Lipid-protein interaction in the photolysis of octopus rhodopsin

Microvillar membranes of octopus photoreceptor cells were treated with phospholipase A₂, phospholipase C, hexane, or their combinations. By these means, various membrane preparations containing qualitatively and quantitatively different lipids were obtained. The lipid composition and phospholipid content of the membrane preparations obtained by the above methods were determined.Photochemical processes in the digitonin extract of the native and treated membranes have been studied by flash photometry. The results suggest that several different variations in the lipids can affect the rates of the photochemical transformations; these are: the content of phospholipid, the amount of unsaturated hydrocarbon chains and free fatty acids.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. II

Frog (Rana catesbeiana) rod outer segment membrane contains cyclic GMP phosphodiesterase (EC 3.1.4.1). Irradiation of dark-adapted rod outer segment membrane increased the enzyme activity by 5–20-fold in the presence of GTP. The phosphodiesterase in rod outer segment membrane is also activated by mixing a photo-product of 11-cis (regenerated), 9-cis or 7-cis rhodopsin which is stable at 0°C. However, neither opsin in the membrane nor all-trans retinal activates the enzyme. The phosphodiesterase in rod outer segment membrane is also activated by irradiation at ?4°C. Thus, we conclude that the phosphodiesterase is activated by a common photolysis intermediate of these rhodopsin isomers, perhaps before metarhodopsin II decays.     

Flash photolysis using a light emitting diode: an efficient, compact, and affordable solution

Flash photolysis has become an essential technique for dynamic investigations of living cells and tissues. This approach offers several advantages for instantly changing the concentration of bioactive compounds outside and inside living cells with high spatial resolution. Light sources for photolysis need to deliver pulses of high intensity light in the near UV range (300-380 nm), to photoactivate a sufficient amount of molecules in a short time. UV lasers are often required as the light source, making flash photolysis a costly approach. Here we describe the use of a high power 365 nm light emitting diode (UV LED) coupled to an optical fiber to precisely deliver the light to the sample. The ability of the UV LED light source to photoactivate several caged compounds (CMNB-fluorescein, MNI-glutamate, NP-EGTA, DMNPE-ATP) as well as to evoke the associated cellular Ca(2+) responses is demonstrated in both neurons and astrocytes. This report shows that UV LEDs are an efficient light source for flash photolysis and represent an alternative to UV lasers for many applications. A compact, powerful, and low-cost system is described in detail.     

Flash photolysis of rhodopsin in the cat retina

The bleaching of rhodopsin by short-duration flashes of a xenon discharge lamp was studied in vivo in the cat retina with the aid of a rapid, spectral-scan fundus reflectometer. Difference spectra recorded over a broad range of intensities showed that the bleaching efficacy of high-intensity flashes was less than that of longer duration, steady lights delivering the same amount of energy. Both the empirical results and those derived from a theoretical analysis of flash photolysis indicate that, under the conditions of these experiments, the upper limit of the flash bleaching of rhodopsin in cat is approximately 90%. Although the fact that a full bleach could not be attained is attributable to photoreversal, i.e., the photic regeneration of rhodopsin from its light-sensitive intermediates, the 90% limit is considerably higher than the 50% (or lower) value obtained under other experimental circumstances. Thus, it appears that the duration (approximately 1 ms) and spectral composition of the flash, coupled with the kinetic parameters of the thermal and photic reactions in the cat retina, reduce the light-induced regeneration of rhodopsin to approximately 10%.     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a³⁺a²⁺₃-CO) complex and the mixed-valence carboxycytochrome c oxidase (a³⁺a²⁺₃-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a²⁺₃-CO complex. This would confirm that the spectral properties of cytochrome a₃ are not affected by the redox state of cytochrome a and Cu_A. When the absorbance changes after photolysis of cytochrome a²⁺₃-CO with a laser flash were followed on a time scale from 1 μs to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7·10³s^?1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a₃. Concomitantly, a decrease of the 830 nm band was observed due to reduction of Cu_A. This demonstrates that in the partially reduced enzyme a pathway is present between Cu_A and the cytochrome a₃-Cu_B pair, via which electrons are transferred rapidly.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. I.

