Katsu Azuma

Circular dichroism of visual pigment analogues containing 3-dehydroretinal and 5,6-epoxy-3-dehydroretinal as the chromophore

Two isomers isolated from irradiated all-trans-3-dehydroretinal were coupled with opsin to form visual pigment analogues. These pigments showed λmax at 517 nm (3-dehydrorhodopsin) and 500 nm (3-dehydroisorhodopsin), respectively. In the case of 5,6-epoxy-3-dehydroretinal also, two isomers were isolated and coupled with opsin to form 5,6-epoxy-3-dehydrorhodopsin and 5,6-epoxy-3-dehydroisorhodopsin. These pigments showed λmax at almost the same wavelength of 465 nm. These synthetic pigments exhibited induced circular dichroism (CD) at the range 300–600 nm like rhodopsin and isorhodopsin. The magnitude of CD or the rotational strength on the α band of 5,6-epoxy-3-dehydrorhodopsin is about equal to that of rhodopsin, but that of 3-dehydrorhodopsin is larger. From the above results and those reported elsewhere, it is proposed that the optical activity of the chromophore in the visual pigment or its analogue is not induced by the preferential selection of an inherently twisted form of retinal.

Circular dichroism of cephalopod rhodopsin and its intermediates in the bleaching and photoregeneration process

In the bleaching process of cephalopod rhodopsin, a new intermediate was found in the conversion process from lumirhodopsin to metarhodopsin. This intermediate of octopus has an absorption peak at about 475 nm and has been named as M475. The circular dichroism value of M475 is too small to be evaluated. On the other hand, lumirhodopsin shows a negative CD at 470 nm, a positive CD at 350 nm and a large positive CD band with three peaks at 280, 287 and 295 nm. Such a large CD band in the ultraviolet region is not observed in rhodopsin, M475 and metarhodopsin. This CD seems to be mainly due to tryptophan and tyrosine residues restricted in free rotation in the protein moiety of lumirhodopsin. The intermediate in the photoregeneration process of cephalopod rhodopsin, P380, has a positive CD band at the main peak, 380 nm, and also a large positive CD band in the ultraviolet region like lumirhodopsin.

Absorption Spectra of Planarian Visual Pigments and Two States of the Metarhodopsin Intermediates

Abstract— Absorption spectra measurements of isolated planarian ocelli by a microspectrophotometer (MSP) and intra‐ocellar recordings of the early receptor potential (ERP) were carried out in order to characterize in situ planarian rhodopsin (pRh) and its photoproducts. The MSP spectra of the isolated ocelli revealed Λmax at about 500 nm. The ERP evoked by a test stimulus was a positive monophasic waveform in the dark but became negative during exposure to violet light. During subsequent darkness, the ERP rapidly reverted to a positive waveform but with a smaller amplitude than before exposure. The ERP amplitude recovered to its initial level upon exposure to red light. The ERP experiments suggest that pRh produces two metarhodopsin intermediates, with Λmax longer than that of pRh: the metastate responsible for the negative ERP converts to another metastate that results in a smaller ERP in the dark‐adapted ocellus.

Studies on cephalopod rhodopsin. Conformational changes in chromophore and protein during the photoregeneration process

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (lamda >520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.

Circular dichroism of squid retinochrome

Abstract Squid retinochrome and its photoproduct (P465) are optically active, indicating that both all- trans and 11- cis retinal become asymmetric upon binding to the retinochrome protein. The protein exhibits a CD spectrum in the far ultraviolet region characteristic of an α-helix conformation. The positive CD bands in the visible region are attributable to the chromophore retinal, and the large negative CD bands at 305 nm and 275 nm are ascribed to residues such as Tyr, Trp and Cys in the protein moiety. On the liberation of retinal from the protein the CD band in the visible range disappears completely and those in the near ultraviolet range decrease remarkably in magnitude. These facts suggest that the chromophore retinal and residues such as Tyr, Trp and Cys in the protein moiety are restricted in free rotation by the binding of the chromophore with retinochrome protein. On the basis of the above results the mechanism of induction of the CD of retinal is discussed.

