Nagashima Teruyoshi, Uematsu Shogo
Department of Physical Biochemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka , 52-1 Yada, Suruga Ward, Shizuoka City, Shizuoka 422-8526, Japan.
Langmuir. 2015 Feb 3;31(4):1479-88. doi: 10.1021/la503906m. Epub 2015 Jan 22.
Phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) nanodomains covered with bound water as well as diacylglycerol 1-palmitoyl-2-oleoyl-sn-glycerol (POG) nanodomains separated from a lipid membrane were studied, using monolayer surfaces of POPC hydrolyzed by phospholipase C (PLC). The investigation was based on the analysis of compression isotherms and on atomic force microscope (AFM) observations of Langmuir-Blodgett (LB) films and Langmuir-Schaefer (LS) films. The results included reaction rate constants obtained by kinetic analysis of phosphocholine at surface pressures from 0.1 to 31 mN/m and determined by a luminol-enhanced chemiluminescence method. Monolayer elastic modulus values and fluorescence microscopic images confirmed that hydrolysis by PLC progressed in the intermediate monolayer between a liquid-expanded (L1) film and a liquid-condensed (L2) film at 2-17 mN/m. Furthermore, the intermediate film was confirmed to consist of L1 film and the POPC nanodomains in the L2 state are covered with bound water, conclusions based on the following AFM results: (1) nanodomains in POPC LS films were catalyzed by PLC, (2) POG nanodomains extended out from LB films of mixed POPC/POG 9/1 (mol/mol) monolayers, and (3) POPC LS films were covered with bound water, as indicated by cross-sectional analysis. At the optimal surface pressure of 10 mN/m, when POPC nanodomains (L2), with internal diameters of ∼75 nm, were hydrolyzed by PLC, they shrank down into pockets of the same size as those that appeared with POG. The resulting pocket sizes on LS films were in agreement with POG nanodomain sizes on LB films. This study demonstrated that PLC reacted with POPC nanodomains (L2) dispersed in L1/L2 mixed phase monolayers selectively and that POG nanodomains were phase-separated from the monolayer as hydrolysis proceeded.
研究了覆盖有结合水的磷脂1-棕榈酰-2-油酰基-sn-甘油-3-磷酸胆碱（POPC）纳米域以及与脂质膜分离的二酰基甘油1-棕榈酰-2-油酰基-sn-甘油（POG）纳米域，使用磷脂酶C（PLC）水解的POPC单层表面进行研究。该研究基于压缩等温线分析以及对朗缪尔-布洛杰特（LB）膜和朗缪尔-谢弗（LS）膜的原子力显微镜（AFM）观察。结果包括通过对表面压力为0.1至31 mN/m的磷酸胆碱进行动力学分析获得的反应速率常数，该常数通过鲁米诺增强化学发光法测定。单层弹性模量值和荧光显微镜图像证实，在2至17 mN/m的表面压力下，PLC水解在液体膨胀（L1）膜和液体凝聚（L2）膜之间的中间单层中进行。此外，基于以下AFM结果证实中间膜由L1膜组成，且L2状态的POPC纳米域被结合水覆盖：（1）POPC LS膜中的纳米域被PLC催化，（2）POG纳米域从混合的POPC/POG 9/1（摩尔/摩尔）单层的LB膜中伸出，（3）横截面分析表明POPC LS膜被结合水覆盖。在最佳表面压力10 mN/m下，当内径约为75 nm的POPC纳米域（L2）被PLC水解时，它们收缩成与POG出现时相同大小的凹坑。LS膜上产生的凹坑尺寸与LB膜上的POG纳米域尺寸一致。本研究表明，PLC与分散在L1/L2混合相单层中的POPC纳米域（L2）选择性反应，并且随着水解的进行，POG纳米域从单层中相分离。