Guanosine 3′,5′-cyclic monophosphate phosphodiesterase (EC 3.1.4.1) in frog rod outer segment prepared by a sucrose stepwise density gradient method was activated by light in the presence of GTP. Rhodopsin in rod outer segment was solubilized with sucrose laurylmonoester and then purified by concavanalin A-Sepharose column. Addition of photo-bleached preparation of the purified rhodopsin to the rod outer segment, which had been prepared by 43% (w/w) sucrose floatation, caused the activation of phosphodiesterase in the dark, while each component of the photo-product eluted from the column (all-trans retinal and opsin) did not. Regenerated rhodopsin prepared from 11-cis retinal and purified opsin activated phosphosdiesterase when it was bleached. From these facts it is suggested that an intermediate or a process of photolysis of rhodopsin causes activation of phosphodiesterase.     

Spatially confined diffusion of calcium in dendrites of hippocampal neurons revealed by flash photolysis of caged calcium

The extent of diffusion of a locally evoked calcium surge in dendrites of cultured hippocampal neurons was studied by flash photolysis of caged EGTA. Cells were transfected with pDsRed for visualization, preincubated with caged NP-EGTA (AM) and Fluo-4 (AM) at room temperature and imaged in a PASCAL confocal microscope. Pulses of UV laser light within an active sphere of about 1 micro m(2) produced a rise of Fluo-4 fluorescence transients in dendrites which peaked at 1 ms and decayed exponentially with a fast (8-10 ms) time constant. A slower decay component was uncovered following incubation with thapsigargin. Lateral diffusion of [Ca(2+)]i did not vary significantly among different size dendrites being symmetric and reaching about 3-3.5 micro mm at a diffusion rate of 0.8 micro mm/ms on both sides of the photolysis center. Fluo-4 was also replaced by the membrane-bound Fluo-NOMO (AM) or by the 'heavy' Calcium Green dextran (CGd) loaded through a patch pipette. Similar rates of diffusion were found in these cases, indicating that the diffusion is not of the dye complexed to calcium but of genuine free calcium ions. Interestingly, presence of a dendritic spine at the focus of photolysis significantly reduced [Ca(2+)]i spread while the focal transient remained unaffected. Finally, [Ca(2+)]i diffused about twice as far from the photolysis sphere in glass tubes of a similar diameter to that of a dendrite, indicating that intrinsic calcium uptake mechanisms in the dendrite determine the diffusion of calcium away from its original site of rise.     

Binding of rhodopsin and rhodopsin analogues to transducin,rhodopsin kinase and arrestin-1

AIM: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. METHODS: Rod outer segments(ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtainedby extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-(3H)-guanosine 5'-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of(γ-32P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. RESULTS: Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover, only a basal amount of the reconstituted rhodopsin and 9-cis-retinal-rhodopsin was phosphorylated by rhodopsin kinase in the dark, whereas the pigment containing the 13-cis-retinal was highly phosphorylated by rhodopsin kinase even in the dark. In addition, arrestin-1 was incubated with rhodopsin, 9-cis-retinal-rhodopsin or 13-cis-retinal-rhodopsin. Experiments were performed using both phosphorylated and non-phosphorylated regenerated pigments. Basal amounts of arrestin-1 interacted with rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin under dark and light conditions. Residual arrestin-1 was also recognized by the phosphorylated rhodopsin and phosphorylated 9-cis-retinal-rhodopsin in the dark. However, arrestin-1 was recognized by phosphorylated 13-cis-retinal-rhodopsin in the dark. As expected, all reformed pigments were capable of activating transducin and being phosphorylated by rhodopsin kinase in a lightdependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. CONCLUSION: In the dark, the rhodopsin analogue containing the 13-cis isomer of retinal appears to fold in a pseudo-active conformation that mimics the active photointermediate of rhodopsin.     

Transient spectra of intermediates in the photolytic sequence of octopus rhodopsin

The intermediate photolytic sequence of octopus rhodopsin was studied at different temperatures and different pH values by means of a flash photolysisrapid scan spectrophotometry near physiological temperature.The first photoproduct in the photolysis of rhodopsin was lumirhodopsin. Transformation of lumirhodopsin → mesorhodopsin took place independently of the pH of the solution. Mesorhodopsin was transformed to acid metarhodopsin in acid solution. In alkaline solution, mesorhodopsin was transformed to transient acid metarhodospsin whose absorption spectrum was similar to acid metarhodopsin. Transient acid metarhodopsin was then transformed to alkaline metarhodopsin reaching a tautomeric equilibrium which was determined by the pH of the solution.     

