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J Pérez-Gil

Publications and source records attributed to J Pérez-Gil.

At least 19 recordsLinked to original sources
PubMed2003

Potassium efflux induced by a new lactoferrin-derived peptide mimicking the effect of native human lactoferrin on the bacterial cytoplasmic membrane.

A 31-amino acid synthetic peptide (NH(2)-FFSASCVPGADKGQFPNLCRLCAGTGENKCA-COOH) was chemically synthesized based on the amino acid sequence of a region of human lactoferrin homologous to other sequences present in the N- and C-lobes of all members of the transferrin family proteins. The peptide, termed kaliocin-1, and lactoferrin showed a bactericidal effect in assays performed in low-ionic-strength conditions. This is the first time that it is shown that the antimicrobial effect of lactoferrin depends on the extracellular cation concentration. The antimicrobial effect of kaliocin-1 was lower than that of human lactoferrin, but their activities were inhibited by Na(+) or K(+) in a cation concentration-dependent manner. In addition, the peptide was able to mimic native lactoferrin, inducing K(+)-efflux and a selective dissipation of the transmembrane electrical potential of Escherichia coli cells without causing extensive damage to the outer and inner bacterial membranes. In contrast, the peptide, but not lactoferrin, was able to permeabilize different ions through liposomal membranes. The hypothetical interaction of kaliocin-1 with a bacterial membrane compound is discussed based in the different ion flux induced on cellular and artificial membranes as well as data from circular dichroism assays. Kaliocin-1 was not cytotoxic and could be a suitable model for the design of analogs able to mimic the antibacterial effect of human lactoferrin.

Amino Acid Sequence↗
PubMed2001

Superficial disposition of the N-terminal region of the surfactant protein SP-C and the absence of specific SP-B-SP-C interactions in phospholipid bilayers.

A dansylated form of porcine surfactant-associated protein C (Dns-SP-C), bearing a single dansyl group at its N-terminal end, has been used to characterize the lipid-protein and protein-protein interactions of SP-C reconstituted in phospholipid bilayers, using fluorescence spectroscopy. The fluorescence emission spectrum of Dns-SP-C in phospholipid bilayers is similar to the spectrum of dansyl-phosphatidylethanolamine, and indicates that the N-terminal end of the protein is located at the surface of the membranes and is exposed to the aqueous environment. In membranes containing phosphatidylglycerol (PG), the fluorescence of Dns-SP-C shows a 3-fold increase with respect to the fluorescence of phosphatidylcholine (PC), suggesting that electrostatic lipid-protein interactions induce important effects on the structure and disposition of the N-terminal segment of the protein in these membranes. This effect saturates above 20% PG molar content in the bilayers. The parameters for the interaction of Dns-SP-C with PC or PG have been estimated from the changes induced in the fluorescence emission spectrum of the protein. The protein had similar K(d) values for its interaction with the different phospholipids tested, of the order of a few micromolar. Cooling of Dns-SP-C-containing dipalmitoyl PC bilayers to temperatures below the phase transition of the phospholipid produced a progressive blue-shift of the fluorescence emission of the protein. This effect is interpreted as a consequence of the transfer of the N-terminal segment of the protein into less polar environments that originate during protein lateral segregation. This suggests that conformation and interactions of the N-terminal segment of SP-C could be important in regulating the lateral distribution of the protein in surfactant bilayers and monolayers. Potential SP-B-SP-C interactions have been explored by analysing fluorescence resonance energy transfer (RET) from the single tryptophan in porcine SP-B to dansyl in Dns-SP-C. RET has been detected in samples where native SP-B and Dns-SP-C were concurrently reconstituted in PC or PG bilayers. However, the analysis of the dependence of RET on the protein density excluded specific SP-B-Dns-SP-C associations.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Selective labeling of pulmonary surfactant protein SP-C in organic solution.

Pulmonary surfactant protein SP-C has been isolated from porcine lungs and treated with dansyl isothiocyanate in chloroform:methanol 2:1 (v/v) solutions,under conditions optimized to introduce a single dansyl group covalently attached to the N-terminalamine group of the protein without loss of its native thioesther-linked palmitic chains. The resulting derivative Dans-SP-C conserves the secondary structure of native SP-C as well as the ability to promote interfacial adsorption of DPPC suspensions and to affect the thermotropic behavior of DPPC bilayers. This derivative can be used to characterize lipid-protein and protein-protein interactions of a native-like SP-C in lipid/protein complexes.

