INTERACTION OF RHODOPSIN, G-PROTEIN AND KINASE IN OCTOPUS PHOTORECEPTORS

Light induced phosphorylation of octopus rhodopsin was greatly enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), suggesting that the kinases are involved in regulating interaction between rhodopsin and G-protein. We determined phosphorylated peptides of octopus rhodopsin in the presence or absence of GTP gamma S. Possible phosphorylation sites for octopus rhodopsin enhanced by GTP gamma S were Thr329, Thr330 and/or Thr336, which suggest that the G-protein associates with cytoplasmic loops including C-terminal peptide in the seventh helix of octopus rhodopsin.     

PHOTOEXCITATION OF RHODOPSIN: CONFORMATION CHANGES IN THE CHROMOPHORE, PROTEIN AND ASSOCIATED LIPIDS AS DETERMINED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— The visual pigment rhodopsin is the major membrane protein in the rod photoreceptor membrane. Rhodopsin's function is to transduce the light induced isomerization (ll-cis to all-trans) of its internally located retinylidene chromophore into transient expression of signal sites at the surface of the protein. Fourier transform infrared (FTIR) difference spectroscopy has been used to study all of the steps in the photobleaching sequence of rhodopsin. Early protein alterations involving the peptide backbone and aspartic and/or glutamic carboxyl groups were detected which increase upon lumirhodopsin formation and spread to water exposed carboxyl groups by metarhodopsin II. The intensified and frequency shifted hydrogen-out-of-plane vibrations of the chromophore that are present in bathorhodopsin are absent in lumirhodopsin. This indicates that by lumirhodopsin, the chromophore has relaxed relative to its more strained all-frans form in bathorhodopsin. Finally, the transition to metarhodopsin II is found to involve perturbation of the acyl tail region of unsaturated phospholipid molecules possibly in response to small changes in the shape of the rhodopsin.     

NEW FLUOROSILOXANE AS A COATING MATERIAL FOR NERVE AGENT SENSORS

Polymer coated quartz crystal microbalance （QCM） sensor based on the frequency shifts due to the adsorption of compounds at the surface of modified quartz crystal electrode is an effective method for detection of sarin （GB） which is a highly toxic nerve warfare agent. A new fluorosiloxane polymer has been synthesized through 6 steps from trifluoromethyl benzene. The synthesis was achieved from trifluoromethyl benzene through nitration, hydrogenation. The obtained m-nitrotrifluoromethyl aniline was treated with NANO2, and then hydrolyzed to m-nitrotrifluoromethyl phenol, m-nitrotrifluoromethyl phenol was reacted with allyl bromide to the ether product. The product was rearranged by Claisen rearrangement, and then reacted with polymethylhydrosiloxane under catalyst of Pt/DVTMS. The fluorosiloxane polymer can be obtained. The polymer has been successfully used as QCM coating material     

识别金属离子客体的荧光传感开关的研究进展

分子识别是超分子化学的核心概念,荧光开关PET(photoinduced electron transfer)体系又是分子识别中的重要组成部分,是超分子化学与光化学学科相结合的成就.荧光开关作为一种全新的客体识别和分析手段,由于其独特的应用价值,近10年来正以惊人的速度在向前发展.     

识别中性分子客体荧光传感和开关的研究进展

分子识别是超分子化学的核心概念,而荧光开关PET(photo-induced electron transfer)体系又是分子识别中的重要组成部分,是超分子化学和光物理学科相结合的成就.本文总结了近年来对中性客体分子的荧光传感和开关的研究进展.     

A COMPARISON OF LIGHT-TRIGGERED SPECTRAL CHANGES IN RHODOPSIN AND ISORHODOPSIN

Abstract— This report describes spectral changes associated with the transformation of metarhodopsin I to metarhodopsin II following light excitation of isorhodopsin and rhodopsin. Irradiated isorhodopsin gives rise to an equilibrium mixture of metarhodopsin I and metarhodopsin II which at 2°C and pH 6.8 favors the former. Isorhodopsin and rhodopsin are converted to metarhodopsin II via metarhodopsin I at very similar rates and activation parameters for the conversions are essentially identical. It is concluded that the initial cis to trans isomerization erases all differences in the two pigments.     

