Studies of Membrane Fusion and Vesicle Fission of Biomembranes using Giant Unilamellar Vesicles and Cubic Phase and Their Applications

(1)采用单巨单α αmel αr囊泡（GUV）方法，我们发现低浓度（低于临界胶束浓度（CMC），但高于其阈值浓度）的两亲物质具有单长烃链（即单长链两亲物质），如溶血磷脂酰胆碱（lyso-PC）、溶血磷脂酸、辛基糖苷、诱导液体有序（lo）相的脂质膜(如双棕榈酰磷脂酰胆碱(DPPC)/胆固醇（6/4）膜的guv发生囊泡裂变。为了阐明在低相膜中包裹DOPC（一种Lα相形成脂质）对DPPC/chol (6/4)-GUV诱导的囊泡裂变的影响，我们研究了低浓度的lyso-PC对DPPC/DOPC/chol膜中guv的影响。随着膜内DOPC浓度的增加，溶索- pc诱导囊泡裂变的阈值浓度增加。在30 mol% DOPC及以上时，囊泡未发生裂变。在以上研究结果的基础上，我们提出了单长链两亲体诱导的低相膜GUV的囊泡裂变机制假说。(2)利用小角x射线散射（SAXS）研究了在生理离子浓度条件下，H+（即pH效应）对带负电荷的脂质-二油基磷脂酰丝氨酸（DOPS）和电中性脂质-单油质（MO）混合物膜相和结构的影响。在中性pH下，含有高浓度DOPS的DOPS/MO膜处于Lα相。首先，通过在中性pH条件下制备的DOPS/MO膜中加入不同的低pH缓冲液，研究了pH对多l αmel αr囊泡（MLV）相和结构的影响。对于20%-DOPS/80%-MO-MLVs，在pH为2.9及以下时，Lα相在1 h内发生向立方相（Q224）的转变，这种相变是可逆的；当膜悬浮液的pH值增加到中性时，立方相转变为原始的Lα相。其次，我们发现pH降低后，在相似αr条件下，LUVs膜融合后，DOPS/MO膜的l α αrge uni - αmel - αr囊泡转变为立方相。提出了低pH诱导相变的机理，并对相变的临界pH值进行了定量分析。这一发现首次证明了pH的变化可以在1小时内诱导脂质膜的l - α相和立方相之间的可逆相变。我们合理地认为pH诱导的立方相和l - α相之间的相互转化也会发生在细胞中。(3)我们提出了一种新的方法——单GUV法来探测生物膜的功能和动力学以及物质与生物膜的相互作用。我们已经成功地揭示了抗菌肽magin2诱导的孔形成，以及抗菌物质(-)-表没食子儿茶素没食子酸酯（ECGg）与脂质膜的相互作用。少

(1) Using the single giant uniLαmelLαr vesicle (GUV) method, we found that low concentrations (less than critical micelle concentration (CMC), but above their threshold concentrations) of amphiphiles with a single long hydrocarbon chain (i.e., single long chain amphiphiles) such as lysophosphatidylcholine (lyso-PC), lyosophosphatidic acid, octylglucoside, induced the vesicle fission of GUVs of lipid membranes in the liquid-ordered (lo) phase such as dipalmitoylphosphatidylcholine(DPPC)/cholesterol(6/4) membranes. To elucidate the effect of the inclusion of DOPC (i.e., an Lα phase forming lipid) in the lo phase-membrane of GUVs on the lyso-PC-induced vesicle fission of the DPPC/chol (6/4)-GUV, we investigated effect of low concentrations of lyso-PC on GUVs of DPPC/DOPC/chol membranes. With an increase in DOPC concentration in the membrane, the threshold concentration of lyso-PC to induce the vesicle fission increased. At and above 30 mol% DOPC, no vesicle fission occurred. On the basis … More of these results, we have proposed a hypothesis of the mechanism of the single long chain amphiphile-induced vesicle fission of the GUV of the lo phase-membrane.(2) We investigated the effect of H+ (i.e. pH effect) on the phase and structure of membranes of a negatively charged lipid, dioleoylphosphatidylserine (DOPS) and an electrically neutral lipid, monoolein (MO) mixtures under a physiological ion concentration condition, using small-angle X-ray scattering (SAXS). At neutral pH, DOPS/MO membranes containing high concentrations of DOPS were in the Lα phase. First, the pH effect on the phase and structure of the multiLαmelLαr vesicles (MLVs) of the DOPS/MO membranes preformed at neutral pH was investigated by adding various low pH buffers into the MLV suspension. For 20%-DOPS/80%-MO-MLVs, at and below pH 2.9, a transition from the Lα to cubic (Q224) phase occurred within 1 h. This phase transition was reversible ; a subsequent increase in pH to a neutral one in the membrane suspension transformed the cubic phase into the original Lα phase. Second, we found that a decrease in pH transformed Lαrge uniLαmelLαr vesicles of DOPS/MO membranes into the cubic phase after the membrane fusion between the LUVs under simiLαr conditions. We have proposed the mechanism of the low-pH induced phase transition, and also made a quantitative analysis on the critical pH of the phase transition. This finding is the first demonstration that a change in pH can induce a reversible phase transition between the Lα and cubic phases of lipid membranes within 1 h. We reasonably consider that pH-induced interconversion between the cubic and the Lα phases would also occur in cells.(3)We have proposed a novel method, the single GUV method to probe functions and dynamics of biomembranes and interactions of substances with biomembranes. We have succeeded in revealing the pore formation induced by antimicrobial peptide, maginin2, and the interactions of antibacterial substance, (-)-epigallocatechin gallate(ECGg) with lipid membranes. Less