Animals↗
PubMed2001

Quantitation of pulmonary surfactant protein SP-B in the absence or presence of phospholipids by enzyme-linked immunosorbent assay.

We have developed an enzyme-linked immunosorbent assay (ELISA) that uses polyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitate purified SP-B in chloroform/methanol and in chloroform/methanol extracts of whole pulmonary surfactant at nanogram levels. This method has been used to explore the effect of the presence of different phospholipids on the immunoreactivity of SP-B. Both polyclonal and monoclonal antibodies produced reproducible ELISA calibration curves for methanolic SP-B solutions with protein concentrations in the range of 20-1000 ng/mL. At these protein concentrations, neither dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, nor phosphatidylcholine or phosphatidylglycerol from egg yolk had significant effects on the binding of antibodies to SP-B up to protein-to-lipid weight ratios of 1:20. Coating of ELISA plates with SP-B concentrations higher than 1 microg/mL produced a substantial decrease in the binding of antibodies to the protein that was prevented by the presence of negatively charged but not zwitterionic phospholipids. Characterization of the secondary structure of SP-B by far-UV circular dichroism showed that phospholipids induced pronounced changes on the conformation of SP-B when the solvent was evaporated and dry lipid-protein films were formed, a necessary step to expose protein to antibodies in ELISA. Under these conditions, negatively charged lipids, but not zwitterionic ones, induced a marked decrease on the ellipticity of SP-B that would be associated with a conformation that is significantly more exposed to antibodies.

Animals↗
PubMed2001

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

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

Animals↗
PubMed2001

Intrinsic structural differences in the N-terminal segment of pulmonary surfactant protein SP-C from different species.

Predictive studies suggest that the known sequences of the N-terminal segment of surfactant protein SP-C from animal species have an intrinsic tendency to form beta-turns, but there are important differences on the probable location of these motifs in different SP-C species. Our hypothesis is that intrinsic structural determinants of the sequence of the N-terminal region of SP-C could define conformation, acylation and perhaps surface properties of the mature protein. To test this hypothesis we have synthesized peptides corresponding to the 13-residue N-terminal sequence of porcine and canine SP-C, and studied their structural behaviour in solution and in phospholipid bilayers and monolayers. In these peptides, leucine at position 1 of both sequences has been replaced by tryptophan in order to allow their study by fluorescence spectroscopy. Far-u.v. circular dichroism spectra of the peptides in aqueous and organic solutions and in phospholipid micelles or vesicles are consistent with predicted conformational differences between the porcine and the canine sequences. Both families of peptides showed changes in their fluorescence emission spectra in the presence of phospholipids that were consistent with spontaneous lipid/peptide interactions. Both canine and porcine peptides were able to form monolayers at air-liquid interfaces, the canine peptides occupying lower area/molecule and being compressible to higher pressures than the porcine sequences. The peptides also shifted the isotherms and perturbed the packing of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers, the effects being always higher in anionic than in zwitterionic lipids, and also substantially higher in films containing canine peptide in comparison to porcine peptide. Acylation of cysteines at the N-terminal end of SP-C may modulate these intrinsic conformational features and the changes induced could be important for the development of its surface activity.

Amino Acid Sequence↗
PubMed2000

Self-aggregation of surfactant protein A.