Carboxyl terminal of rhodopsin kinase is required for the phosphorylation of photo—activated rhodopsin

Human rhodopsin kinase (RK) and a carboxyl terminus-truncated mutant RK lacking the last 59 amino acids (RKC) were expressed in human embryonic kidney 293 cells to investigate the role of the carboxyl terminus of RK in recognition and phosphorylation of rhodopsin.RKC,like the wild-type RK,was detected in both plasma membranes and cytosolic fractions.The Cterminal truncated rhodopsin kinase was unable to phosphorylate photo-activated rhodopsin,but possesses kinase activity similar to the wild-type RK in phosphorylation of small peptide substrate.It suggests that the truncation did not disturb the gross structures of RK catalytic domain.Our results also show that RKC failed to translocate to photo-activated rod out segments.Taken together,our study demonstrate the carboxyl terminus of RK is required for phosphorylation of photo-activated rhodopsin and strongly indicate that carboxyl-terminus of RK may be involved in interaction with photo-activated rhodopsin.     

Principal absorption axes of rhodopsin and prelumirhodopsin

The experimental data on the absorption of plane polarized light by a solution of cattle rhodopsin at –196 C have been theoretically analysed to model the directional absorption properties of rhodopsin and prelumirhodopsin. It has been found that rhodopsin and prelumirhodopsin are planar absorbers having ratios of about 1007 and 1004, respectively, between the extinction coefficients along the long axis and perpendicular to it. These results support that the chromophore in prelumirhodopsin is more linear than the chromophore in rhodopsin.Work partially supported by Department of Science and Technology (India)Associated with the Biochemistry Cell     

Flash photolysis and low temperature photochemistry of bovine rhodopsin with a fixed 11-ene.

Nonbleachable rhodopsins containing retinal moieties with fixed 11-ene structures have been prepared. When the nonbleachable rhodopsin analogue corresponding to the natural pigment was flash-photolysed at 20.8 degrees C, no absorption changes occurred at the monitoring wavelengths of 380, 480, and 580 nm for the time range of 2 microseconds--10 s. This observation is in contrast to that of natural rhodopsin which showed the formation of metarhodopsin I and its decay to meta II. Irradiation of the artificial rhodopsin, 77 K, with light of 460 and 540 nm, also gave no spectral changes; in the case of natural rhodopsin, however, the irradiation leads to formation of the red-shifted intermediate bathorhodopsin. The absence of photochemistry in the artificial pigment shows that an 11-cis to trans photoisomerization of the retinal moiety is a crucial step in inducing the chain of events in te photolysis of rhodopsin.     

Transient light-induced conformational changes in rhodopsin

Arguments are presented which support the possibility that the unfolding of the rhodopsin molecule during photolysis up to the stage of metarhodopsin II is followed by a spontaneous refolding of the protein, once the isomerized retinaldehyde has left its original binding site. Such a transient conformational change might imply a very similar conformation for rhodopsin and opsin, apart from the presence of the chromophore.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Isoelectric focusing of phosphorylated cattle rhodopsin

³²P-rhodopsin was partially separated by isoelectric focusing into several fractions of different phosphorylation extent. It was found that the incorporated phosphate is not uniformly distributed in a population of rhodopsin molecules. In a preparation with an average phosphorylation extent of 2.4 moles of phosphate per mole of rhodopsin, most of the ³²P-phosphate was found in fractions where 4–5 phosphates are bound per rhodopsin, whereas a large fraction of the total rhodopsin was not phosphorylated at all. The maximum number of phosphate binding sites in rhodopsin appears to be at least five.Abbreviations used P/Rh moles of phosphate per mole of rhodopsin - ROS rod outer segments Presented in part at the EMBO workshop on Transduction Mechanism of Photoreceptors, held in Jülich, Germany, on 4–8 October, 1976     

