Tomiyuki Hara

[108] Cephalopod retinochrome

Publisher Summary This chapter discusses the cephalopod retinochrome. The cephalopod retina has a dual system of photosensitive chromoproteins with retinaldehyde as chromophores rhodopsin and retino-chrome. Rhodopsin is associated only with microvillar membranes of the outer segments of visual cells, whereas retinochrome is found in both inner and outer segments. The two photopigments differ from each other in the stereoisomeric form of their chromophore retinal, which is 11- cis in rhodopsin but all- trans in retinochrome. Retinochrome can be extracted also from the released fragments of outer segments. In the squid Loligo pealei , it is contained equally in both the inner and outer segments, and in Todarodes pacificus, much more is found in the outer segments. As retinochrome in the outer segments increases markedly during light adaptation of animal, it seems likely that some retinochrome protein moves forward from the inner to the outer segments and reacts there with metarhodopsin.

Retinal-binding protein as a shuttle for retinal in the rhodopsin-retinochrome system of the squid visual cells.

The molluscan visual cell is characterized by having two photopigment systems, rhodopsin and retinochrome. In connection with these systems, located separately in the rhabdomal microvilli and in the nucleated cell bodies, the physiological role of retinal-binding protein (RALBP) was investigated in the squid (Todarodes pacificus) by using 3-dehydroretinal (retinal 2) as a tracer for retinal chromophore. In dark-adapted eyes, squid RALBP is combined abundantly with 11-cis-retinal. However, upon incubation with an excess of all-trans-retinal or retinol, RALBP took up great amounts of each of them, releasing its native retinoid ligands. When an all-trans-retinal-rich RALBP thus produced was incubated in the dark with metaretinochrome 2-carrying membranes, the RALBP released all-trans-retinal to the membranes to regenerate retinochrome, taking up 11-cis-retinal 2 from metaretinochrome 2. Upon further incubation of this 11-cis-retinal 2-rich RALBP with metarhodopsin-carrying membranes, the RALBP released the 11-cis-retinal 2 to the membranes to form rhodopsin 2, receiving all-trans-retinal from metarhodopsin. These findings show that squid RALBP is capable of serving as a shuttle during the recycling of retinal in the rhodopsin-retinochrome conjugate system to maintain the photoreceptive function of the visual cells.

Cloning and nucleotide sequence of cDNA for retinochrome, retinal photoisomerase from the squid retina

The Rhodopsin‐retinochrome system is essential for the visual photoreception of molluscs. cDNA coding for retinochrome of the squid (Todarodes pacificus) was cloned and the nucleotide sequence has been determined. The sequence (2.1 kb) covers the whole coding region of 903 bp. The deduced primary sequence suggests that retinochrome contains seven transmembrane spanning domains. The homology with bovine rhodopsin and the possible retinal binding site are also discussed.

RECONSTITUTION OF SQUID RHODOPSIN IN RHABDOMAL MEMBRANES

Abstract— Squid opsin which is capable of combining with 11‐cis or 9‐cis retinal to reconstitute photo‐pigment has been prepared by irradiation of rhabdomal membranes with orange light (> 530 nm) in the presence of 0.2 M hydroxylamine. When the irradiation is carried out either at concentrations of hydroxylamine higher than 0.2 M or with light of wavelength shorter than 530 nm, rhodopsin in the membranes is bleached quickly, but the ability of the resultant opsin to form rhodopsin is greatly reduced.

Isolation and characterization of a retinal-binding protein from the squid retina

A retinal-binding protein (RALBP) was isolated from the squid retina, and purified by anion-exchange and size-exclusion chromatography. The molecular weight was determined to be 51,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration. The purified sample showed absorption maxima at about 330 and 400 nm in addition to a protein band, indicating the occurrence of retinol and retinal, respectively. The relative heights of these two peaks varied from preparation to preparation, depending on retinoid ligands. Irradiation of RALBP caused no marked change in absorption, but the amount of 11-cis-retinal decreased to form a photosteady state mixture with all-trans- and 13-cis-retinals. RALBP was fairly stable even in the presence of hydroxylamine (100 mM), but was affected by sodium borohydride (30 mM) or borane dimethylamine (400 mM), with the retinal reduced to retinol. When incubated with metaretinochrome-carrying membranes in the dark, RALBP specifically took up 11-cis-retinal and lost all-trans-retinol. Upon further incubation of this RALBP with opsin-containing membranes, rhodopsin was progressively formed in the dark. Squid RALBP may act as a shuttle in transferring the 11-cis-retinal from metaretinochrome to opsin in the visual cells.

Histochemical localization of retinochrome and rhodopsin studied by fluorescence microscopy

SummaryRetinochrome is readily reduced by sodium borohydride into an N-retinyl protein that emits visible fluorescence upon irradiation with near-ultraviolet light. Rhodopsin is also converted to a similar fluorescent product, but only when denatured with formaldehyde before reduction. Based upon this difference, retinochrome was discriminated from rhodopsin on frozen sections. The distribution of these two photopigments in various photosensitive tissues was examined by means of epifluorescence microscopy.In the octopus retina (Octopus vulgaris), the yellow-green fluorescence of reduced retinochrome was observed in both the basal regions of the outer segments and throughout the inner segments of the visual cells, while the fluorescence of reduced rhodopsin was restricted to within the rhabdomal layer of the outer segments. In the squid parolfactory vesicles (Todarodes pacificus), rhodopsin was present in the central lumen, which contains the distal processes of the photoreceptor cells, while retinochrome was detected in the myeloid bodies scattered within the vesicular wall. In the slug retina (Limax flavus), rhodopsin was found in the microvilli, and retinochrome appeared to be concentrated in the photic vesicles of the visual cells.