Environmental factors of physiological relevance such as pH, calcium, ionic strength, and temperature can affect the state of self-aggregation of surfactant protein A (SP-A). We have studied the secondary structure of different SP-A aggregates and analyzed their fluorescence characteristics. (a) We found that self-aggregation of SP-A can be Ca(2+)-dependent. The concentration of Ca(2+) needed for half-maximal self-association (K(a)(Ca)()2+) depended on the presence of salts. Thus, at low ionic strength, K(a)(Ca)()2+ was 2.3 mM, whereas at physiological ionic strength, K(a)(Ca)()2+ was 2.35 microM. Circular dichroism and fluorescence measurements of Ca(2+)-dependent SP-A aggregates indicated that those protein aggregates formed in the absence of NaCl are structurally different from those formed in its presence. (b) We found that self-aggregation of SP-A can be pH-dependent. Self-aggregation of SP-A induced by H(+) was highly influenced by the presence of salts, which reduced the extent of self-association of the protein. The presence of both salts and Ca(2+) attenuated even more the effects of acidic media on SP-A self-aggregation. (c) We found that self-aggregation of SP-A can be temperature-dependent. At 20 degrees C, SP-A underwent self-aggregation at physiological but not at low ionic strength, in the presence of EDTA. All of these aggregates were dissociated by either adding EDTA (a), increasing the pH to neutral pH (b), or increasing the temperature to 37 degrees C (c). Dissociation of Ca(2+)-induced protein aggregates at low ionic strength was accompanied by an irreversible loss of both SP-A secondary structure and SP-A-dependent lipid aggregation properties. On the other hand, temperature-dependent experiments indicated that a structurally intact collagen-like domain was required for either Ca(2+)- or Ca(2+)/Na(+)-induced SP-A self-aggregation but not for H(+)-induced protein aggregation.

Acrylamide↗
PubMed2000

Microstructure and dynamic surface properties of surfactant protein SP-B/dipalmitoylphosphatidylcholine interfacial films spread from lipid-protein bilayers.

Suspensions of dipalmitoylphosphatidylcholine (DPPC) bilayers containing 5, 10 or 20% (w/w) surfactant protein SP-B have been reconstituted and spread at air-liquid interfaces. Compression isotherms of DPPC/SP-B monolayers spread from these preparations were qualitatively comparable to the isotherms of the corresponding DPPC/SP-B monolayers spread from solvents. SP-B was squeezed-out at higher pressures from vesicle-spread films than from solvent-spread monolayers. SP-B caused a marked decrease on the rate of relaxation of DPPC collapse phases to equilibrium pressures in all the lipid/protein films assayed. This stabilizing effect was higher in vesicle-spread than in solvent-spread monolayers. Inclusion in the films of traces of the fluorescent probe NBD-PC (1 mol%) and use of a fluorescent derivative of SP-B labeled with a rhodamine derivative, Texas Red, allowed for direct observation of protein and lipid domains at the interface by epifluorescence microscopy. Upon compression, SP-B altered the packing of phospholipids in the bilayer-spread films, observed as a SP-B-induced reduction of the area of liquid-condensed domains, in a way similar to its effect in solvent-spread monolayers. SP-B was not associated with condensed regions of the films. Fluorescence images from vesicle-spread films showed discrete fluorescent aggregates that could be consistent with the existence of lipid-protein vesicles in close association with the monolayer. Both the retention of SP-B at higher surface pressures and the greater stability of collapse phases of DPPC/SP-B films prepared by spreading from liposomes in comparison to those spread from solvents can be interpreted as a consequence of formation of complex bilayer-monolayer interacting systems.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract.

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.

4-Chloro-7-nitrobenzofurazan↗
PubMed1999

Interactions of pulmonary surfactant protein A with phospholipid monolayers change with pH.

The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the pi-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the pi-A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Rotational dynamics of spin-labelled surfactant-associated proteins SP-B and SP-C in dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol bilayers.

Pulmonary surfactant proteins SP-B and SP-C have been isolated from porcine lungs and selectively labelled with 2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPO)-isothiocyanate at their N-terminal amine ends, to analyse the mobility of both proteins on the nanosecond time scale using electron spin resonance (ESR) spectroscopy. Reconstitution of the labelled forms of these proteins in bilayers of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) results in much broader and anisotropic ESR spectra, indicating a large restriction in rotational mobility of the protein-attached probe when inserted in membranes. Distinctive differences were found between the ESR spectra of the two polypeptides, that were consistent with intrinsic differences in mode of interaction of SP-B and SP-C with phospholipid bilayers. The mobility of the protein spin probes was sensitive to temperature on the time scale of conventional spin-label ESR. Both proteins, TEMPO-SP-B and TEMPO-SP-C, showed considerable increases in mobility at temperatures above the pretransition of pure DPPC. Finally, the mobility of the spin probes attached to both SP-B and SP-C was more restricted in DPPG than in DPPC bilayers, demonstrating that electrostatic interactions of the positively charged residues at the protein surface influence the rotational dynamics of the proteins in anionic lipid bilayers. Although some residual segmental mobility of the thiourea-linked probes cannot be discounted, the results clearly reflect preferential differences in overall protein dynamics in gel and fluid phases of the two phospholipids that could be important for the biophysical properties of surfactant bilayers and monolayers.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Depth profiles of pulmonary surfactant protein B in phosphatidylcholine bilayers, studied by fluorescence and electron spin resonance spectroscopy.

