Worthman L A, Nag K, Rich N, Ruano M L, Casals C, Pérez-Gil J, Keough K M
Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3X9, Canada.
Biophys J. 2000 Nov;79(5):2657-66. doi: 10.1016/S0006-3495(00)76504-0.
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.
采用落射荧光显微镜研究肺表面活性蛋白A（SP-A）与猪表面活性脂质提取物（PSLE）铺展单层的相互作用，PSLE铺展在含有0、0.13或0.16μg/ml SP-A以及0、1.64或5 mM氯化钙的盐下层（145 mM氯化钠，5 mM Tris-HCl，pH 6.9）上，且PSLE含有1 mol%的荧光探针（NBD-PC）。在没有SP-A的情况下，无论有无钙离子，PSLE单层均未观察到差异。在低表面压力（π约为5 mN/m）下，在荧光背景上观察到圆形的探针排除（暗）区域，且这些区域的大小随π的增加而增大。在25 mN/m以上，区域大小随π的增加而减小。在π约为25 mN/m时，可观察到的暗相量在总膜面积的18%时达到最大值，然后在π约为40 mN/m时降至约3%。在盐下层中添加0.16μg/ml SP-A以及0或1.64 mM钙离子会导致暗区域聚集成松散网络，并且在10 - 28 mN/m的π之间，暗相总量增加到约25%。在存在5 mM钙离子和SP-A的情况下，单层特征与在没有SP-A时铺展的情况没有实质性差异，这可能是由于在较高浓度钙离子存在下SP-A的自缔合和聚集。PSLE膜铺展在含有共价结合德克萨斯红（TR-SP-A）的0.16μg/ml SP-A的盐下层上。在没有钙离子的情况下，TR-SP-A与重组的暗相相关（如用脂质探针所见）。5 mM钙离子的存在导致TR-SP-A出现在流体相中，并在单层的流体/凝胶相边界处出现聚集体。这项研究表明，SP-A与PSLE单层相关，特别是与凝聚或固相脂质相关，并导致表面活性剂单层中刚性相脂质的一些重组。