PHOTOCHEMISTRY OF RETINOCHROME

Abstract— Retinochrome is a photopigment found in the visual cells of cephalopods. It has been considered to act as a supplier of the 11‐cis‐retinal required for synthesis of rhodopsin, because its all‐trans chromophore is isomerized to 11‐cis form in the light. Light and thermal reactions of squid retinochrome were investigated by low‐temperature spectrophotometry.

Rhodopsin and retinochrome in the retina of a marine gastropod, Conomulex luhuanus

Photopigments in the conch retina were examined with special attention given to the photic vesicles characteristic of gastropod photoreceptors. Three different fractions of visual cell fragments were prepared from the retina: the MV-fraction containing the rhabdomal microvilli, and the PVH- and PVL-fractions containing the photic vesicles located in the visual cell body. Rhodopsin was found in the MV-fraction (lambda max = 474 nm), and yielded a photoequilibrium mixture with metarhodopsin (lambda max = 512 nm) on irradiation with blue light. Retinochrome was found in both of the PVH- and PVL-fractions (lambda max = approximately 510 nm), and was bleached into metaretinochrome by exposure to orange light, showing no marked shift of the absorption peak. Unlike the PVH-fraction, the PVL-fraction contains much aporetinochrome in addition to retinochrome, suggesting that the large mass of photic vesicles around the nucleus may serve as storage for retinal in retinochrome and for newly synthesized aporetinochrome. The total amount of retinochrome in the retina was several times higher than that of rhodopsin, distinguishing the gastropod eye from the cephalopod eye.

Reconstitution of squid and cattle rhodospin by the use of metaretinochrome in their respective membranes

Abstract Retinochrome is, even in membranes, converted to metaretinochrome by exposure to orange light. Upon incubation of metaretinochrome in membranes with cattle opsin in rod outer segment membranes, cattle rhodopsin is reconstituted in the dark. When opsin is present in molar excess to metaretinochrome, about 80% of the prosthetic retinal of retinochrome present initially is utilized for the reconstitution of cattle rhodopsin. One reason why all of the prosthetic retinal is not used for the rhodopsin reconstitution is that metaretinochrome transforms slowly to retinochrome during incubation in the dark and another is that metaretinochrome is in a photoequilibrium mixture with a trace of retinochrome after exposure to orange light. Squid rhodopsin is reconstituted when a mixture of metaretinochrome and squid opsin in their respective membranes is incubated in the dark. The reconstituted rhodopsin is converted to acid or alkaline metarhodopsin by exposure to orange light at neutral or alkaline pH, respectively. Three possible mechanisms for the transference of 11- cis retinal from metaretinochrome in a membrane to opsin in a different membrane were considered: (1) the migration of 11- cis retinal through an aqueous medium between the separate membranes, (2) the migration of 11- cis retinal from metaretinochrome to opsin in a fused membrane and (3) the transfer of retinal from membrane to membrane in close contact. In conclusion, the first two mechanisms were inapplicable and the third appeared to explain the present experimental findings. The possibility is discussed that the photoproduct of retinochrome may contribute to the rhodopsin synthesis as an effective donor of 11- cis retinal to opsin in the squid retina.

Squid retinochrome. Configurational changes of the retinal chromophore.

The configurations of the retinal chromophore in light and dark reactions of squid retinochrome were investigated by means of high-performance liquid chromatography. Orange light isomerized the chromophore of retinochrome, all-trans-retinal, mainly to the 11-cis configuration in metaretinochrome. Irradiation with shorter-wavelength lights not only accelerates the photoreversal of metaretinochrome to retinochrome but also leads to a slight production of isoretinochrome (13-cis-retinochrome), yielding a photoequilibrium mixture of three kinds of retinochrome. 13-cis- and 9-cis-retinochromes are photosensitive, and are converted into metaretinochrome upon irradiation with orange light. When steadily exposed to orange light in the presence of a trace of retinochrome-protein, all of the all-trans-, 13-cis-, and 9-cis-retinals are catalytically isomerized only to the 11-cis form, although the reaction rate is reduced in the order of the retinals listed above. In the dark, 9-cis-retinochrome, like retinochrome, remains unchanged, but both meta- and 13-cis-retinochromes slowly change to retinochrome. The chromophore of 13-cis-retinochrome changes directly to the all-trans form, whereas the 11-cis chromophore of metaretinochrome goes to all-trans mainly through the 13-cis form. The direct isomerization from 11-cis to all-trans hardly occurs at temperatures as low as 20 degrees C, and shows high values of the activation enthalpy and entropy changes. Based upon these findings, the role of retinochrome in the photoreception of the visual cells is discussed.