Takaharu Seki

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.

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.

[7] Isolation and purification of squid rhabdoms

Publisher Summary This chapter discusses the isolation and purification of squid rhabdoms. The technique for isolating rhabdomeric membranes is based on the method of Hubbard and St. George. All procedures are performed under dim red light at 0–4° unless otherwise specified. Eyes previously frozen and stored in the freezer are used as starting material. If the squid eye was not previously frozen or if octopus eyes are being used, the retinular outer segments are not as readily detached from the underlying structures. The retina is peeled off from the eye and ground in the saline solution. The homogenate is then filtered through a double layer of gauze. The chapter illustrates the purity of the squid rhabdoms. The purity of the rhabdomeric membrane preparation is determined by examination of absorbance spectra, by electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gels, and by analysis for phospholipid content.

3-Dehydroretinal in the eye of a bioluminescent squid, Watasenia scintillans.

3-Dehydroretinal was found in the retina of a bioluminescent squid, Watasenia scintillans. The possible vitamin A2-visual pigment was localized in the ventral part of the eye and its proportion was about 15% of the total visual pigment.

Identification of 3,4-Didehydroretinal Isomers in the Xenopus Tadpole Tail Fin Containing Photosensitive Melanophores

It is well characterized that melanophores in the tail fin of Xenopus laevis tadpoles are directly photosensitive. In order to better understand the mechanism underlying this direct photosensitivity, we performed a retinal analysis of the tail fins and eyes of Xenopus tadpoles at stages 51-56 using high performance liquid chromatography (HPLC). Following the extraction of retinoids by the formaldehyde method, a fraction containing retinal and/or 3,4-didehydroretinal isomers from the first HPLC analysis were collected. These isomers were then reduced by sodium borohydride to convert retinal and/or 3,4-didehydroretinal isomers into the corresponding retinol isomers to prepare for a second HPLC analysis. Peaks of 11-cis and all-trans 3,4-didehydroretinol were detected in the eyes and tail fins containing melanophores, but they were not detected in the tail fins without melanophores. The amounts of 11-cis and all-trans 3,4-didehydroretinol were 27.5 and 5.7 fmol/fin, respectively, and the total quantity of 3,4-didehydroretinal was calculated at approximately 5×106 molecules/melanophore. These results strongly suggest the presence of 11-cis and all-trans 3,4-didehydroretinal in melanophores of the tadpole tail fin, which probably function as the chromophore of photoreceptive molecules.

Dark regeneration of squid rhodopsin and isorhodopsin

The visual cells of the squid contain two kinds of photopigment. rhodopsin and retinochrome. Rhodop sin has the I l-c13 form of retinal as chromophore, whereas retinochrome has all-trans form. When retinochrome absorbs light, its chromophore retinal is isomerized from all-rruns to I I-cis form (Hara and Hara, 1967). As this change is just the reverse of what occurs on the photoisomerization of rhodopsin, we have suggested that the 1 I-cis retinal formed on the bleaching of retinochrome may be used to regenerate rhodopsin (Hara, and Hara, 1968). An important approach to this probtem is to observe the synthesis of squid rhodopsin in the dark. Unlike cattle rhodop sin. squid rhodopsin is converted by light to the photoproduct metarhodopsin which is stable at physiological temperatures; no one has succeeded so far in obtaining the protein part, opsin, which is active .enough to recombine with I I-cis retinal. Squid opsin has been supposed to be so labile that it readily denatures by the removal of the chromophore (Hubbard and St. George, 1958). In the present study,* we tried to remove the chromophore from rhodopsin which was retained in the rhabdomal membranes, and successfully prepared active opsin which was capable of reconstituting rhodopsin and isorhodopsin during dark incubation with 1 I-cis and 9-cis retinal, respectively. The dark-adapted eyes of the squid, Todarodes pacificus, were hemisected and the eye-cups were shaken in a 0.4 M NaCl solution in phosphate buffer (67 mM, pH 6.5) so as to detach the outer segments of the visual cells (Hara and Ham, 1972). They were collected by centrifugation, homogenized in a 45% solution of sucrose in buffered saline and spun at 12,000 rpm to sediment ommin granules. The rhabdom fragments in the supematant were then pelleted in a large volume of buffered saline by centrifuging, and subjected further to two successive flotations with 40% sucrose in buffered saline to reduce contamination. Finally. the pellet was suspended in 40% sucrose buffered at pH 6.5 and centrifuged at 45,000