The increase in sensitivity following light illumination in frog photoreceptors

Abstract Dark-adaptation of photoreceptors was studied by recording fast PIII responses of the isolated bull frog retina superfused with Conways solution containing 5 mM sodium aspartate. When a dark-adapted retina is illuminated by an adapting light, the amplitude of the response decreases. Initially on turning off this light, the amplitude increases over that of the dark-adapted response. This phenomenon, “hypersensitivity” is thought to be due to an increase in sensitivity of red rods. The hypersensitivity occurs following several minutes illumination with relatively weak light if [Ca 2+ out is low. Intense light and higher concentration of Ca 2+ inhibit the hypersensitivity. Possible mechanisms for the hypersensitivity are discussed.

Reversible spectral change of squid retinochrome by salts.

THE cephalopod retina contains two kinds of photosensitive pigments, rhodopsin and retinochrome. Retinochrome was first found in the inner segment of squid visual cells1. More recently it was reported that the outer segment might also contain retinochrome together with rhodopsin2. The chromophore of retinochrome, all-trans retinal, is predominantly converted into the 11-cis form by illumination. It has been suggested that retinochrome supplies 11-cis retinal for the regeneration of cephalopod rhodopsin in the dark, by acting as an isomerase1. The validity of the mechanism, however has not yet been established in vitro or in situ.

Light-induced Extracellular Changes of Calcium and Sodium Concentrations in the Planarian ocellus

Ion-selective double-barreled microelectrodes inserted into a planarian ocellus were used to monitor the ocellus potential and the changes in extracellular concentrations of Ca2+ (Ca(o)) and Na+ (Na(o)) caused by a 0.5-sec light flash or sustained (120s) illumination. Ca(o) and Na(o) were slightly decreased following a flash. Sustained illumination caused a biphasic change in Ca(o) (a rapid decrease followed by a slow increase) and a tonic decrease in Na(o). When Na+ in the planarian saline was replaced by Li+ or choline+, the increase in Ca(o) was prevented: sustained illumination induced only a decrease in Ca(o). These results suggest that illumination induces influxes of both Ca+ and Na+ into planarian photoreceptors, and that the Ca2+ influx is rapidly followed by a Na-dependent Ca2+ efflux due to Na-Ca exchange.

Extracellular and intracellular ion concentration of Octopus visual cells and ion permeability of the cell membranes

Abstract Sodium, potassium, chloride, calcium, magnesium and sulphate concentrations in the vitreous humour and retina of Octopus vulgaris were measured by flame photometry. The density and dry mass fraction of the dark-adapted retina were determined to be 1.074 ± 0.027 g cm−3 and 22.4% ± 0.6% respectively. Those of the vitreous humour were 1.033 ± 0.002 g cm−3 and 3.0% ± 0.2% respectively. Subsequent calculations yielded an interstitial space of the retina of 19% (v/v). Using this value of the interstitial space, the intracellular ion concentrations of the dark-adapted and illuminated retinas were determined. These ion concentrations were used, together with the membrane potentials obtained from electrophysiological experiments, to calculate the ion permeability of Octopus visual cell membranes in the dark and on flash illumination.

ABSORBANCE and CIRCULAR DICHROISM SPECTRA OF 1‐C1S PHOTOPRODUCT FORMED BY IRRADIATING FROG RHODOPSIN

Abstract— We showed by spectrophotometry and HPLC that a photoproduct having 7‐cis retinal (1‐cis photoproduct) can be derived from the photoisomerization of frog lumirhodopsin (L) and metarhodopsin I (M I). The efficiency of the isomerization was higher in M I than in L. The absorption maximum of the 1‐cis photoproduct at ‐20°C is at 455 nm, and its maximum absorbance 1.1 times as large as that of rhodopsin. The photoproduct exhibited two positive CD bands at 450 nm α‐band) and 320 nm (β‐band); the molecular ellipticity at a‐band ([θ] = 73000) being larger than that of rhodopsin ([θ] = 61000). Re‐examination of the absorption spectra of rhodopsin intermediates gave the absorption maxima of L. M 1 and M 111 to be 522, 482 and 475 nm, respectively.