Tôru Yoshizawa

Carboxyl methylation and farnesylation of transducin gamma-subunit synergistically enhance its coupling with metarhodopsin II.

A heterotrimeric G‐protein in vertebrate photoreceptor cells is called transducin (T alpha beta gamma), whose gamma‐subunit is a mixture of two components, T gamma‐1 and T gamma‐2. T gamma‐2 is S‐farnesylated and partly carboxyl methylated at the C‐terminal cysteine residue, whereas T gamma‐1 lacks the modified cysteine residue. To elucidate the physiological significance of the double modifications in T gamma, we established a simple chromatographic procedure to isolate T gamma‐1, methylated T gamma‐2 and non‐methylated T gamma‐2 on a reversed phase column. Taking advantage of the high and reproducible yield of T gamma from the column, we analyzed the composition of T gamma subspecies in the T alpha‐T beta gamma complex which did not bind with transducin‐depleted rod outer segment membranes containing metarhodopsin II. The binding of T alpha‐T beta gamma with the membranes was shown to require the S‐farnesylated cysteine residue of T gamma, whose methylation further enhanced the binding. This synergistic effect was not evident when T alpha was either absent or converted to the GTP‐bound form which is known to dissociate from T beta gamma. Thus we concluded that a formation of the ternary complex, T alpha‐T beta gamma‐metarhodopsin II, is enhanced by the farnesylation and methylation of T gamma. This suggests that the double modifications provide most efficient signal transduction in photoreceptor cells.

Differentiation of both rod and cone types of photoreceptors in the in vivo and in vitro developing pineal glands of the quail

The avian pineal is a photo-endocrinal organ and is considered to synthesize and secrete melatonin in an intrapineal rhythm which can be modified by direct light stimulation of the pineal photoreceptors. Since the avian retina contains numerous different types of photoreceptors, at least 6 types in the quail retina, it is interesting to ask how many types of photoreceptors are present in the avian pineal. In the present study, we have identified two types of photoreceptors in the quail pineal organ, one appears rod-like and the other cone-like, using an immunohistochemical method with highly specific anti-chicken rhodopsin and anti-iodopsin monoclonal antibodies. Rhodopsin-immunoreactive (Rho-I) cells were much larger in number than iodopsin-immunoreactive (Iodo-I) cells. During pineal development, Rho-I cells were first observed at embryonic day 13 (E13: 13 days of incubation), whereas Iodo-I cells were found at day E15. Rho-I cells showed numerous neurite-like processes, but Iodo-I cells had few, if any, processes. We developed a new culture system for avian pineal cell differentiation by seeding cells on nitrocellulose membrane filters. By this method both types of pineal photoreceptors differentiated in vitro: Rho-I cells were much larger in number and had much more fine processes than Iodo-I cells, similar to those seen in the intact developing pineal. With the new culture system the relation between pineal photoreceptor differentiation and sympathetic innervation was examined in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

PHOTOSENSITIVITIES OF IODOPSIN AND RHODOPSINS

Abstract— The relative photosensitivity and the molar extinction coefficient of a highly purified iodopsin (chicken red sensitive cone visual pigment) solubilized in a mixture of 3–[(3‐cholamidopropyI)‐dimethylammonio]‐1‐propanesulfonate and phosphatidylcholine (CHAPS‐PC) were measured using bovine rhodopsin solubilized in 2% digitonin as a standard and compared with those of chicken and bovine rhodopsins. The photosensitivity obtained (1.08) was close to those of rhodopsins (chicken, 1.04; bovine, 0.99) in CHAPS‐PC. The molar extinction coefficient of iodopsin (47200) was 1.15–1.17 times higher than those of rhodopsins (chicken, 40500; bovine, 41200). The oscillator strength of iodopsin (0.60) calculated from the extinction coefficient was nearly identical to that of chicken rhodopsin (0.61), suggesting that the chromophore of iodopsin is similar in configuration to rhodopsin. In contrast, the difference in quantum yield between iodopsin (0.62) and chicken rhodopsin (0.70) suggests that the chromophore‐opsin interaction after absorption of a photon by the chromophore may be different.

