Chicken Iodopsin Research Articles

Does the Chromophore’s Ring Move after Photoexcitation of Rhodopsin?

By comparing the shift of the absorption maxima when a visual pigment is converted to its lumirhodopsin photointermediate for two classes of pigments, we can infer whether or not the pigment's beta-ionone ring has left its binding site. We compare this shift for the long-wavelength sensitive visual pigment of chicken iodopsin (lambdamax = 571 nm), which has polar residues in the ring binding site that interact with the ring, with that for three pigments, which do not. We conclude that by the time the Lumi product of the pigment is formed, the ring has moved away from the ring binding site.

O-Glycosylation of G-protein-coupled receptor, octopus rhodopsin : Direct analysis by FAB mass spectrometry

In addition to the N-glycan that is evidently conserved in G-protein-coupled receptors (GPCRs), O-glycans in the N-terminus of GPCRs have been suggested. Using a combination of enzymatic and manual Edman degradation in conjunction with G-protein coupled receptor mass spectrometry, the structure and sites of O-glycans in octopus rhodopsin are determined. Two N-acetylgalactosamine residues are O-linked to Thr4 and Thr5 in the N-terminus of octopus rhodopsin. Further, we found chicken iodopsin, but not bovine rhodopsin, contains N-acetylgalactosamine. This is the first direct evidence to determine the structure and sites of O-glycans in GPCRs.

Structure and photobleaching process of chicken iodopsin

Iodopsin, a dominant cone pigment in a chicken retina, has an absorption spectrum in longer wavelength region than rhodopsin. To account for this red-shift of iodopsin, we had proposed a structural model from retinal analogue experiments, in which iodopsin would have a relatively long distance between the protonated Schiff base nitrogen and the counterion. This was confirmed by a resonance Raman spectroscopy. The photochemical properties of iodopsin were studied and compared with those of rhodopsin, which revealed the following differences. The regeneration rate of iodopsin with 11- cis-retinal was 240 times faster than rhodopsin. Meta-iodopsin II, the signalling state of iodopsin, decayed about 100 times faster than meta-rhodopsin II. The K m value of meta-iodopsin II and rhodopsin kinase was lower than meta-rhodopsin II. These results are in consistent with rapid adaptation and low photosensitivity of cones relative to those of rods.

Visual pigments in the pineal complex of the Japanese quail, Japanese grass lizard and bullfrog: Immunocytochemistry and HPLC analysis

We investigated localization of visual pigments in the pineal complex of Japanese quail, Japanese grass lizards and bullfrogs immunocytochemically by use of the antiserum against bovine rhodopsin (Rh-As) and monoclonal antibodies against chicken iodopsin (Io-mAb). We also analyzed retinoids, chromophores of visual pigments, by a high performance liquid chromatography (HPLC). The outer segments and cell membranes of some photoreceptor cells in the pineal organ of the Japanese quail exhibited immunoreactivity to Rh-As, but there are also many immunonegative cells. The number of immunoreactive cells among individuals varied. Immunoreactivity to Io-mAb was weak or did not exist. The HPLC analysis revealed peaks of 11-cis and all-trans isomers of retinal in the oxime extracts of the pineal organ of Japanese quail and chickens. In the pineal of Japanese grass lizards, the outer segments of some cells were immunopositive to Io-mAb, but there were no cells immunoreactive to Rh-As. The parietal eye exhibited a well-developed lens and photoreceptor cells, but the outer segments of photoreceptor cells were immunonegative to both Rh-As and Io-mAb. In bullfrogs, three types of cells were identified in both the pineal and frontal organ; (1) immunopositive to Rh-As, (2) immunopositive to Io-mAb and (3) immunonegative to either of the antibodies. In the pineal organ of bullfrogs, 11-cis and all-trans retinal and 11-cis 3-dehydroretinal were detected, and 11-cis and all-trans retinal were also detected in the frontal organ. We detected 11-cis and all-trans retinal in the ventral part of diencephalon including the hypothalamus. Thus, the chromophore is the same between the retinal and pineal visual pigments, but the expression of opsins is different between the retina and pineal complex, which probably reflects the different function of each organ.

