is enriched in DPPC-d62. At high levels of protein, both lipid components are selected. The applicability of vibrational spectroscopy for determination of the partitioning preferences of membrane proteins into regions of particular chemical structure or physical order in a complex lipid environment is demonstrated.     

Effects of cholesterol on the interaction of Ca2(+)-ATPase with 1-palmitoyl-2-oleoylphosphatidylethanolamine. An FTIR study

Ca2(+)-ATPase from rabbit skeletal muscle has been isolated, purified, and reconstituted into vesicles containing binary mixtures of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/cholesterol. Fourier transform infrared spectroscopy (FTIR) was used to investigate the effect of protein on the thermotropic behavior of POPE in these reconstituted ternary complexes. The CH2 symmetric stretching modes of the phospholipid acyl chains near 2850 cm-1 served as an index of the melting process. The thermotropic transition of the POPE component in a 103:12:1 (POPE/cholesterol/Ca2(+)-ATPase) complex was shifted to lower temperatures compared with a protein-free binary lipid mixture of the same relative proportions. When combined with differential scanning calorimetric (DSC) data for the binary (POPE/cholesterol) lipid systems, this observation suggests that Ca2(+)-ATPase preferentially sequesters 15-35 molecules of POPE from the lipid mixture and therefore excludes cholesterol from its immediate environment. Higher levels of cholesterol in ternary complexes progressively eliminate the cooperative POPE melting event.     

A comparison of differential scanning calorimetric and Fourier transform infrared spectroscopic determination of mixing behavior in binary phospholipid systems

Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry (DSC) have been used to elucidate the phase behavior of two binary lipid mixtures, acyl chain perdeuterated 1,2-dipalmitoylphosphatidylethanolamine (DPPE-d62)/1,2-dielaidoylphosphatidylcholine (DEPC) and acyl chain perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d62)/1,2-dimyristoylphosphatidylethanolamine (DMPE). The former shows gel state immiscibility over most of the composition range. The FT-IR data indicate that one of the solid phases is essentially pure DEPC, while the other solid phase contains both lipids. The DPPC-d62/DMPE pair are miscible over the entire composition range. The use of deuterated lipids as one component in the mixture permits the melting characteristics of each component to be separately determined in the FT-IR experiment. The FT-IR data are used to assign the endotherms observed in the DSC to particular molecular components. For the DPPE-d62/DEPC system, two endotherms are observed at compositions between 10 and 67 mol% DPPE-d62. The lower transition is assigned to the DEPC component, while the higher event contains contributions to the enthalpy from both lipids in the mixture. The midpoint of the DEPC melting occurs substantially below that for DPPE-d62. For the miscible pair, each of the lipids melt over approximately the same temperature range. The complementary and consistent nature of the information available from FT-IR and from DSC is demonstrated from the current work.     

Probing perturbation of bovine lung surfactant extracts by albumin using DSC and 2H-NMR

Lung surfactant (LS), a lipid-protein mixture, forms films at the lung air-water interface and prevents alveolar collapse at end expiration. In lung disease and injury, the surface activity of LS is inhibited by leakage of serum proteins such as albumin into the alveolar hypophase. Multilamellar vesicular dispersions of a clinically used replacement, bovine lipid extract surfactant (BLES), to which (2% by weight) chain-perdeuterated dipalmitoylphosphatidycholine (DPPG mixtures-d(62)) had been added, were studied using deuterium-NMR spectroscopy ((2)H-NMR) and differential scanning calorimetry (DSC). DSC scans of BLES showed a broad gel to liquid-crystalline phase transition between 10-35 degrees C, with a temperature of maximum heat flow (T(max)) around 27 degrees C. Incorporation of the DPPC-d(62) into BLES-reconstituted vesicles did not alter the T(max) or the transition range as observed by DSC or the hydrocarbon stretching modes of the lipids observed using infrared spectroscopy. Transition enthalpy change and (2)H-NMR order parameter profiles were not significantly altered by addition of calcium and cholesterol to BLES. (2)H-NMR spectra of the DPPC-d(62) probes in these samples were characteristic of a single average lipid environment at all temperatures. This suggested either continuous ordering of the bilayer through the transition during cooling or averaging of the DPPC-d(62) environment by rapid diffusion between small domains on a short timescale relative to that characteristic of the (2)H-NMR experiment. Addition of 10% by weight of soluble bovine serum albumin (1:0.1, BLES/albumin, dry wt/wt) broadened the transition slightly and resulted in the superposition of (2)H-NMR spectral features characteristic of coexisting fluid and ordered phases. This suggests the persistence of phase-separated domains throughout the transition regime (5-35 degrees C) of BLES with albumin. The study suggests albumin can cause segregation of protein bound-lipid domains in surfactant at NMR timescales (10(-5) s). Persistent phase separation at physiological temperature may provide for a basis for loss of surface activity of surfactant in dysfunction and disease.     