THE ROAD TO COLOR VISION: STRUCTURE, EVOLUTION AND FUNCTION OF CHICKEN AND GECKO VISUAL PIGMENTS

It is indeed a great honor for me to have an opportunity to give an Honorary Lecture as the final talk of “The US-Japan Cooperative Seminar”. I would like to express my deep appreciation to the organizers, Drs. R. A. Mathies, R. S. H. Liu and T. Kakitani. When I was invited to the seminar by Dr. Kakitani, he gave me a title of my lecture “The Road to Color Vision”. Under the same title, I had delivered a talk as the final lecture at Kyoto University, where I retired under the agp, limit at the end of last March. At that time, I gave a historical review of my vision research. Dr. Kakitani did not want me to give such a retrospective story, simply because the seminar had been planned to integrate the recent achievements in the field of retinal proteins analyzed in physico-chemical aspects and to facilitate the further progress in research. Thus I would like to review our recent studies on cone visual pigments which have been carried out mainly in the last few years.

Localization of iodopsin and rod-opsin immunoreactivity in the retina and pineal complex of the river lamprey, Lampetra japonica

The aim of this study has been to examine whether iodopsin immunoreaction exists in the photoreceptor cells of the retina of the river lamprey, Lampetra japonica, and whether this immunoreaction also appears in the photoreceptors of the pineal complex. The lamprey retina possesses long and short photoreceptor cells that display iodopsin immunoreactivity and rod-opsin immunoreactivity, respectively. In the pineal organ, iodopsin immunoreaction has been observed in the peripheral region and the dorsal wall of the end-vesicle. Immunoreactivity is also found in the atrium and the pineal stalk. The iodopsin-immunoreactive outer segments are smaller than those displaying rod-opsin immunoreactivity. In the parapineal organ, iodopsin immunoreactivity is distributed in both dorsal and ventral portions. Double immunostaining has been employed to investigate whether iodopsin and serotonin immunoreactivities are colocalized in one and the same cell. This approach has revealed that the iodopsin-immunoreactive outer segments belong to serotonin-immunopositive and to serotonin-immunonegative photoreceptor cells. These results demonstrate that rod-type or cone-type visual pigments are contained in both typical and modified pineal photoreceptors.

Molecular basis for color vision.

Amino acid sequences of four kinds of chicken cone pigments and two kinds of nocturnal gecko visual pigment were determined. Calculations of amino acid identities indicate that gecko pigments should be cone pigments. A phylogenetic tree of visual pigments constructed demonstrated that cone pigments evolved earlier than rod pigments (rhodopsins), indicating that daylight vision including color vision appeared earlier than twilight vision. The divergence of cone pigments to rhodopsins would be caused by replacing basic amino acid residues to acidic ones according to net charge calculations. A comparison between chicken rhodopsin and cone pigments (chicken green and red) displayed that the cone pigments are faster in regeneration from 11-cis retinal and opsin, faster in formation of meta II-intermediate and shorter in lifetime of meta II-intermediate than rhodopsin. These facts would partly explain the rapid dark adaptation, the rapid light response and the low photosensitivity of cones compared with rods. In comparison with di- and tri-chromatic color visions, chicken tetra-chromatic vision was discussed on the basis of both absorption spectra of cone pigments and filtering effect of oil droplets.

