表面压与紫膜含量对紫膜LB膜质量的影响

本文报导了用表面轮廓测量仪测量了不同表面压和不同紫膜含量下制备的紫膜LB膜中紫膜碎片的厚度。实验结果表明:单个紫膜碎片在紫脂LB膜中的厚度为50左右,相当数量的紫膜碎片之间有重叠。当表面压为30mN/m或紫膜碎片与大豆磷脂之重量比为20:1时,紫膜碎片容易进入到水相或碎片之间相互重叠变得更加严重。     

pH对紫膜表面电位的影响

用荧光标记物1,8-AMS与紫膜结合,测量了能化态和非能化态下紫膜表面电位随介质pH的变化.在pH5.5以下,紫膜表面电位随pH的降低而下降,但囊泡中的紫膜表面电位变化幅度较大;在pH5.5以上,处于非能化态的紫膜(无论是紫膜碎片还是处于囊泡中)的表面电位都没有明显变化,处于能化态时,紫膜碎片的表面电位在pH9.2出现一个峰.     

紫膜与溶剂的相互作用

本文研究了溶剂正己烷,正十六烷,甲苯和二甲基甲酰胺(DMF dimethyl formamide)与紫膜的相互作用.吸收光谱,园二色谱和紫膜光循环中间产物M412的动力学过程的测量表明,在不同条件下,溶剂与紫膜能相互作用而影响到紫膜的光谱特性和光化学循环动力学过程.结果说明,在制作紫膜LB膜时,正己烷和正十六烷是合适的,使用二甲基甲酰胺时必须防止强光照射,甲苯则不能采用.     

PM-SP-LB多层膜中BR分子的有序定向排列

用垂直转移法在石英片上制成的PM—SP—LB多层膜的可见区吸收谱表明吸收峰峰位与成膜液一致,均在574nm左右,比PM水悬浮液的吸收峰位略有红移;稳态线二色性表明,除PM碎片平躺在多层膜平面内外,在提拉时的竖直方向存在BR的取向优势,优势率约为0.51左右;同时还表明,25mN/m条件下制备的PM—SP—LB多层膜中BR分子的视黄醛生色团的跃迁矩与膜平面法向所成的角接近于天然紫膜中的值。     

紫膜碎片的电二色性研究

悬浮在水中的嗜盐菌紫膜碎片,在外电场作用下产主定向排列.在20℃时,568nm的电二色性研究表明:外加电场为2kV/m时取向程度可达60%以上;大于5.5kV/m时,取向作用趋于饱和状态;饱和时简约电二色性为-0.437左右,视黄醛生色团的跃迁矩方向与电偶极矩方向形成60.9°夹角;紫膜的永久偶极短为9.2×10~(-24)C、M,剩余电极化率为3.0×10~(-27)m~2;紫膜的旋转扩散常数为0.53秒~(-1).曲线拟合分析表明,感应偶极对紫膜碎片的定向的贡献应予考虑.本文对紫膜碎片的定向机理进行了讨论.     

付里叶变换偏振红外光谱法研究紫膜LB膜内菌紫质的取向

用付里叶变换偏振红外法测量了紫膜LB膜内菌紫质的酰胺Ⅰ、Ⅱ和A带的二色性,计算了菌紫质的α-螺旋取向.实验表明,紫膜内菌紫质的α-螺旋轴同膜法线的平均夹角为13°,即与膜平面的平均夹角为77°.     

温度对紫膜bR结构的影响

温度升高使天然紫膜在可见差分吸收光谱中650nm处出现一吸收峰,这意味着紫膜去离子兰膜的形成,而这在脱氧胆酸处理后的紫膜中并未出现.天然紫膜与脱脂紫膜在630nm处的吸收与温度间的关系进一步表明:只有天然紫膜在温度大于68℃才有膜结合离子的释放,释放的离子结合于移去的膜脂之上.紫膜结合离子的释放改变了天然紫膜中bR的构象.热致离子的释放还受介质pH值及介质中离子的影响.pH5.03时,热致离子释放温度提前至温度46℃,而pH7.00,10mM MgCl_2及pH9.00时都没有观察到紫膜离子释放.温度对紫膜在不同pH值时表面电位影响的不一致性进一步表明这是由于膜表面电荷参与的结果.     