Pulmonary surfactant-associated protein B (SP-B) has been isolated from porcine lungs and reconstituted in bilayers of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) to characterize the extent of insertion of the protein into phospholipid bilayers. The parameters for the interaction of SP-B with DPPC or PC using different reconstitution protocols have been estimated from the changes induced in the fluorescence emission spectrum of the single protein tryptophan. All the different reconstituted SP-B-phospholipid preparations studied had similar Kd values for the binding of the protein to the lipids, on the order of a few micromolar. The depth of penetration of SP-B into phospholipid bilayers has been estimated by the parallax method, which compares the relative efficiencies of quenching of the protein fluorescence by a shallow or a deeper spin-labeled phospholipid probe. SP-B tryptophan was found to be located 10-13 A from the center of bilayers, which is consistent with a superficial location of SP-B in phosphatidylcholine membranes. Parallax experiments, as well as resonance energy transfer from SP-B tryptophan to an acceptor probe located in the center of the bilayer, indicate that there are significant differences in the extent of insertion of the protein, depending on the method of reconstitution. SP-B reconstituted from lipid/protein mixtures in organic solvents is inserted more deeply in PC or DPPC bilayers than the protein reconstituted by addition to preformed phospholipid vesicles. These differences in the extent of insertion lead to qualitative and quantitative differences in the effect of the protein on the mobility of the phospholipid acyl chains, as studied by spin-label electron spin resonance (ESR) spectroscopy, and could represent different functional stages in the surfactant cycle.

Animals↗
PubMed1998

Effect of acidic pH on the structure and lipid binding properties of porcine surfactant protein A. Potential role of acidification along its exocytic pathway.

Pulmonary surfactant protein A (SP-A) is synthesized by type II cells and stored intracellularly in secretory granules (lamellar bodies) together with surfactant lipids and hydrophobic surfactant proteins B and C (SP-B and SP-C). We asked whether the progressive decrease in pH along the exocytic pathway could influence the secondary structure and lipid binding and aggregation properties of porcine SP-A. Conformational analysis from CD spectra of SP-A at various pH values indicated that the percentage of alpha-helix progressively decreased and that of beta-sheet increased as the pH was reduced. The protein underwent a marked self-aggregation at mildly acidic pH in the presence of Ca2+, conditions thought to resemble those existing in the trans-Golgi network. Protein aggregation was greater as the pH was reduced. We also found that both neutral and acidic vesicles either with or without SP-B or SP-C bound to SP-A at acidic pH as demonstrated by co-migration during centrifugation. However, the binding of acidic but not neutral vesicles to SP-A led to 1) a striking change in the CD spectra of the protein, which was interpreted as a decrease of the level of SP-A self-aggregation, and 2) a protection of the protein from endoproteinase Glu-C degradation at pH 4.5. SP-A massively aggregated acidic vesicles but poorly aggregated neutral vesicles at acidic pH. Aggregation of dipalmitoylphosphatidylcholine (DPPC) vesicles either with or without SP-B and/or SP-C strongly depended on pH, being progressively decreased as the pH was reduced and markedly increased when pH was shifted back to 7.0. At the pH of lamellar bodies, SP-A-induced aggregation of DPPC vesicles containing SP-B or a mixture of SP-B and SP-C was very low, although SP-A bound to these vesicles. These results indicate that 1) DPPC binding and DPPC aggregation are different phenomena that probably have different SP-A structural requirements and 2) aggregation of membranes induced by SP-A at acidic pH is critically dependent on the presence of acidic phospholipids, which affect protein structure, probably preventing the formation of large aggregates of protein.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Differential partitioning of pulmonary surfactant protein SP-A into regions of monolayers of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol.