CONFORMATIONAL ANALYSIS OF THE RHODOPSIN CHROMOPHORE USING BICYCLIC RETINAL ANALOGUES

Abstract— For investigation of the chromophore conformation around the trimethyl cyclohexene ring and of the origin of the induced β‐circular dichroism band in rhodopsin, two C6‐C7 single bond‐fixed retinal analogues, 6s‐cb‐ and 6s‐trans‐locked bicyclic retinals (6 and 7, respectively) were synthesized and incorporated into bovine opsin in CHAPS‐PC mixture. 6s‐cb‐ and 6s‐tram‐Locked rhodopsin analogues (8 and 9) with A max at 539 and 545 nm, respectively, were formed. Interestingly, both 8 and 9 displayed α‐ and β‐circular dichroism bands. The ellipticity of α‐bands are similar in each other, while the β‐band of 8 was about three times stronger than that of 9. Irradiation of 6s‐trans‐locked rhodopsin, 9, in the presence of hyroxylamine, resulted in the formation of only one of the enantiomers of 6s‐rrans‐locked retinal oxime showing a positive circular dichroism signal at around 390 nm. This fact strongly suggests that the retinal binding site of rhodopsin shows a chiral discrimination. From these experimental results, the interactions between the trimethyl cyclohexene ring portion in the chromophore and the neighbouring protein moiety in the rhodopsin molecule are discussed.

PHOTORECEPTOR CELL TYPES IN THE RETINA OF VARIOUS VERTEBRATE SPECIES: IMMUNOCYTOCHEMISTRY WITH ANTIBODIES AGAINST RHODOPSIN AND IODOPSIN

Abstract— Types of photoreceptor cells in the retinas of 36 species of vertebrates (5 classes, 14 orders) were investigated immunocytochemically with monoclonal antibodies against chicken iodopsin (Io‐mAb) and antiserum against bovine rhodopsin (Rh‐As). In mammals, Rh‐As labeled the outer segments of some photoreceptor cells in striped squirrels (a diurnal mammal) and those of most photoreceptor cells in mice (a nocturnal mammal), while Io‐mAb did not label any photoreceptor cells in either of them. In all species of birds studied, Io‐mAb labeled the principal and accessory members of double cones and single cones with a red oil droplet. Rh‐As labeled single cones with a yellow or clear oil droplet in addition to rods. In turtles, both Rh‐As and Io‐mAb labeled single cones with a red or clear oil droplet and the principal (with a yellow oil droplet) and accessory members of double cones. This suggests that the visual pigments in these cones of turtles have common epitopes with bovine rhodopsin and chicken iodopsin. In Japanese grass lizards, single cones with a yellow oil droplet and double cones were immunoreactive to both Rh‐As and Io‐mAb. In snakes, rods and cones could not be distinguished but both positively and negatively stained cells were observed by the use of each antibody. In geckos, however, all photoreceptor cells were immunonegative to Io‐mAb. In all species studied in amphibians, Rh‐As labeled rods but not cones. Neither rods nor cones reacted with Io‐mAb. In fishes, almost all species studied had well developed cones, and some of these cones were labeled by Rh‐As. However, Io‐mAb labeled the outer segments of some cones only in loaches. Rh‐As labeled photoreceptor cells in all species of fishes studied. Thus, Rh‐As recognized the outer segments of rods in all species studied from fishes to mammals, whereas the epitope recognized by Io‐mAb is conserved in some species of fishes, most species of reptiles and all species of birds studied.

Changes in structure of the chromophore in the photochemical process of bovine rhodopsin as revealed by FTIR spectroscopy for hydrogen out-of-plane vibrations

The hydrogen out-of-plane bending (HOOP) vibrations were studied in the difference Fourier transform infrared spectra of lumirhodopsin and metarhodopsin I by use of a series of specifically deuterated retinal derivatives of bovine rod outer segments. The 947 cm-1 band of lumirhodopsin and the 950 cm-1 band of metarhodopsin I were assigned to the mode composed of both 11-HOOP and 12-HOOP vibrations. This result suggests that the perturbation near C12-H of the retinal in the earlier intermediate, bathorhodopsin (Palings, van den Berg, Lugtenburg and Mathies, Biochemistry, 28 (1989) 1498-1507), is extinguished in lumirhodopsin and metarhodopsin I. Unphotolyzed rhodopsin and metarhodopsin I exhibited the 14-HOOP bands in the 12-D derivatives at 901 and 886 cm-1, respectively. Lumirhodopsin, however, did not show the 14-HOOP in the 12-D derivatives. The result suggests a change in geometrical alignment of the C14-H bond in lumirhodopsin with respect to the N-H bond of the Schiff base.