Identification of the α-Subunits of Rod and Cone Transducin in Chicken Photoreceptor Cells

The chicken retina has several types of cone photoreceptor cells, each of which contains a visual pigment, chicken red (iodopsin), green, blue or violet. Although biochemical and photochemical properties of these cone pigments have been well characterized, no information is available about the chicken photoreceptor G-protein, transducin, which couples with the visual pigment to convert a photon signal into a cellular response. To identify α-subunits of chicken rod and cone transducins (T rα and T cα, respectively), we produced two site-directed antibodies which discriminate between bovine T rα and T cα. Immunohistochemical studies on chicken retinas revealed that the antibody against bovine T rα specifically stained the rod outer segments. On the other hand, the antibody against bovine T cα uniformly stained the outer segments of the double cones and all types of single cones, while the single cones were immunohistochemically classified into three types by using a combination of antibodies against bovine rhodopsin and chicken iodopsin. Immuno-blot analyses demonstrated that the antibody against T cα recognized a single band of chicken photoreceptor protein, whose molecular weight (42000) was in good agreement with that of bovine T cα (41000). The antibody against T rα recognized a protein having the same molecular weight as that of bovine T rα (39000). These observations strongly suggested that all types of chicken cone cells have a single common T cα (42 kDa) structurally related to bovine T cα, though each cone cell type has a distinct visual pigment.

Phosphorylation of iodopsin, chicken red-sensitive cone visual pigment

The amino acid sequence has been determined for the carboxyl-terminal 41 amino acids of chicken red-sensitive cone pigment, iodopsin. This sequence is distinct from but structurally homologous to that of other visual pigments. It contains a region rich in the hydroxy amino acids serine and threonine. In the related rod cell visual pigment, rhodopsin, such serines and threonines have previously been identified as sites for phosphorylation by rhodopsin kinase. Phosphorylation of photolyzed rhodopsin serves to terminate its ability to function in visual transduction as an activator of G-protein. We have purified and reconstituted both chicken rhodopsin and chicken iodopsin and shown them to be phosphorylated by bovine rhodopsin kinase. Chicken iodopsin has a Km and Vmax similar to but distinguishably different from that for bovine rhodopsin. These results, in conjunction with other data, suggest that visual pigments in cone cells, upon absorption of light, undergo functional processes similar to those of the visual pigments in rod cells.

Effects of chloride on chicken iodopsin and the chromophore transfer reactions from iodopsin to scotopsin and B-photopsin

Spectroscopic properties of chicken iodopsin were investigated in correlation with the concentration of chloride in digitonin extracts. When chloride in the extract was depleted by extensive dialysis, chloride-depleted iodopsin (absorption maximum, 512 nm) was formed. It was converted to chloride-bound iodopsin (absorption maximum, 562 nm) by the addition of chloride in the extract. There existed an equilibrium between two forms of iodopsin with a dissociation constant of 0.8 mM chloride. The chromophore-transfer reaction from iodopsin to scotopsin or B-photopsin, the protein moiety of chicken rhodopsin or chicken blue-sensitive cone pigment, respectively, in digitonin extract was also investigated in correlation with the concentrations of chloride, other monovalent and divalent anions, and detergent. The chromophore of chloride-depleted iodopsin was easily transferred to scotopsin in the extract, resulting in formation of rhodopsin. On the other hand, chloride-bound iodopsin was fairly stable even in the presence of scotopsin, indicating that the reaction is inhibited by binding of chloride to iodopsin. The chromophore-transfer reaction to B-photopsin was also observed from chloride-depleted iodopsin but not from chloride-bound iodopsin. The reaction was observable in the 10% digitonin extract as well as in the 2% digitonin extract. The reaction was also observed when 25 mM Na2SO4 was present in the mixture instead of NaCl, but was not when 67 mM NaNO3 was present. All these facts suggest that the chloride binding site of iodopsin does not accept a divalent anion such as SO4(2+), but does accept a monovalent anion such as Cl- or NO3-, which causes inhibition of the chromophore transfer.

Monoclonal antibodies to chicken iodopsin

The protein moiety of chicken iodopsin, R-photopsin, was purified from the chicken retina using a sucrose flotation method followed by two steps of column chromatography. Apparent molecular weights of R-photopsin and scotopsin (the protein moiety of chicken rhodopsin), which was partly purified in the process of purification of R-photopsin, were estimated to be 34,000 and 36,000, respectively, by sodium docecylsulfate-polyacrylamide gel electrophoresis. Using the purified R-photopsin as an antigen, four kinds of hybridoma cells which secreted monoclonal antibodies specific for R-photopsin and iodopsin were prepared. The antibodies thus obtained reacted with neither other chicken cone visual pigments nor rhodopsin as analyzed by immunoblots and immunoprecipitation methods. All the monoclonal antibodies stained the majority of the cone outer segments in chicken retina, while an antiserum raised against cattle rhodopsin stained the rod outer segments as well as some cone outer segments in the retina.