Fourier transform infrared studies of secondary structure and orientation of pulmonary surfactant SP-C and its effect on the dynamic surface properties of phospholipids

SP-C, a highly hydrophobic, 3.7-kDa protein constituent of lung surfactant, has been isolated from bovine lung lavage, purified, and reconstituted into binary lipid mixtures of 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylglycerol (DPPG). Fourier transform infrared (FT-IR) spectroscopy has been applied to examine SP-C secondary structure, the average orientation of alpha-helical segments relative to the bilayer normal in membrane films, and the effect of protein on the thermotropic properties of the phospholipid acyl chains. In addition, dynamic surface measurements were made on phospholipid films at the A/W interface in the presence and absence of SP-C. SP-C (0.5 mol %) was found to possess about 60% alpha-helical secondary structure in lipid vesicles. Higher levels (1.5 mol %) of SP-C resulted in a slight increase of beta-forms, possibly resulting from protein aggregation. The helical segments exhibited an average angle of orientation of about 24 degrees with respect to the bilayer normal, suggesting a trans-bilayer orientation of the peptide. The observation that 70% of the peptide bond hydrogens are hard to exchange in D2O further reflects the hydrophobic nature of the molecule. SP-C produced little effect on the thermotropic properties of the binary lipid mixture, as measured from acyl chain C-H and C-D stretching frequencies. However, the presence of 1 mol % protein markedly reduced the viscance and increased the elasticity of surface films suggesting a mechanism by which SP-C facilitates the spreading of phospholipids on an aqueous surface. The possible physiological consequences of these observations are discussed.     

Thermal stability and DPPC/Ca2+ interactions of pulmonary surfactant SP-A from bulk-phase and monolayer IR spectroscopy.

Surfactant protein A (SP-A), the most abundant pulmonary surfactant protein, is implicated in multiple biological functions including surfactant homeostasis, biophysical activity, and host defense. SP-A forms ternary complexes with lipids and Ca2+ which are important for protein function. The current study uses infrared (IR) transmission spectroscopy to investigate the bulk-phase interaction between SP-A, 1,2-dipalmitoylphosphatidylcholine (DPPC), and Ca2+ ions along with IR reflection-absorption spectroscopy (IRRAS) to examine protein secondary structure and lipid orientational order in monolayer films in situ at the air/water interface. The amide I contour of SP-A reveals two features at 1653 and 1636 cm(-1) arising from the collagen-like domain and a broad feature at 1645 cm(-1) suggested to arise from the carbohydrate recognition domain (CRD). SP-A secondary structure is unchanged in lipid monolayers. Thermal denaturation of SP-A in the presence of either DPPC or Ca2+ ion reveals a sequence of events involving the initial melting of the collagen-like region, followed by formation of intermolecular extended forms. Interestingly, these spectral changes were inhibited in the ternary system, showing that the combined presence of both DPPC and Ca2+ confers a remarkable thermal stability upon SP-A. The ternary interaction was revealed by the enhanced intensity of the asymmetric carboxylate stretching vibration. The IRRAS measurements indicated that incorporation of SP-A into preformed DPPC monolayers at a surface pressure of 10 mN/m induced a decrease in the average acyl chain tilt angle from 35 degrees to 28 degrees. In contrast, little change in chain tilt was observed at surface pressures of 25 or 40 mN/m. These results are consistent with and extend the fluorescence microscopy studies of Keough and co-workers [Ruano, M. L. F., et al. (1998) Biophys. J. 74, 1101-1109] in which SP-A was suggested to accumulate at the liquid-expanded/liquid-condensed boundary. Overall these experiments reveal the remarkable stability of SP-A in diverse, biologically relevant environments.     