紫膜表面电荷数的测量

以CAT₁₂为自旋探针,用ESR自旋标记法测量了不同表面pH时酰化前后紫膜表面负电荷密度,以酰化所屏蔽的表面电荷数为标准,计算了表面pH4-11时单位菌紫质紫膜两侧的总负电荷数.结果表明:表面pH5-9时为9,表面pH≥10及表面pH < 5时增大.结果有力支持了膜表面5个二价阳离子结合位点的模型.     

紫膜BLM的光电位与非质子离子迁移

嵌入紫膜碎片的BLM系统在不同离子强度的介质中产生不同的光电位信号。本文通过在介质中加入离子载体或离子通道剂对紫膜的光电响应信号进行了系统的研究。结果表明:上述光电位信号的差异主要是由紫膜的非质子离子的迁移引起的。与紫膜的质子跨膜迁移相比,这种非质子离子迁移也是由光驱动的,随介质的离子强度的增强而显著增强,但它的迁移速率更快,且产生反向的光电位。用通常方法所获得的光电位信号主要是这两种迁移信号的迭加。三价阳离子(La~(3+))可使紫膜质子泵反向并能抑制质子的吸收,但它似乎并不参与紫膜的非质子离子迁移。     

水在菌紫质光循环和质子泵中的作用

用圆二色仪和闪光动力学先谱仪分别测量了空气干燥紫膜薄层的圆二色谱及紫膜LB膜的M_(412)的衰减过程.在于燥紫膜的圆二色谱上出现412nm的正峰,它是光循环中间体M_(412)的特征峰.在无水介质中,紫膜LB膜中的BR仍能进行先化学循环而检测到中间体M_(412),但M_(412)的衰减速度减慢,产生M_(412)的堆积,质子化过程受阻.在有水的介质中,只要有足够的H~+存在,紫膜LB膜中的BR的中间体M_(412)的衰减速度明显加快.说明水介质的H~+是完成正常光化学循和质子化过程必不可少的.     

Structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films

A suspension of purple membrane fragments in a solution of soya phosphatidyl-choline in hexane is spread at an air-water interface. Surface pressure and surface potential measurements indicate that the membrane fragments and lipids organize at the interface as an insoluble film. Electron microscopy of shadow-cast replicas of the film reveal that in the bacteriorhodopsin to soya PC weight ratio range of 2:1 to 10:1, these films consist of nonoverlapping membrane fragments which occupy approximately 35% of the surface area and are separated by a lipid monolayer. Furthermore, the membrane fragments are oriented with their intracellular surface towards the aqueous subphase. Nearly all the bacteriorhodopsin molecules at the interface are spectroscopically intact and exhibit visible spectral characteristics identical to those in aqueous suspensions of purple membrane and in intact bacteria. In addition, bacteriorhodopsin in air-dried interface films show spectral changes upon dark-adaptation and upon flash illumination similar to those observed in aqueous suspensions of purple membrane, but with slower kinetics. The kinetics of the spectral changes in interface films can be made nearly the same as in aqueous suspension by immersing the films in water.     

Oriented immobilization of bacterial photosynthetic membrane

Summary We have examined a method for oriented immobilization of photosynthetic membrane fragments on a solid surface by specific avidin-biotin interaction. Photosynthetic membrane fragments from the purple non-sulphur photosynthetic bacterium Rhodopseudomonas viridis, of which the H-subunit of the photosynthetic reaction centre was biotinylated, was immobilized on an avidin-adsorbed plate. Orientation of the immobilized membrane on the plastic plate was checked by an antisera binding assay that could react to the respective sides of the membrane: the H-subunit side was selectively adsorbed on the plate. Light-induced potential and current responses could be measured when the membrane immobilized on the SnO₂-coated glass plate was dried and sandwiched with a counter electrode of Hg. The electrical response in the immobilized membrane was much improved in comparison with the control (membranes were simply adsorbed on the plate), supporting the idea that the membranes have an orientation on solid surfaces.Offprint requests to: M. Hara     

Monomolecular surface film and tubular myelin figures of the pulmonary surfactant in hamster lung

Summary Perfusion fixation via pulmonary trunk was applied to the alveolar lining layer in situ at different lung volumes using a fixative containing tannic acid-ferrocyanide osmium. The monomolecular surface film and hypophasic tubular myelin figures were enhanced. In the range of transpulmonary pressure (1–10 cmH₂O), the surface film appeared in the form of a single, electron-dense leaflet, 2.7±0.6 nm (M±SD) in thickness while trilaminar membrane structure was retained in all parts of the tubular myelin figures of the hypophase. The surface film was attached underneath at right angles with trilaminar membranes which formed the outermost parts of the tubular myelin. Such structural continuity was taken to support a view that the phospholipid unit membrane of the tubular myelin figure would be transformed at the hydrophobic phase into a pair of monomolecular leaflets, eventually forming the surface film.     