Interaction of bovine rhodopsin with calcium ions

The formation of metarhodopsin II in various bovine rhodopsin preparations (rod outer segment (ROS) suspensions and rhodopsin-detergent solutions) was measured by means of flash spectrophotometry. The half-lifetime and formation of metarhodopsin II in ROS did not depend on the calcium concentration in the range of less than 10^–9 M (using EGTA or EDTA) to 15×10^–3 M calcium at pH values of 5.0, 7.1, and 9.0 (Table 1).The regeneration of rhodopsin from opsin by adding 11-cis retinal to ROS-suspensions and rhodopsin digitonin solutions was measured spectrophotometrically. It was not substantially different in either saline, one containing less than 10^–7 M calcium (by adding EGTA), the other containing 10^–3 M calcium (Table 2).Abbreviations A absorption - A absorption change - CTAB N-Cetyl-N,N,N-trimethylammoniumbromide - E₇₀₀ extinction at =700 nm - EDTA ethylenediamine-NNNN-tetraacetic acid - EGTA 2,2-ethylenedioxybis [ethyliminodi (acetic acid)] - MI metarhodopsin I - MII metarhodopsin II - Rh rhodopsin - ROS rod outer segment This work is based upon a Ph. D. dissertation (Nöll, 1974) and was presented in part at the Jahrestagung der Deutschen Gesellschaft für Biophysik, Freiburg, Germany, October 1974     

Spectral studies on the conformation of rhodopsin

A summary is given of visible and ultraviolet spectral studies which deal with the conformation of rhodopsin in situ and solubilized with detergent. Emphasis is placed on studies which give specific information about the macromolecular structure and which set quantitative limits on the magnitude of light-induced conformational changes.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Specific photoisomerization of retinal in squid rhodopsin and metarhodopsin

The composition of retinal isomers in the photosteady-state mixtures formed from squid rhodopsin and metarhodopsin was determined by high-pressure liquid chromatography. A large amount of 9-cis-retinal was obtained at liquid N₂ temperature when rhodopsin was irradiated with orange light, but only small quantities of 9-cis-retinal were obtained at 15°C. Scarcely any 9-cis-retinal was produced from metarhodopsin by irradiation at liquid N₂ temperature. A large quantity of 7-cis-retinal was found in the photoproduct of rhodopsin irradiated at solid carbon dioxide temperature, but not at 15°C and liquid N₂ temperature. 7-cis-Retinal was not produced from metarhodopsin at any temperatures. These results indicate that the photoisomerization of retinal is regulated by the structure of the retinal-binding site of this protein. The formation of 9-cis- and 7-cis-retinals is forbidden in the metarhodopsin protein.     

Two forms of intermediates of frog rhodopsin in rod outer segments

Using frog rod outer segments, we measured changes of the absorption spectrum during the conversion of rhodopsin to a photosteady-state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (440 nm) at ? 190°C and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (718 nm) at ? 190°C. The reaction kinetics was expressed by one exponential in the former case and by two exponentials in the latter. These results suggest that rhodopsin is composed of a single molecular species, while bathorhodopsin is composed of two kinds of molecular species designated as batho₁-rhodopsin and batho₂-rhodopsin. On warming the two forms of bathorhodopsin, each bathorhodopsin converted to its own lumirhodopsin, metarhodopsin I and finally a free all-trans-retinal plus opsin. The absorption spectra of the two forms of bathorhodopsin, lumirhodopsin and metarhodopsin I were measured at ? 190°C. We infer that a rhodopsin molecule in the excited state relaxes to either batho₁-rhodopsin or batho₂-rhodopsin, and then converts to its own intermediates through one of the two parallel pathways.     

Mutational analysis of the rhodopsin gene in Chinese ADRP families by conformation sensitive gel electrophoresis

Retinitis pigmentosa is a very heterogeneous group of retinal degenerations, with multiple genes identified in each mode of inheritance. For autosomal dominant retinitis pigmentosa (ADRP), the most common gene is the rhodopsin (RHO) gene, mutations in which contribute to about 25% of ADRP in Caucasian population. To investigate the frequency and pattern of RHO point mutations in Chinese patients with ADRP, we have screened the five coding exons and splice sites of the RHO gene in 50 unrelated probands from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing. Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified each in one family, thus the frequency of RHO mutations among ADRP families in this study is less than 14% (2/50=4%, 95% confidence interval: 1-14%), lower than that in Europe and North America, which may reflect an ethnic difference between Chinese and Caucasian populations. Loss of all phosphorylation sites at the C-terminus and a highly conserved sequence QVS(A)PA may occur because of Pro327(1-bp del). CSGE was found to be a sensitive, simple and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.