Intermolecular cross-links mediate aggregation of phospholipid vesicles by pulmonary surfactant protein SP-A

As the most abundant glycoprotein component of pulmonary surfactant, SP-A (Mr = 30,000-36,000) plays a central role in the organization of phospholipid bilayers in the alveolar air space. SP-A, isolated from lung lavage, exists in oligomeric forms (N = 6, 12, 18, ...), mediated by collagen-like triple helices and intermolecular disulfide bonds. These protein-protein interactions, involving the amino-terminal domain of SP-A, are hypothesized to facilitate the alignment of surfactant lipid bilayers into unique tubular myelin structures. SP-A reorganization of surfactant lipid was assessed in vitro by quantitating the calcium-dependent light scattering properties of lipid vesicle suspensions induced by SP-A. Accelerated aggregation of unilamellar vesicles required SP-A and at least 3 mM free calcium. The initial rate of aggregation was proportional to the concentration of canine SP-A over lipid:protein molar ratios ranging from 200:1 to 5000:1. Digestion with bacterial collagenase or incubation with dithiothreitol (DTT) completely blocked lipid aggregation activity. Both treatments decreased the binding of SP-A to phospholipids. The conditions used in the DTT experiments (10 mM DTT, nondenaturing Tris buffer, 37 degrees C) resulted in the selective reduction and 14C-alkylation of the intermolecular disulfide bond involving residue 9Cys, whereas the four cysteines found in the noncollagenous domain of SP-A were inefficiently alkylated with [14C]-iodoacetate. HPLC analysis of tryptic SP-A peptides revealed that these four cysteine residues participate in intramolecular disulfide bond formation (138Cys-229Cys and 207Cys-221Cys). Our data demonstrate the importance of the quaternary structure (triple helix and intermolecular disulfide bond) of SP-A for the aggregation of unilamellar phospholipid vesicles.     

Physiological and inflammatory response to instillation of an oxidized surfactant in a rat model of surfactant deficiency.

Pulmonary surfactant is a mixture of phospholipids ( approximately 90%) and surfactant-associated proteins (SPs) ( approximately 10%) that stabilize the lung by reducing the surface tension. One proposed mechanism by which surfactant is altered during acute lung injury is via direct oxidative damage to surfactant. In vitro studies have revealed that the surface activity of oxidized surfactant was impaired and that this effect could be overcome by adding SP-A. On the basis of this information, we hypothesized that animals receiving oxidized surfactant preparations would exhibit an inferior physiological and inflammatory response and that the addition of SP-A to the oxidized preparations would ameliorate this response. To test this hypothesis, mechanically ventilated, surfactant-deficient rats were administered either bovine lipid extract surfactant (BLES) or in vitro oxidized BLES of three doses: 10 mg/kg, 50 mg/kg, or 10 mg/kg + SP-A. When instilled with 10 mg/kg normal surfactant, the rats had a significantly superior arterial Po2 responses compared with the rats receiving oxidized surfactant. Interestingly, increasing the dose five times mitigated this physiological effect, and the addition of SP-A to the surfactant preparation had little impact on improving oxygenation. There were no differences in alveolar surfactant pools and the indexes of pulmonary inflammation between the 10 mg/kg dose groups, nor was there any differences observed between either of the groups supplemented with SP-A. However, there was significantly more surfactant and more inflammatory cytokines in the 50 mg/kg oxidized BLES group compared with the 50 mg/kg BLES group. We conclude that instillation of an in vitro oxidized surfactant causes an inferior physiological response in a surfactant-deficient rat.     

Effect of surfactant-associated protein-A (SP-A) on the activity of lipid extract surfactant

The properties of natural bovine surfactant and its lipid extract have been examined with a pulsating bubble surfactometer which assesses the ability of surfactant lipids to adsorb to the air/liquid interface and reduce the surface tension to near 0 dynes/cm during dynamic compression. Studies conducted at 1 mg/ml phospholipid revealed that the surface activity (i.e., the ability to produce low surface tensions) of lipid extracts could be enhanced by incubating the sample at 37 degrees C for 120 min or by addition of CaCl2. In contrast, incubation at 37 degrees C only slightly improved the biophysical activity of natural surfactant and the addition of CaCl2 had a more modest effect than with lipid extracts. With 20 mM CaCl2, the surfactant activity of lipid extract surfactant was similar to that of natural surfactant. Incubation with EDTA reduced the biophysical activity of natural surfactant. Experiments in which increasing amounts of lipid extract were replaced by natural surfactant revealed that small amounts of natural surfactant enhanced the surfactant activity of lipid extract. The biophysical activity of lipid extract surfactant was also increased by the addition of soluble surfactant-associated protein-A (SP-A) (28-36 kDa) purified from natural bovine surfactant. These results indicate that SP-A (28-36 kDa) improves the surfactant activity of lipid extracts by enhancing the rate of adsorption and/or spreading of phospholipid at the air/liquid interface resulting in the formation of a stable lipid monolayer at lower bulk concentrations of either phospholipid or calcium.     

