两种化学修饰菌紫质的光化循环和光电位

本文分别用两种氮氧自由基对菌紫质(bR)中的两种氨基酸残基——赖氨酸和丝氨酸进行了修饰,并对修饰后的bR光循环中间产物M_(412)动力学过程、质子泵效率及光电响应信号进行了测量。通过与正常紫膜进行比较,可以看出:赖氨酸被修饰后,M_(412)快、慢组分的衰减及质子的吸收过程均减慢,M_(412)和质子的产额、跨膜光电位信号均有不同程度的提高;丝氨酸被修饰后,M_(412)和质子的动力学过程的变化与赖氨酸基本相同,但M_(412)和质子的产额及跨膜光电位提高很大,且光电位出现缓慢反向现象。结果表明赖氨酸不大可能直接参与质子的传递过程,其对紫膜质子泵的影响主要是通过其所带电荷对表面电位的贡献;而丝氨酸却似乎对bR的功能影响很大并对维持质子通道构象环境的稳定起着很大作用。     

菌紫质人工膜的光电压与离子强度的非线性关系

本文研究了Ｋ＋、Ｍｇ２＋和Ｌａ３＋离子强度对菌紫质人工膜的光电压的影响。研究结果表明,在实验所测定的离子强度范围内菌紫质人工沉淀膜的光电压与上述离子的离子强度间具有非线性关系,并随离子价数的提高其响应峰值的位置向低浓度发展。本文研究结果将对菌紫质光化学反应循环、质子迁移机制的研究,以及菌紫质光电池的设计、和以菌紫质为基础的视觉功能模拟和人工视觉研究提供有价值的数据 。     

菌紫质人工膜的光电压与离子强度的非线性关系

本文研究了Ｋ＾＋,Ｍｇ＾２＋和Ｌａ＾３＋离子强度对菌紫质人工膜的光电压的影响。研究结果表明,在实验所测定的离子强度范围的内菌紫质人工沉淀膜的光电压与上述离子的离子强度间具有非线性关系,并随离子价数的提高其响应峰值的位置向低浓度发展。本文研究结果将对菌紫紫质光化学反应循环,质子迁移机制的研究,以及菌紫质光电池的设计,和以菌紫质为基础的视觉功能模拟和人工视觉研究提供有价值的数据。     

有取向紫膜薄层光电位及其与光强度的关系

本文报道利用电泳法得到有取向紫膜薄层,并测定了在连续光照下由此薄层所产生的光电位.研究了相对湿度对光电位的影响.文中还从光电位和M产物对光强度的依赖关系,研究了二者间的相关关系.一些事实说明在M衰减的数个组分中,并不是每一组分都与光电位相关的.在M衰减过程中,可能存在有电荷沿膜平面的运动,它对M产物是有贡献的,但对膜电位却无贡献.     

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.     

酰化菌紫质的动力学光谱及光电特性研究

用人工双分子膜(BLM)技术及动力学光谱研究了赖氨酸残基在紫膜的结构和功能中所起的作用.酰化基团与菌紫质(bR)分子中的赖氨酸残基的ε氨基作用,使光照后bR的跨膜质子迁移信号及膜的充放电速度变慢,光循环中间产物M412的产量下降,半衰期延长.但UV/Vis吸收光谱表明酰化并未破坏bR中视黄醛的构象环境.在高pH或高盐浓度下,酰化的影响降低.这些结果表明:赖氨酸残基并不是泵出质子的提供者,没有直接参与质子的跨膜输运,而是通过表面电位来影响bR的质子泵功能.     

离子强度、温度及表面活性剂Triton X-100对紫膜质子泵效率的影响

用毫秒级闪光动力学光谱仪研究了离子强度、温度及表面活性剂Triton X-100对紫膜质子泵效率的影响。结果表明:紫膜溶液中适量离子的加入可使其质子泵效率(H~+/M_(412))显著提高(纯水中,H~+/M_(412)—0.56,而在150mM KCl中可达—1.3),高价离子的影响显然远大于低价离子;在5℃—50℃的较大温度范围内,紫膜的质子泵效率变化不大,但当温度升至60℃以上时,紫膜的质子泵效率迅速下降直至为零;非离子表面活性剂Triton X-100的加入对紫膜的质子泵效率影响不大,仅随Triton量的增加而略微下降。以上实验现象,直接或间接地说明了离子在紫膜质子泵功能中的重要作用。就此对离子及脂在紫膜质子泵中的作用机制进行了进一步的探讨。     

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.     

