Cruz A, Casals C, Keough K M, Pérez-Gil J
Departamento de Bioquímica y Biología Molecular I, Facultad Biología, Universidad Complutense, 28040 Madrid, Spain.
Biochem J. 1997 Oct 1;327 ( Pt 1)(Pt 1):133-8. doi: 10.1042/bj3270133.
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.
肺表面活性物质相关蛋白B（SP - B）已通过两种不同方法被整合到二棕榈酰磷脂酰胆碱（DPPC）或蛋黄磷脂酰胆碱（PC）囊泡中，以表征脂蛋白相互作用对重组方法的依赖性。在方法A中，蛋白质溶解在少量甲醇或60%（v/v）乙腈中，并注入含有磷脂囊泡的水相中。在方法B中，囊泡通过注入溶解在甲醇或乙腈水溶液中的磷脂和SP - B的混合物来制备。两种重组方法都导致SP - B与PC双层膜发生广泛相互作用，这通过离心过程中的共迁移、对蛋白水解的显著保护、SP - B荧光发射强度的变化以及丙烯酰胺对SP - B色氨酸荧光淬灭的保护得以证明。无论蛋白质重组方法如何，SP - B都能促进DPPC在气/液界面的快速吸附。然而，通过方法B重组的SP - B的界面吸附活性在数小时内保持稳定，而通过方法A制备的SP - B的界面吸附活性随时间降低。电子显微镜显示，将SP - B注入含有PC或DPPC囊泡的水相中（方法A）会导致囊泡快速聚集。相比之下，当通过共注入SP - B和磷脂来重组蛋白质时（方法B），检测囊泡聚集需要更长时间。通过方法B制备的DPPC双层膜中存在5%（w/w）的SP - B会拓宽差示扫描量热法热谱图并降低转变焓。相反，将SP - B注入预先形成的DPPC囊泡中（方法A）不会影响DPPC双层膜的凝胶-液相转变。综上所述，这些结果表明SP - B与表面活性剂磷脂的相互作用模式和程度取决于脂质/蛋白质样品的制备条件，并且在解释重组系统中这些表面活性剂蛋白在肺泡空间作用的研究结果时应谨慎。
