Reiko 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.

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.

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.

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.

Biochemical Properties of Retinochrome

The cephalopod retina has two kinds of photosensitive pigments. These pigments have been examined in various squids and octopi (1–4), and are called rhodopsin and retinochrome(5). Rhodopsin is present in the outer segments of the visual cells, and catches the light for visual excitation. On the other hand, retinochrome is abundantly contained in the inner segments of the visual cells. When the outer and inner portions of the retina are collected separately, each photopigment can be extracted by digitonin. The absorption spectrum of squid retinochrome is shown in Fig. 1. The shapes of the spectra of retinochrome and rhodopsin are very similar. Retinochrome is a chromoprotein that includes retinaldehyde as prosthetic group. However, retinochrome is different from rhodopsin not only in its location in the retina but also in photochemical behaviour, molecular architecture, and other chemical properties (5–7). The most essential difference is in the stereoisomeric configuration of the prosthetic group. Rhodopsin has the 11-cis isomer of retinal, whereas retinochrome has the all-trans. The outline of our earlier research on retinochrome is presented in several reviews (8–10).

Squid m-retinochrome: Two forms of metaretinochrome

When retinochrome absorbs light, it bleaches to m-retinochrome, which may act as a direct supplier of 11-cis-retinal to protein opsin to form rhodopsin. The present experiments were aimed at further elucidating the molecular state of m-retinochrome. Retinochrome and m-retinochome were mixed each with sodium borohydride, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resultant fluorescence bands were examined. The reduced retinochrome showed only one band due to N-retinyl protein, whereas the reduced m-retinochrome had two bands. When extracted with successive volumes of n-hexane, m-retinochrome released the 11-cis-retinal chromophore in a time-course consisting of fast and slow phases. These findings indicated that m-retinochrome exists in two forms, with loose and tight coupling of the chromophore to the protein moiety. Those forms were usually balanced in a molar ratio of about 1:2, and the proportion of the tight form of m-retinochrome was increased in the presence of excess 11-cis-retinal. Based upon these findings, the role of m-retinochrome in the visual cells is discussed.

Dependency of absorption characteristics of retinochrome on pH and salts

Abstract It has been suggested that, in the inner segments of the visual cells of cephalopods, retinochrome may be located in lamellated structures, often called myeloid bodies. When the outer segment-free retinas are homogenized and divided into light and heavy fractions by centrifuging in 43% sucrose, most of the myeloid bodies are concentrated into the light fraction, from which one can easily prepare fine extract of retinochrome. According to this improved method of preparation, the yield of retinochrome from the light fraction reaches more than 80% of the total contained in the inner segments. Within a pH range of about 5–7, the absorption maximum ( λ max ) of retinochrome remains near 495 nm, irrespective of the pH of the medium. In the presence of salt, however, its λ max moves with changes of pH, as shifted to 476 nm in 1 m -NaCl by lowering the pH from 6·2 to 4·8. The mere addition of a variety of 1 m -salt (NaCl, KCl, MgCl 2 and KBr) also causes the λ max 495 nm at about pH 5·5 to shift 10–20 nm toward shorter wavelengths, maximally to 476 nm. In any case, these blue shifts can be reversed by raising the pH and an isosbestic point always appears near 477 nm. On irradiation with orange light, the acid extract of retinochrome is usually bleached exponentially in the absence of salt. With increasing salt concentration, the photobleaching becomes slow, and its overall process is more or less deviated from the first-order kinetics. When the sample finally has λ max 476 nm, the bleaching again follows a first-order process, although its rate is greatly reduced. The present experiments shows that retinochrome at weakly acid pH exists in two tautomeric forms with their own λ max at about 495 nm and 476 nm, depending on ionic conditions.

[29] Cephalopod retinochrome

Publisher Summary This chapter discusses the cephalopod retinochrome. The cephalopod retina has a dual system of photosensitive chromo-proteins 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. The visual cell is very lanky, and separated by a narrow neck into inner and outer segments; the boundary between the two segments forms a basement membrane parallel to the surface of the retina. The outer segment carries the rhabdomeres, which are made up of compact piles of rhodopsin-containing microvilli. Rhodopsin is the key substance for vision. Retinochrome can also be extracted from the released fragments of outer segments. According to experiments with retinochrome antibody, a fluorescein isothiocyanate (FITC) fluorescence pattern clearly showed that retinochrome is abundant in the basal parts of the outer segments and in the inner segments.