酶切菌紫质(bR)C端对紫膜光循环和质子泵效率的影响

本文研究紫膜悬浮液经低剂量木瓜蛋酶处理去掉菌紫质(Bacteriorhodopsin简写bR)分子C-末端后。其光循环产物和质子泵效率的变化。实验发现经酶切后,M_(412)产物中慢衰减组份M_(412)降低了20％,O_(640)降低了50％,而质子泵效率降低了70％。双光脉冲实验表明酶解作用并不影响光循环周期。这些事实说明了去C-端所引起的质子泵效率降低,不是通过光循环的途径而产生的。介质中离子强度对正常紫膜和酶解紫膜的质子泵效率有明显不同的影响 说明了C端在不同盐浓度中的构象对质子泵行为有很重要的作用。     

光电响应对紫膜外表面[k~ ]依赖性与质子泵效率相关

本文研究发现,不锈钢／电沉积紫膜薄膜／含水胶（电介质）／铜型菌紫质光电池的光电响应对紫膜外表面的钾离子浓度的依赖性与质子泵效率的这种依赖性成相关性。这种相关性说明：１．膜中菌紫质的光电响应主要来自质子泵运而非钾离子直接的贡献;２．存在一合适的离子浓度使菌紫质的光电响应有极大值;３．作用在外表面的离子主要影响质子释放结构域     

Abrupt onset of large scale nonproton ion release in purple membranes caused by increasing pH or ionic strength.

The abrupt onset of large scale nonproton ion release by photo-excited purple membrane suspensions has been observed near neutral pH using transient conductivity measurements. At pH 7 and low ionic strength, the conductivity transients due to proton and nonproton ions are of comparable magnitude but of opposite sign: fast proton release and ion uptake, followed by slow proton uptake and ion release. By increasing either the pH or the NaCl concentration, the amplitude of the conductivity transient increases sharply and the signal is then dominated by nonproton ion release. These results can be understood in terms of light-induced changes in the population of counterions condensed at the purple membrane surface caused by changes in the surface charge density. The critical charge density required for condensation to occur is evidently achieved near neutral pH by ionizing dissociable groups on the membrane by either titration (increasing the pH) or shifting their pKs (increasing the ionic strength).     

Large scale nonproton ion release and bacteriorhodopsin's state of aggregation in lipid vesicles. I. Monomers.

Light-induced conductivity transients have been observed in preparations of bacteriorhodopsin (bR) in phospholipid vesicles at high lipid/protein molar ratios. Under these conditions, bR is known to be dissolved as monomers in the lipid bilayer. The conductivity transients are due mostly to proton movements, including a trans-membrane component. Kinetic resolution of the conductance change due to proton ionophore-induced leakage through the vesicle membrane provides a novel method to quantitate the number of protons pumped, even in heavily buffered solutions. Some of the transient signal seen on the timescale of the bR photocycle is due to nonproton ions but is smaller than that observed in native purple membranes at pH 7 in low salt. Furthermore, when the pH is raised to 8, the very large transient nonproton ion release seen in purple membranes is not seen in the vesicles. This correlates well with previous results (Marinetti, T., and D. Mauzerall, 1986, Biophys. J., 50:405-415), in which the nonproton ion movements observed with native purple membranes were abolished by solubilization in Triton X-100. Thus, the nonproton ion release appears to be a property of bR in the native aggregated state.     

Proton transport by bacteriorhodopsin through an interface film

Summary Interface films of purple membrane and lipid containing spectroscopically intact and oriented bacteriorhodopsin have been used as a model system to study the function of this protein. Small positive charges in surface potential (<1 mV) are detected upon illumination of these films at the air-water interface. These photopotentials, are not affected by overlaying the interface film with a thin layer (0.3 mm) of decane. However, they are dramatically increased when lipid soluble proton carriers FCCP or DNP are added to the decane. The polarity of the photopotential indicates that, in the light, positive charges are transported through the interface from the aqueous to the organic phase. The action spectrum of the photopotential is identical to the absorption spectrum of bacteriorhodopsin. Since bacteriorhodopsin molecules are oriented with their intracellular surface towards the aqueous subphase, the characteristics of the photopotential indicate that in the light bacteriorhodopsin translocates protons from its intracellular to its extracellular surface. The kinetics of the photopotential reveal that the rate and extent of proton transport are proportional both to the fraction of bacteriorhodopsin molecules excited and to the concentration of proton acceptor. The photopotentials result from changes in the ionic distribution across the decane-water interface and can be cancelled by lipid soluble anions.     

A nonproton ligand sensor in the acid-sensing ion channel

Acid-sensing ion channels (ASICs) have long been considered as extracellular proton (H(+))-gated cation channels, and peripheral ASIC3 channels seem to be a natural sensor of acidic pain. Here, we report the identification of a nonproton sensor on ASIC3. We show first that 2-guanidine-4-methylquinazoline (GMQ) causes persistent ASIC3 channel activation at the normal pH. Using GMQ as a probe and combining mutagenesis and covalent modification analysis, we then uncovered a ligand sensor lined by residues around E423 and E79 of the extracellular "palm" domain of the ASIC3 channel that is crucial for activation by nonproton activators. Furthermore, we show that GMQ activates sensory neurons and causes pain-related behaviors in an ASIC3-dependent manner, indicating the functional significance of ASIC activation by nonproton ligands. Thus, natural ligands beyond protons may activate ASICs under physiological and pathological conditions through the nonproton ligand sensor, serving for channel activation independent of abrupt and marked acidosis.     