LIGHT-INDUCED CONFORMATIONAL CHANGES IN CATTLE RHODOPSIN AS PROBED BY MEASUREMENTS OF THE INTEFFACE POTENTIAL

Abstract— A novel technique of capacitative coupling of oriented rhodopsin at a polar/apolar interface allows the time resolved investigation of conformational changes following a flash. Electric signals arise as a consequence of changes of the interface potential. A signal occurring within milliseconds behaves like the R₂-phase of the "early receptor potential" (= ERP). This response is interpreted as a conformational change of rhodopsin. No correlation of this signal is found to the spectroscopically defined metarhodopsin I-metarhodopsin II transition.
The temperature dependence of the conformational change coincides with the temperature dependence of the latency of the 'a-wave' of the electroretinogram, reported by Arden and Ikeda (1968). It is suggested that the command step of visual excitation is the conformational change and not one of the spectroscopically defined photolysis steps of rhodopsin.
Analysis of slower electrical signals following the fast response is in accordance with the model of a light-induced pore formation.     

LIGHT INDUCED INTERACTION BETWEEN RHODOPSIN AND GTP DEPENDENT PROCESSES IN ROD OUTER SEGMENTS—I. KINETIC ANALYSES OF LIGHT SCATTERING TRANSIENTS

Abstract— A fast light-induced light scattering transient in the presence of Mg²⁺ and GTP is observed from bovine rod outer segment suspension. This transient is studied at various temperatures, degrees of photo-bleaching and GTP concentration. The kinetic analysis of this signal at low photo-bleaching (∼ 5%) reveals two different processes which are consecutive rather than simultaneous. The rate of both processes depends upon the extent of photo-bleaching and indicates that each process requires a photolyzed rhodopsin intermediate. At low temperature, the rate constant of both the processes increases with the extent of photo-bleaching while at higher temperatures it decreases. The calculated activation energy of both processes decreases with increasing photo-bleaching. It has also been observed that with decreasing GTP concentration, the rates of both processes decrease indicating that the two processes though first order are most likely bimolecular, first order in both GTP and the photolytic rhodopsin intermediate (metarhodopsin II)     

EXISTENCE OF HYPSORHODOPSIN AS THE FIRST INTERMEDIATE IN THE PRIMARY PHOTOCHEMICAL PROCESS OF CATTLE RHODOPSIN

It has long been believed that bathorhodopsin is the first intermediate of visual process for cattle rhodopsin. In the present paper hypsorhodopsin is shown to be the first intermediate by the use of picosecond spectropic technique. The main first intermediate, hypsorhodopsin, is formed with the time constant of 15 ± 5 ps. The time constant of the formation of bathorhodopsin from hypsorhodopsin is 50 ± 20 ps. Bathorhodopsin intermediates formed directly from excited state rhodopsin and those formed indirectly through hypsorhodopsin are 71/2^#% and 93%, respectively, of total bathorhodopsin intermediates in octylglucoside buffered solution. Batho intermediates formed directly and indirectly are 0% and 100%. respectively, of total batho intermediates in LDAO buffered solution.     

LIGHT-INDUCED PERTURBATION OF AROMATIC RESIDUES IN BOVINE RHODOPSIN AND BACTERIORHODOPSIN*

Light-induced changes in the UV absorption spectrum of bovine rod outer segment membranes were measured by conventional difference spectroscopy and by flash photolysis methods. Different thermal intermediates of rhodopsin (lumirhodopsin, metarhodopsin I, metarhodopsin II, and meta-rhodopsin III) have absorption spectra in the ultraviolet which differ from the rhodopsin spectrum and from each other. The spectra associated with metarhodopsin I, metarhodopsin II, and metarhodopsin III are characteristic of perturbation of a small number of tyr. and/or trp residues, most likely one trp residue. These aromatic residues are in the neighborhood of the retinal Schiff base and undergo coordinated changes of interaction with retinal during the bleaching sequence. At the metarhodopsin II stage, the magnitude of the UV spectral changes is consistent with the exposure of a previously shielded trp residue to an aqueous environment. The present results are consistent with previous spectral studies which limit the extent of light-induced conformational changes to regions of the protein in the neighborhood of the retinal Schiff base. An analogous study was made on light-adapted purple membranes of Halobacterium halobium. The UV absorption spectrum associated with the deprotonated Schiff base intermediate of the trans-bacteriorhodopsin cycle is indicative, in part, of aromatic residue perturbation. However, significant changes in the secondary and tertiary structures of the bacterio-rhodopsin protein characteristic of a delocalized conformational change are unlikely at this intermediate stage.     