Monoclonal antibodies specific to chicken iodopsin

Monoclonal antibodies (mABs) from hyridoma cells were raised and screened with a purified cone pigment, iodopsin, from the chicken retina. Four different methods were used to test these antibodies: (1) dot-immunobinding assay; (2) enzyme-linked immunoabsorbent assay (ELISA); (3) one dimensional immunoblotting (4) two dimensional immunoblotting. Three classes of antibody producing cell lines were identified. One class produces a mAB specific to iodopsin. The mAB from the second class crossreacts with iodopsin and probably one of the other three cone pigments. The mAB from the third class probably crossreacts with all the four cone pigments in the chicken retina. The mABs from all these classes of hybridoma cell lines were selected so that they do not crossreact with rhodopsin. Two dimensional immunoblotting indicated that iodopsin has a much higher isoelectric point than rhodopsin, as suggested from the known amino acid sequences of human rod and cone pigments.

Activation of phosphodiesterase by chicken iodopsin

Activation of guanosine 3′,5′-cyclic monophosphate phosphodiesterase in outer-segment membrane of chicken retina was investigated. Irradiation of dark-adapted chicken outer segment membrane for bleaching of iodopsin increased the enzyme activity twice as much as that in the dark in the presence of GTP. Further irradiation of the sample for bleaching of rhodopsin in the membrane induced some additional activation of the enzyme. However, chicken iodopsin activated the enzyme in frog rod outer segment membrane without irradiation, while chicken rhodopsin did not. Irradiation of chicken iodopsin increased the enzyme activity twice as much as that in the dark.

THE POSSIBLE PRESENCE OF A THIRD INTERMEDIATE FOR IODOPSIN AT ‐195°C*

Abstract— From the theoretical analysis of the time variation of the change in absorbance of chicken iodopsin solution (at—195°C), the formation of a new intermediate during bleaching has been suggested.

Is proton transfer the initial photochemical process in vision?

THERE has been renewed effort1 to test the so-called proton transfer hypothesis (PTH)2 or hydrogen shift1 formulation for the initial photochemical step in vision. This hypothesis proposes an alternative to photochemical cis to trans isomerisation of rhodopsin, while attempting to account for some of the spectral properties of bathorhodopsin (previously called prelumirhodopsin), particularly certain low frequency lines at 920, 877 and 856 cm−1. Spectral lines in this region are not unique to the resonance Raman spectrum of bathorhodopsin3, however, for similar spectral absorption has been found with 11-cis retinal crystals4. The discovery that the batho- intermediate formed by irradiating chicken iodopsin at −195 °C spontaneously reverted to iodopsin at −140 °C by a dark, thermal process5, rather than decaying to lumiiodopsin, also seemed to require such an alternative hypothesis2 (Fig. 1). Although an attempt to demonstrate proton transfer by means of tritium incorporation into the retinylidene chromophore failed2, Fransen et al. apparently now confirm the PTH by their report1 of a small, though significant incorporation of deuterium into retinal. Their newer experiments are similar in design to the earlier ones, differing mainly in their use of deuterium rather than tritium and their method of analysis of labelled retinal. The experiments described here bear directly on the proposed PTH and were performed with an analogue of retinal, 5-desmethyl (dm) retinal: They show that the initial compound produced by irradiating a visual pigment, the batho-intermediate, cannot result from the transfer of a proton from the chromophore to opsin.

Photic reactions of chicken iodopsin and rhodopsin at liquid helium temperature

Irradiation of chicken rhodopsin at liquid helium temperature (4°K) yields hypsorhodopsin. which goes to bathorhodopsin on warming above 50° K in the dark. The absorption maximum of chicken hypsorhodopsin is at about 430 nm, as in cattle. The irradiation of chicken iodopsin at 4°K did not form hypsoiodopsin. The behaviour of iodopsin at 4°K is similar to that at 77°K. Whether the production of the hypso-intermediate occurs or not depends on the structure of opsin.