Large transient nonproton ion movements in purple membrane suspensions are abolished by solubilization in Triton X-100.

Light-induced release/uptake of both protons and other ions cause transient changes in conductivity in suspensions of purple membrane (PM) fragments (Marinetti, Tim, and David Mauzerall, 1983, Proc. Natl. Acad. Sci. USA, 80:178-180). We find that the release/uptake of nonproton ions with quantum yield greater than 1 is observed at most pHs and ionic strengths. Only at both low pH and low ionic strength is the conductivity transient mostly due to protons. Our hypothesis is that during the photocycle, changes occur in the PM's dense surface charge distribution that result in changes in the number of counterions bound or condensed at the membrane surface. To test this, the PM structure was perturbed with the nonionic detergent Triton X-100. Immediately after addition, Triton does not abolish the nonproton ion movements; in fact at low detergent concentrations (0.02% vol/vol) the signal amplitudes increased considerably. However, when PM is completely solubilized into monomers in Triton, the conductivity transients are due to protons alone, though at lower quantum yield compared with native PM. These results suggest that changes in the surface charge distribution in native PM's photocycle could contribute to proton transfer between the aqueous phase and bR itself.     

Osmotic activity and ionic permeability of membrane vesicles from Streptococcus faecalis and Micrococcus lysodeikticus cells]

Membrane fractions containing osmotically active vesicles with sufficiently low membrane permeability for K+, Na+ and Cl- ions typical for the intact cell membrane were isolated from the cells of the glycolyzing bacterium Streptococcus faecalis. In their osmotic properties and ionic permeability the membrane fractions of S. faecalis were found similar to those of the respiring bacterium Micrococcus lysodeikticus, which are capable of the energy-dependent potassium transport. It may be thus assumed that the S. faecalis fractions obtained may be used to study ionic transport. The removal of proton-dependent ATPase of the S. faecalis membrane preparations did not affect the permeability of membranes for K+ ions which is indicative of different mechanisms of proton and potassium translocation.     

紫膜在含水凝胶中的定向和状态

用几种光谱学方法研究了紫膜凝胶的定向度、生色团视黄醛的状态以及其光循环中间产物M的动力学过程。结果表明:用通常采用的制备方法所得到的紫膜凝胶虽然能得到光电响应信号,但其定向度并不理想,生色团视黄醛的构象受到较大扰动,光循环中间产物M的产出及衰减也受到抑制,部分样品甚至由于其视黄醛的脱落而完全失去颜色,其质子泵功能也随之丧失。这说明虽然紫膜凝胶是目前研究紫膜质子泵机理(光电响应测量)和构造分子电子器件较好的和有希望的人工膜系统之一,但由于该系统对紫膜结构和功能的扰动仍然较大,紫膜的定向较难控制,所以,我们仍需在凝胶的形成体系及方法的改进上作大量工作。     

The protonation state of the Glu-71/Asp-80 residues in the KcsA potassium channel: a first-principles QM/MM molecular dynamics study

Although a few x-ray structures of the KcsA K(+) channel have been crystallized several issues concerning the mechanisms of the ionic permeation and the protonation state of the selectivity filter ionizable side chains are still open. Using a first-principles quantum mechanical/molecular mechanical simulation approach, we have investigated the protonation state of Glu-71 and Asp-80, two important residues located in the vicinity of the selectivity filter. Results from the dynamics show that a proton is shared between the two residues, with a slight preference for Glu-71. The proton is found to exchange on the picosecond timescale, an interesting phenomenon that cannot be observed in classical molecular dynamics. Simulations of different ionic loading states of the filter show that the probability for the proton transfer is correlated with the filter occupancy. In addition, the Glu-71/Asp-80 pair is able to modulate the potential energy profile experienced by a K(+) ion as it translates along the pore axis. These theoretical predictions, along with recent experimental results, suggest that changes of the filter structure could be associated with a shift in the Glu-Asp protonation state, which in turn would influence the ion translocation.     

Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles

The substrate and ionic requirements of ATP and inorganic pyrophosphate (PPi) hydrolysis by tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were compared with the requirements of ATP-and PPi-dependent proton translocation by the same material. Both ATP hydrolysis and ATP-dependent proton translocation are most stimulated by Cl⁻ and inhibited by NO₃⁻. NaCl and KCl support similar rates of ATP hydrolysis and ATP-dependent proton translocation while K₂SO₄ supports lesser rates for both. PPi hydrolysis and PPi-dependent proton translocation are most stimulated by K⁺. KCl and K₂SO₄ support similar rates of PPi hydrolysis and PPi-dependent proton translocation but NaCl has only a small stimulatory effect on both. Since PPi does not inhibit ATP hydrolysis and ATP does not interfere with PPi hydrolysis, it is inferred that the two phosphohydrolase and proton translocation activities are mediated by different tonoplast-associated enzymes. The results indicate the presence of an energy-conserving proton-translocating pyrophosphatase in the tonoplast of red beet.