Elucidation of the mechanism of ion transport by bacteriorhodopsin and halorhodopsin by electrical measuements

通过电学测量阐明细菌视紫红质和盐菌视紫红质的离子传输机制

• 批准号: 09833001
• 负责人: MUNEYUKI Eiro
• 金额: $ 1.73万
• 依托单位: TOKYO INSTITUTE OF TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09833001/
• 关键词: bacteriorhodopsin; halorhodopsin; active transport; electrical measurment

In the first year, I constructed an experimental system which enabled to measure the fast photoelectric response of the membrane fractions containing bacteriorhodopsin (bR) or halorhodopsin (hR) by adsorbing them onto a thin polymer film, In the second year, I examined the photocurrent of bR and its mutant, two kinds of hRs from different halophytic bacteria as a function of pH or Cl-concentrations. The result suggested that the pH dependency of the photoelectric current by bR reflected the affinities of the entrance and exit of the proton pathway of bR for protons. similar results were obtained for hRs and it was suggested that the way of ion translocation in the pump proteins were similar to th at of a multiion channel. By using a pulsed laser flash, I could measure the charge movement within the pump protein in a single turnover. The charge movement in bR occurred in the musec and msec time region whereas the charge movement in two hRs occurred in the msec and sub-msec time region. By comparing the data obtained in the electrical measurements with the data obtained in the photochemical cycle, it was suggested that the charge movement occurs during the formation and decay of the N intermediate.On the other hand, I carried out a theoretical model study based on the properties of proton translocation by bR The model assumes three ion binding sites and the affinity change induce the uni-directional ion translocation. Furthermore, the distribution of the high affinity state and low affinity state must deviate from equilibrium distribution in order to induce active transport. I propose this is the critical difference between ion pumps and ion channels. The experimental results obtained in the second year fairly matches this idea. Thus it seems reasonable to assume that an ion pump is a multiion channel in which the affinity change for transported ion induces active transport.

第一年，我构建了一个实验系统，通过将含有细菌视紫红质（bR）或盐生视紫红质（hR）的膜组分吸附到聚合物薄膜上，能够测量它们的快速光电响应。第二年，我检查了bR及其突变体（来自不同盐生细菌的两种hR）的光电流与pH或Cl浓度的函数关系。结果表明，bR 光电流的 pH 依赖性反映了 bR 质子路径入口和出口对质子的亲和力。 hRs 也获得了类似的结果，这表明泵蛋白中离子易位的方式与多离子通道的方式相似。通过使用脉冲激光闪光，我可以测量单周转内泵蛋白内的电荷运动。 bR 中的电荷移动发生在 musec 和 msec 时间区域，而两个 hR 中的电荷移动发生在 msec 和 sub-msec 时间区域。通过将电测量中获得的数据与光化学循环中获得的数据进行比较，表明电荷运动发生在N中间体的形成和衰变过程中。另一方面，我基于bR的质子易位性质进行了理论模型研究。该模型假设三个离子结合位点，亲和力变化诱导单向离子易位。此外，高亲和力状态和低亲和力状态的分布必须偏离平衡分布才能诱导主动运输。我认为这是离子泵和离子通道之间的关键区别。第二年得到的实验结果与这个想法相当吻合。因此，假设离子泵是多离子通道，其中传输离子的亲和力变化引起主动传输似乎是合理的。

项目成果

Okuno, D.et al.: "Chloride concentration dependency of the electrogenic activity of halorhodopsin" Biochemistry. in press. (1999)

Okuno, D.et al.：“盐视紫红质的生电活性的氯化物浓度依赖性”生物化学。

Muneyuki, E.et al.: "A Single Mutation at the Catalytic Site of TF_1-α_3β_3γ Complex Switches the Kinetics of ATP Hydrolysis from Negative to Positive Cooperativity" FEBS Letters. 413. 55-60 (1997)

Muneyuki, E. 等人：“TF_1-α_3β_3γ 复合物催化位点的单一突变将 ATP 水解动力学从负协同性转变为正协同性” FEBS Letters 413. 55-60 (1997)。

Tsunoda,S.et al.: "Cross-linking of Two Subunits in the Closed Conformation in F1-ATPase" J.Biol.Chem.274. 5701-5703 (1999)

Tsunoda,S.et al.：“F1-ATPase 中闭合构象中两个亚基的交联”J.Biol.Chem.274。

Muneyuki, E at al.: "A new system for the measurement of electrogenicity producned by ion pumps using a thin polymer film : Examination of wild type bR and the D96N mutant over a wide pH range" FEBS Letters. 427. 109-114 (1998)

Muneyuki, E 等人：“一种使用聚合物薄膜测量离子泵产生的生电性的新系统：在宽 pH 范围内检查野生型 bR 和 D96N 突变体”FEBS Letters。

Yokoyama, K.et al.: "V-ATPase of Thermus thermophilus is inactivated during ATP hydrolysis but can synthesize ATP" J.Biol.Chem.273. 20504-20510 (1998)

Yokoyama, K.等人：“嗜热栖热菌的 V-ATP 酶在 ATP 水解过程中失活，但可以合成 ATP”J.Biol.Chem.273。