ON THE STATE OF CHROMOPHORE PROTONATION IN RHODOPSIN: IMPLICATION FOR PRIMARY PHOTOCHEMISTRY IN VISUAL PIGMENTS

Resonance Raman multicomponent spectra of bovine rhodopsin, isorhodopsin, and bathorhodopsin are obtained at low temperature. Application of the double beam, 'pump-probe' technique allows an extraction of the rhodopsin and bathorhodopsin spectra in both protonated and deuterated media. Our results show that the Schiff bases of both rhodopsin and bathorhodopsin are fully protonated and the degree of protonation is unaffected by the rhodopsin-bathorhodopsin transformation. Further, the data support the concept or cis-trans isomerization as occurring in this transition. The effect of these results on various models for the primary photochemical event in vision is discussed.     

RHODOPSIN-PHOSPHOLIPID INTERACTION IN DETERGENT AND IN THE DISK

Abstract— Solubilization of cattle disk membrane in deoxycholate shifted the fluorescence emission maximum from 324 to 331 nm without changing the intensity. Tryptophyl residues are probably located at the hydrophobic interface between rhodopsin and phospholipid. Depletion of deoxycholate concentration from the solubilized disk by Sephacryl 200 column chromatography produced rhodopsin-phospholipid complexes with different characteristics that are the intermediate stages of membrane formation from homogeneous molecular solution. Association of rhodopsin takes place in a two-dimensional way even in the appreciably low content of phospholipid.
Sedimentation velocity studies showed that reassociation of lipid and rhodopsin occurs in 0.2% deoxycholate as well as in 0.05% sodium dodecylsulfate.
By using Sephacryl column we can now prepare, within 60 min, the rhodopsin-lipid complex that can form large vesicles in response to the addition of MgCl₂ without dialysis. This type of lipoprotein complex will be useful to the study of the mechanism of the two dimensional membrane formation.     

A POSSIBLE MECHANISM FOR THE ROLE OF N-RETINYLIDENE PHOSPHATIDYL ETHANOLAMINE IN THE REGENERATION OF RHODOPSIN

Abstract— A series of molecular orbital calculations on a model Schiff base comparable to protonated N -retinylidene phosphatidyl ethanolamine isomers has been made. The effect of the charged oxygen atoms of the phosphate moiety on the distribution of positive charge along the polyene chain of these isomers has been calculated. The stabilizing coulombic energy of interaction of these opposite charges and the possibility of free rotation around carbonxarbon double bonds in the electronically excited state has led to the conclusion that an 11- cis Schiff base isomer is the most probable product of the photoisomerization of an all-trans Schiff base.
The formation of a stable unprotonated all-trans Schiff base in aqueous detergent dispersion and its subsequent conversion to the protonated form, both with absorption spectra in conformity with the literature, is demonstrated.     

甲基橙为光谱探针研究聚电解质与表面活性剂相互作用

聚电解质与两亲分子间相互作用近年备受关注^[1,2].其原因之一是这种作用与作为形成生物膜基础的类脂间的相互作用极其相似,并可看作细胞中蛋白质-核酸相互作用的一种模式^[3].对众多水溶性高分子尤其是聚电解质与表面活性剂间相互作用研究表明,这种作用不仅有重要的学术意义,而且在实际应用方面也非常重要,如应用于泥浆凝聚,油井采油以及膜分离技术^[4]等.     

聚丙烯酸水溶液的粘度和构象变化

本文测定了不同中和度聚丙烯酸在不同离子强度盐溶液中的特性粘数,研究了聚丙烯酸的分子形态及构象对聚合物浓度、电荷密度及溶液离子强度的依赖性,得到聚离子的构象变化与溶液盐浓度的对数成反比,与电荷密度的平方根成正比等规律。     

普鲁士蓝在化学传感器中的研究及应用

综述了普鲁士蓝化学传感器的研究和应用进展。介绍了传感器敏感膜的制备方法，在电化学传感器和光学传感器中的应用及机理研究方面的成果。普鲁士蓝优越的氧化还原性质使普鲁士蓝化学传感器对于许多物质的检测有着重要的意义。     