Effects of palmitoylation on dynamics and phospholipid-bilayer-perturbing properties of the N-terminal segment of pulmonary surfactant protein SP-C as shown by 2H-NMR

It has been proposed that palmitoylation of the N-terminal segment of surfactant protein SP-C is important for maintaining association of pulmonary surfactant complexes with interfacial films compressed to high pressures at the end of expiration. In this study, we examined surfactant membrane models containing palmitoylated and nonpalmitoylated synthetic peptides, based on the N-terminal SP-C sequence, in dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (7:3, w/w) by ²H-NMR. Perturbations of lipid properties by the peptide versions were compared in samples containing chain- and headgroup-deuterated lipid (DPPC-d₆₂ and DPPC-d₄ respectively). Also, deuterated peptide palmitate chains were compared with those of DPPC in otherwise identical lipid-protein mixtures. Palmitoylated peptide increased average DPPC-d₆₂ chain orientational order slightly, particularly for temperatures spanning gel and liquid crystalline coexistence, implying penetration of palmitoylated peptide into ordered membrane. In contrast, the nonpalmitoylated peptide had a small disordering effect in this temperature range. Both peptide versions perturbed DPPC-d₄ headgroup orientation similarly, suggesting little effect of palmitoylation on the largely electrostatic peptide-headgroup interaction. Deuterated acyl chains attached to the SP-C N-terminal segment displayed a qualitatively different distribution of chain order, and lower average order, than DPPC-d₆₂ in the same membranes. This likely reflects local perturbation of lipid headgroup spacing by the peptide portion interacting with the bilayer near the peptide palmitate chains. This study suggests that SP-C-attached acyl chains could be important for coupling of lipid and protein motions in surfactant bilayers and monolayers, especially in the context of ordered phospholipid structures such as those potentially formed during exhalation, when stabilization of the respiratory surface by surfactant is the most crucial.     

Domains of surfactant protein A that affect protein oligomerization, lipid structure and surface tension

Surfactant protein A (SP-A) is an abundant protein found in pulmonary surfactant which has been reported to have multiple functions. In this review, we focus on the structural importance of each domain of SP-A in the functions of protein oligomerization, the structural organization of lipids and the surface-active properties of surfactant, with an emphasis on ultrastructural analyses. The N-terminal domain of SP-A is required for disulfide-dependent protein oligomerization, and for binding and aggregation of phospholipids, but there is no evidence that this domain directly interacts with lipid membranes. The collagen-like domain is important for the stability and oligomerization of SP-A. It also contributes shape and dimension to the molecule, and appears to determine membrane spacing in lipid aggregates such as common myelin and tubular myelin. The neck domain of SP-A is primarily involved in protein trimerization, which is critical for many protein functions, but it does not appear to be directly involved in lipid interactions. The globular C-terminal domain of SP-A clearly plays a central role in lipid binding, and in more complex functions such as the formation and/or stabilization of curved membranes. In recent work, we have determined that the maintenance of low surface tension of surfactant in the presence of serum protein inhibitors requires cooperative interactions between the C-terminal and N-terminal domains of the molecule. This effect of SP-A requires a high degree of oligomeric assembly of the protein, and may be mediated by the activity of the protein to alter the form or physical state of surfactant lipid aggregates.     

Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant

Pulmonary surfactant forms a surface film that consists of a monolayer and a monolayer-associated reservoir. The extent to which surfactant components including the main component, dipalmitoylphosphatidylcholine (DPPC), are adsorbed into the monolayer, and how surfactant protein SP-A affects their adsorptions, is not clear. Transport of cholesterol to the surface region from dispersions of bovine lipid extract surfactant [BLES(chol)] with or without SP-A at 37 degrees C was studied by measuring surface radioactivities of [4-(14)C]cholesterol-labeled BLES(chol), and the Wilhelmy plate technique was used to monitor adsorption of monolayers. Results showed that transport of cholesterol was lipid concentration dependent. SP-A accelerated lipid adsorption but suppressed the final level of cholesterol in the surface. Surfactant adsorbed from a dispersion with or without SP-A was transferred via a wet filter paper to a clean surface, where the surface radioactivity and surface tension were recorded simultaneously. It was observed that 1) surface radioactivity was constant over a range of dispersion concentrations; 2) cholesterol and DPPC were transferred simultaneously; and 3) SP-A limited transfer of cholesterol.These results indicate that non-DPPC components of pulmonary surfactant can be adsorbed into the monolayer. Studies in the transfer of [1-(14)C]DPPC-labeled BLES(chol) to an equal or larger clean surface area revealed that SP-A did not increase selective adsorption of DPPC into the monolayer. Evaluation of transferred surfactant with a surface balance indicated that it equilibrated as a monolayer. Furthermore, examination of transferred surfactants from dispersions with and without prespread BLES(chol) monolayers revealed a functional contiguous association between adsorbed monolayers and reservoirs.     

