Studies on Retinoid Proteins in Membranes of Halobacterium halobium

(1)我们前期的研究表明，在halobium Halobacterium的细胞质膜中存在第四类维甲酸蛋白。我们将其命名为磷视紫红质（简称pR）。本研究揭示了利用闪光灯光解的pR的光循环。照射后，形成350 nm吸收的光化学中间体，在室温下以140 msec的半时间转变为530 nm吸收的中间体。530 nm处的中间吸收以大约300 msec的一半时间衰减，并恢复到原始的pR.(2)H。盐藻被长波长的光吸引，这种行为被称为正趋光性。为了证明正趋光性受体是感觉视紫红质（sR）的假设，我们仔细测量了正趋光性的作用光谱，并将其与sR的吸收光谱进行了比较，两者的光谱吻合良好，支持了这一假设。(3)细菌视紫红质（bR）仅为一种蛋白质，在紫色膜中含量较高。当介质pH低于3.2或bR通过阳离子交换柱时，bR呈蓝色。我们发现，在中性溶液中，四（4-氟苯基）硼亲脂阴离子加入bR也能形成蓝色膜。在去离子化的蓝色膜上加入亲脂性阳离子，使其颜色恢复为紫色。分析表明亲水性离子结合的疏水区存在，该区域的电子电荷调节bR的颜色。(4)测定光致膜电位是测定盐藻膜中光驱动离子泵活性的方法之一。对于小细胞或小泡，应采用间接估计膜电位的方法。亲脂离子在细胞内外的分布是常用的。估计值有歧义，这是由亲脂离子结合引起的。我们开发了一种校正结合的方法来估计真正的膜电位。少

(1)Our previous study has shown the existence of the fourth retinoid protein in the cytoplasmic membrane of Halobacterium halobium. We named it as phoborhodopsin (abbreviated as pR). The present study revealed the photocycle of pR with use of flash-photolysis. Upon an illumination, a photochemical intermediate absorbing at 350 nm is formed, which transforms to an intermediate absorbing at 530 nm with a half time of 140 msec at room temperature. The intermediate absorbing at 530 nm decays with a half time of ca 300 msec and returns to the original pR.(2)H. halobium is attracted to the long wavelength light and this behavior is called positive photo-taxis. In order to prove the hypothesis that the receptor of positive photo-taxis is sensory rhodopsin (sR), we measured carefully the action spectrum of positive photo-taxis and compared it with the absorption spectrum of sR. The finding that both spectra agree well each other supported the hypothesis.(3)Bacteriorhodopsin (bR) is only protei … More n in purple membrane. When pH of the medium is lowered than 3.2 or when bR is passed through cation exchanger column, bR turns blue. We found that addition of tetrakis(4-fluorophenyl)boron, lipophilic anion to bR formed blue membrane even in neutral solutions. Addition of lipophilic cation to the deionized blue membrane restored the color to purple. Analysis suggested the existence of hydrophobic region where lipophilic ions bind and the electronic charges of this region regulate the color of bR.(4)One of ways for the estimation of activity of light-driven ion-pumps in the membrane of H. halobium is to measure the photo-induced membrane potential. For small cells or vesicles, indirect method of the estimation of membrane potential should be employed. Distribution of lipophilic ions inside and outside the cells are frequently used. Ambiguity of the estimated values has been suggested which are caused by the binding of lipophilic ions. We developed the method to correct the binding to estimate the true membrane potential. Less