Preparation and characterization of monolayer and multilayer Langmuir-Blodgett films of a series of 6-O-alkylcelluloses

Monolayer and multilayer Langmuir-Blodgett (LB) films of 6-O-alkylcelluloses with various chain lengths were prepared and studied. The surface pressure (pi)-area (A) isotherms of 6-O-alkylcelluloses exhibited characteristic behaviors depending on the length of the alkyl chain and temperature. 6-O-Stearylcellulose on the subphase formed a homogeneous monolayer at 10 mN m(-1). By transfer ratio, FT-IR, and contact angle measurements, it was proved that the monolayer of 6-O-stearylcellulose on the water surface was transferred successfully onto a substrate by a vertical dipping method to form a Z-type LB film. The transmission and reflection absorption IR spectrum indicated that the hydrocarbon chains had all-trans rotamers and were oriented nearly perpendicular to the surface in the film. AFM section analysis revealed that the thickness per layer was calculated to be 2.35 nm. These results suggested that the hydrocarbon chains were inclined at an angle of about 25.3 degrees to have high packing density in the alkyl region.     

X-ray diffraction from a single layer of purple membrane at the air/water interface

X-ray diffraction patterns have been recorded from a single layer of purple membrane ( approximately 50 A thickness) at the air/water interface in a Langmuir trough. Grazing-incidence X-ray diffraction is demonstrated to be a promising method for obtaining structural information on membrane proteins under physiological conditions. The method is so sensitive that diffraction can be measured from samples with only 10(13) protein molecules in the beam. Diffraction from hexagonal crystals of purple membrane with a lattice constant of 61. 3 A was observed up to the order {h,k}={4,3}, corresponding to a resolution of approximately 9 A. The work reported here is a first step towards a new way of protein crystallography using grazing-incidence X-ray diffraction at the air/water interface.     

Following single antibody binding to purple membranes in real time

Antibody binding to surface antigens in membranes is the primary event in the specific immune defence of vertebrates. Here we used force microscopy to study the dynamics of antibody recognition of mutant purple membranes from Halobacterium salinarum containing a genetically appended anti-Sendai recognition epitope. Ligation of individual anti-Sendai antibodies to their antigenic epitopes was observed over time. Their increase in number within a small selected area revealed an apparent kinetic on-rate. The membrane-bound antibodies showed many different conformations that ranged from globular to V- and Y-like shapes. The maximum distance of two Fab fragments of the same antibody was observed to be approximately 18 nm, indicating an overall strong intrinsic flexibility of the antibody hinge region. Fab fragments of bound anti-Sendai antibodies were allocated to antigenic sites of the purple membrane, allowing the identification and localization of individual recognition epitopes on the surface of purple membranes.     

Estimation of disk membrane lateral pressure and molecular area of rhodopsin by the measurement of its orientation at the nitrogen-water interface from an ellipsometric study

The internal lateral pressure of a bilayer has been estimated by numerous investigators. Most of these measurements were made by using the monolayer technique. In our approach, the disk membrane lateral pressure was estimated by assuming that this value is equal to the surface pressure necessary to maintain the transmembrane orientation of rhodopsin. The orientation of rhodopsin at the nitrogen-water interface was determined by using ellipsometry, which can measure the thickness of the film. By examining surface pressure and ellipsometric isotherms of intact and partially hydrolyzed rhodopsin, we have determined that a lateral pressure of 38 mN/m is necessary to give rhodopsin its natural transmembrane orientation and that surface pressures exceeding 45 mN/m lead to the formation of multilayers in the disk membrane film. At 38 mN/m, pure rhodopsin is found to have a molecular area of 2300 A2.