Enhancement of biophysical activity of lung surfactant extracts and phospholipid-apoprotein mixtures by surfactant protein A

The effects of surfactant protein (SP)-A on the dynamic surface tension lowering and resistance to inhibition of dispersions of calf lung surfactant extract (CLSE) and mixtures of synthetic phospholipids combined with SP-B,C hydrophobic apoproteins were studied at 37 degrees C and rapid cycling rate (20 cycles/min). Addition of SP-A to CLSE, which already contains SP-B and -C, gave a slight improvement in the time course of surface tension lowering on an oscillating bubble apparatus in the absence of inhibitory protein molecules such as albumin or hemoglobin. However, when these proteins were present at concentrations of 10-50 mg/ml, SP-A substantially improved the resistance of CLSE to their inhibitory effects. The beneficial effect of SP-A required the presence of Ca2+ ions, and disappeared when EDTA was substituted for this divalent cation in the subphase. The effect was also retained when SP-A was heated to 50 degrees C prior to addition to CLSE, but was abolished by heating SP-A to 99 degrees C. Additional studies showed that similar improvements in resistance to inhibition were found when SP-A was added to synthetic mixtures of dipalmitoyl phosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (80:20 by weight) reconstituted with 1% SP-B or SP-B and -C, but not to phospholipid mixtures containing only SP-C. The requirements for SP-B and calcium for the beneficial effects of SP-A on surface activity suggest that the formation of ordered, larger phospholipid-apoprotein aggregates may be involved in the process. The finding that SP-A enhances the ability of CLSE and other surfactant mixtures containing SP-B to resist inhibition is an advantage that will need to be weighed against other factors such as increased antigenicity and heat sensitivity in therapeutic applications in surfactant replacement therapy.     

Effect of calcium on phospholipid interaction with pulmonary surfactant protein C.

Porcine pulmonary surfactant-associated protein SP-C was incorporated into bilayers of chain-perdeuterated dipalmitoylphosphatidylglycerol (DPPG-d62) and chain-perdeuterated dipalmitoyl-phosphatidylcholine (DPPC-d62) and into bilayers containing 70 mol% dipalmitoyl-phosphatidylcholine (DPPC) and 30 mol% DPPG-d62 or 70 mol% DPPC-d62 and 30 mol% dipalmitoylphosphatidylglycerol (DPPG). The effect of SP-C on the phase behavior, lipid chain order, and dynamics in these bilayers was examined by using deuterium nuclear magnetic resonance. SP-C was found to have a similar effect on the chain order and phase behavior of DPPC-d62 and DPPG-d62 in bilayers with a single lipid component. In gel phase DPPC/DPPG (7:3) bilayers with one or the other lipid component chain-perdeuterated, SP-C was found to affect first spectral moment more strongly for DPPG-d62 than for DPPC-d62. This may indicate that SP-C induced a nonrandom lateral distribution in the mixed lipid bilayer. SP-C was also found to influence motions responsible for deuteron transverse relaxation in both the gel and liquid crystalline phases. The presence of 5 mM Ca2+ in the aqueous phase substantially altered the effect of SP-C on transverse relaxation in the bilayer.     

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)     

The effect of hydrophobic analogues of the type I winter flounder antifreeze protein on lipid bilayers

The effect of four synthetic analogues of the 37-residue winter flounder type I antifreeze protein (AFP), which contain four Val, Ala or Ile residues in place of Thr residues at positions 2, 13, 24 and 37 and two additional salt bridges, on the binary lipid system prepared from a 1:1 mixture of the highly unsaturated DGDG and saturated DMPC has been determined using FTIR spectroscopy. In contrast to the natural protein, which increases the thermotropic phase transition, the Thr, Val and Ala analogues decreased the thermotropic phase transitions of the liposomes by 2.2 degrees Celsius, 3.4 degrees Celsius and 2.4 degrees Celsius, while the Ile analogue had no effect on the transition. Experiments performed using perdeuterated DMPC showed that the Ala and Thr peptides interacted preferentially with the DGDG in the lipid mixture, while the Val peptide showed no preference for either lipid. The results are consistent with interactions involving the hydrophobic face of type I AFPs and model bilayers, i.e. the same face of the protein that is responsible for antifreeze properties. The different effects correlate with the helicity of the peptides and suggest that the solution conformation of the peptides has a significant role in determining the effects of the peptides on thermotropic membrane phase transitions.