The interaction of the pulmonary surfactant protein SP-A fluorescently labeled with Texas Red (TR-SP-A) with monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/dipalmitoylphosphatidylglycerol 7:3 w/w has been investigated. The monolayers were spread on aqueous subphases containing TR-SP-A. TR-SP-A interacted with the monolayers of DPPC to accumulate at the boundary regions between liquid condensed (LC) and liquid expanded (LE) phases. Some TR-SP-A appeared in the LE phase but not in the LC phase. At intermediate surface pressures (10-20 mN/m), the protein caused the occurrence of more, smaller condensed domains, and it appeared to be excluded from the monolayers at surface pressure in the range of 30-40 mN/m. TR-SP-A interaction with DPPC/dipalmitoylphosphatidylglycerol monolayers was different. The protein did not appear in either LE or LC but only in large aggregates at the LC-LE boundary regions, a distribution visually similar to that of fluorescently labeled concanavalin A adsorbed onto monolayers of DPPC. The observations are consistent with a selectivity of interaction of SP-A with DPPC and for its accumulation in boundaries between LC and LE phase.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Increase of C-reactive protein and decrease of surfactant protein A in surfactant after lung transplantation.

In this study, we asked whether the serum acute-phase protein C-reactive protein (CRP) increased in large surfactant aggregates after lung transplantation and analyzed the changes in composition and interfacial adsorption activity of those aggregates. Single left lung transplantation was performed in weight-matched pairs of dogs. A double-lung block from the donor animal was flushed with either modified Euro-Collins solution (EC) (n = 6) or University of Wisconsin solution (UW) (n = 6) at 4 degrees C followed by immersion in cold EC or UW for 22 h. The left donor lung was transplanted. The recipient dog was then reperfused for 4.5 h. Irrespective of the preservation fluid, gas exchanged was impaired in the transplanted lung after 4.5 h of reperfusion. Large surfactant aggregates obtained from this lung showed reduced ability to rapidly adsorb to an air-liquid interface. Phospholipid (PL) content and PL composition of surfactant from lung transplants was similar to that of the control lungs. However, the content of surfactant protein A decreased after reperfusion. In addition, Western blot analyses showed that levels of CRP increased in surfactant from transplanted but not from donor lungs. The addition of human CRP to control surfactant (CRP:PL weight ratio, 0.01:1) caused a decrease of surfactant adsorption. We conclude that the impairment of adsorption facilities of surfactant from transplanted lungs may be correlated with decreased levels of surfactant protein A and increased levels of CRP. The presence of elevated levels of CRP in bronchoalveolar lavage could be a very sensitive marker of lung injury.

Adsorption↗
PubMed1997

Different modes of interaction of pulmonary surfactant protein SP-B in phosphatidylcholine bilayers.

Pulmonary surfactant-associated protein B (SP-B) has been incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) by two different procedures to characterize the dependence of lipid-protein interactions on the method of reconstitution. In method A the protein was dissolved in a small volume of either methanol or 60% (v/v) acetonitrile and injected into an aqueous phase containing phospholipid vesicles. In method B the vesicles were prepared by injection of a mixture of phospholipid and SP-B dissolved in methanol or aqueous acetonitrile. Both methods of reconstitution led to the extensive interaction of SP-B with PC bilayers as demonstrated by co-migration during centrifugation, marked protection against proteolysis, change in the fluorescence emission intensity of SP-B, and protection of SP-B tryptophan fluorescence from quenching by acrylamide. SP-B promoted the rapid adsorption of DPPC on an air/liquid interface irrespective of the method of protein reconstitution. However, the interfacial adsorption activity of SP-B reconstituted by method B remained stable for hours, but that of SP-B prepared by method A decreased with time. Electron microscopy showed that the injection of SP-B into an aqueous phase containing PC or DPPC vesicles (method A) induced a rapid aggregation of vesicles. By contrast, a much longer time was required for detecting vesicle aggregation when the protein was reconstituted by co-injection of SP-B and phospholipids (method B). The presence of 5% (w/w) SP-B in DPPC bilayers prepared by method B broadened the differential scanning calorimetry thermogram and decreased the enthalpy of the transition. In contrast, the injection of SP-B into preformed DPPC vesicles (method A) did not influence the gel-to-liquid phase transition of DPPC bilayers. Taken together, these results indicate that the mode and extent of interaction of SP-B with surfactant phospholipids depends on the conditions of preparation of lipid/protein samples, and that care should be taken in the interpretation of findings from reconstituted systems on the role of these surfactant proteins in the alveolar space.

1,2-Dipalmitoylphosphatidylcholine↗