Taneva S G, Keough K M
Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3X9, Canada.
Biophys J. 2000 Oct;79(4):2010-23. doi: 10.1016/S0006-3495(00)76449-6.
Epifluorescence microscopy combined with a surface balance was used to study monolayers of dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (PG) (8:2, mol/mol) plus 17 wt % SP-B or SP-C spread on subphases containing SP-A in the presence or absence of 5 mM Ca(2+). Independently of the presence of Ca(2+) in the subphase, SP-A at a bulk concentration of 0.68 microg/ml adsorbed into the spread monolayers and caused an increase in the molecular areas in the films. Films of DPPC/PG formed on SP-A solutions showed a pressure-dependent coexistence of liquid-condensed (LC) and liquid-expanded (LE) phases. Apart from these surface phases, a probe-excluding phase, likely enriched in SP-A, was seen in the films between 7 mN/m < or = pi < or = 20 mN/m. In monolayers of SP-B/(DPPC/PG) spread on SP-A, regardless of the presence of calcium ions, large clusters of a probe-excluding phase, different from probe-excluding lipid LC phase, appeared and segregated from the LE phase at near-zero surface pressures and coexisted with the conventional LE and LC phases up to approximately 35 mN/m. Varying the levels of either SP-A or SP-B in films of SP-B/SP-A/(DPPC/PG) revealed that the formation of the probe-excluding clusters distinctive for the quaternary films was influenced by the two proteins. Concanavalin A in the subphase could not replace SP-A in its ability to modulate the textures of films of SP-B/(DPPC/PG). In films of SP-C/SP-A/(DPPC/PG), in the absence of calcium, regions consisting of a probe-excluding phase, likely enriched in SP-A, were detected at surface pressures between 2 mN/m and 20 mN/m in addition to the lipid LE and LC phases. Ca(2+) in the subphase appeared to disperse this phase into tiny probe-excluding particles, likely comprising Ca(2+)-aggregated SP-A. Despite their strikingly different morphologies, the films of DPPC/PG that contained combinations of SP-B/SP-A or SP-C/SP-A displayed similar distributions of LC and LE phases with LC regions occupying a maximum of 20% of the total monolayer area. Combining SP-A and SP-B reorganized the morphology of monolayers composed of DPPC and PG in a Ca(2+)-independent manner that led to the formation of a separate potentially protein-rich phase in the films.
结合表面天平的落射荧光显微镜用于研究在含有或不含5 mM Ca(2+)的情况下，在含有表面活性蛋白A（SP-A）的亚相中铺展的二棕榈酰磷脂酰胆碱（DPPC）/ 鸡蛋磷脂酰甘油（PG）（8:2，摩尔/摩尔）加17 wt% SP-B或SP-C的单层膜。无论亚相中是否存在Ca(2+)，本体浓度为0.68 μg/ml的SP-A都会吸附到铺展的单层膜中，并导致膜中分子面积增加。在SP-A溶液上形成的DPPC/PG膜显示出液相凝聚（LC）相和液相扩张（LE）相的压力依赖性共存。除了这些表面相外，在7 mN/m≤π≤20 mN/m的膜中还观察到一个可能富含SP-A的探针排斥相。在铺展于SP-A上的SP-B/(DPPC/PG)单层膜中，无论是否存在钙离子，在接近零表面压力下都会出现与脂质LC相不同的大的探针排斥相簇，并从LE相中分离出来，直至约35 mN/m时都与传统的LE相和LC相共存。改变SP-B/SP-A/(DPPC/PG)膜中SP-A或SP-B的水平表明，四元膜特有的探针排斥簇的形成受到这两种蛋白质的影响。亚相中的伴刀豆球蛋白A在调节SP-B/(DPPC/PG)膜的织构方面无法替代SP-A。在SP-C/SP-A/(DPPC/PG)膜中，在没有钙的情况下，除了脂质LE相和LC相外，在2 mN/m至20 mN/m的表面压力下还检测到由可能富含SP-A的探针排斥相组成的区域。亚相中的Ca(2+)似乎将该相分散成微小的探针排斥颗粒，可能由Ca(2+)聚集的SP-A组成。尽管它们的形态明显不同，但含有SP-B/SP-A或SP-C/SP-A组合的DPPC/PG膜显示出相似的LC相和LE相分布，LC区域最多占单层膜总面积的20%。结合SP-A和SP-B以与Ca(2+)无关的方式重组了由DPPC和PG组成的单层膜的形态，导致在膜中形成一个单独的潜在富含蛋白质的相。
