title: Different modes of interaction of pulmonary surfactant protein SP-B in phosphatidylcholine bilayers.
abstract: 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.
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad Biología, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Cruz
authors.forename: C
authors.initials: C
authors.lastname: Casals
authors.forename: K M
authors.initials: KM
authors.lastname: Keough
authors.forename: J
authors.initials: J
authors.lastname: Pérez-Gil
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000327:Adsorption; D000818:Animals; D002152:Calorimetry, Differential Scanning; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008168:Lung / Q000737:chemistry; D008854:Microscopy, Electron; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D011402:Pronase / Q000378:metabolism; D011485:Protein Binding; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine; D014461:Ultracentrifugation
chemical_list: D000178:Acrylamides; D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D011510:Proteolipids; D011663:Pulmonary Surfactants; D020106:Acrylamide; D015060:1,2-Dipalmitoylphosphatidylcholine; D011402:Pronase