RHODOPSIN REGENERATION, CALCIUM, AND THE CONTROL OF THE DARK CURRENT IN VERTEBRATE RODS

Abstract— The dark current of retinal rods is suppressed for an extended period when their rhodopsin is bleached. An 8% bleach completely suppresses the current for 8 min and after 35 min it is fully recovered. The dark current can recover fully from a bleaching flash without any rhodopsin being regenerated. Moreover the recovery can be hastened either by lowering the activity of calcium ions surrounding the rods or by regenerating the rhodopsin. The recovery of the dark current following these bleaches showed zero order kinetics, regardless of whether the recovery was hastened by low calcium, 11- cis retinaldehyde or not. If all the rhodopsin is bleached in the retina, the dark current does not recover; the addition of 11- cis retinaldehyde, but not all- trans retinaldehyde, to the bleached retina causes the dark current to begin its recovery as early as 10 min after the addition with zero order kinetics (1.3% min^-1). In two of three similar experiments, the dark current recovered 100%. When the recovery rate of the dark current from the retina showing the earliest response is compared with the rate of the regeneration of rhodopsin in the plasma and disc membranes, the dark current begins its recovery after the plasma membrane rhodopsin is fully regenerated and the disc rhodopsin is half regenerated. When the disc rhodopsin is fully regenerated, the dark current is recovered 75%, and 20 min later it is completely recovered.     

QUANTUM EFFICIENCY AND PHOTOSENSITIVITY OF THE RHODOPSIN ⇌ METARHODOPSIN CONVERSION IN CRAYFISH PHOTORECEPTORS

Photosensitivity (K_λ) of a visual pigment is the product of the molecular absorption coefficient (α_λ) and the quantum efficiency for photoconversion (γ). Among the invertebrates, many visual pigments are stable not only in the rhodopsin (R) conformation but also as the photoproduct, metarhodopsin (M), We here employ a method for determining the photosensitivities of the two stable pigments of a rhodopsin-metarhodopsin pair, using kinetic analysis of fluorescence from metarhodopsin combined with measurements of spectral absorption made before and after saturation at the isosbestic wavelength of the pigment pair. A curve fitting technique, in which a theoretical function is scaled for best fit to the measured absorption spectrum of the photosteady-state mixture, yields values for the photosensitivity of rhodopsin at λ._max, the ratio of quantum efficiencies for rhodopsin—metarhodopsin interconversion, and the fractional composition of the steady-state mixture. With knowledge of the molecular extinction coefficient, the absolute values of quantum efficiency can be calculated. For crayfish ( Orconectes, Procambarus ) rhodopsin, measured in isolated rhabdoms, K_max= 1.05 x 10^-16 cm² at 535 nm with >7λR→M0.69. These values are similar to the photosensitivity and quantum efficiency of bleaching of vertebrate rhodopsins in digitonin solution (Dartnall, 1972). For the metarhodopsin, K_max= 1.02 x 10^-16 cm² at 510 nm, and λ_M-R= 0.49.     

正离子表面活性剂与负离子表面活性剂在水溶液中的相互作用

本工作研究了碳氢链较短的溴化辛基三甲铵(简写为C₈NMe₃Br)及辛基硫酸钠(简写为C₈SNa)混合水溶液的一些表面化学性质:在表面及“油-水”界面上的混合吸附、在表面上的气泡寿命和在界面上的液滴寿命以及溶液在石蜡表面上的润湿性能.从这些性质了解正、负离子表面活性剂的相互作用.结果表明:(1)混合表面活性剂有很高的表面活性.1:1C₈NMe₃Br-C₈SNa混合物的临界胶团浓度(cmc)为～7.5×10^-3m,远小于单一表面活性剂.在此浓度时,表面张力低达～23达因/厘米;溶液-正庚烷的界面张力更低,达～0.2达因/厘米,亦远低于单一表面活性剂;(2)混合表面活性剂溶液有很高的气泡寿命及液滴寿命,亦即有较大的表面膜及界面膜强度;(3)混合溶液比单一表面活性剂的润湿性能好;(4)不同比例的C₈NMe₃Br-C₈SNa混合溶液的表(界)面总吸附量与1:1混合溶液的相近,当浓度较大时,吸附层中两者比例大多接近1:1,且表(界)面张力亦甚低:(5)与一般离子型表面活性剂的情形完全不同,无机盐对1:1混合溶液的表(界)面张力影响很小.计算吸附量时,Gibbs公式应取1RT形式,而不采用对一般离子型表面活性剂适用的2RT形式.由此得出的最小平均“分子”面积为27～29Ų,表明吸附层中表面活性离子排列紧密,膜凝聚性强.上述结果充分说明正、负离子表面活性剂在水溶液中有强烈的相互作用,其本质主要在于附加的正、负电荷的库仑引力,由此导致正、负离子表面活性剂混合溶液的一系列表面化学特性.