Masami 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.

Elevated concentrations of linoleic acid in erythrocyte membrane phospholipids in patients with inflammatory bowel disease

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn disease (CD), is a disorder characterized by diffuse inflammation of the gastrointestinal tract. The immune response and inflammation are mediated by polyunsaturated fatty acids and influenced by dietary fats and lipid metabolism. This study examined the qualitative and quantitative fat intake of IBD patients and healthy controls on plasma phospholipid and erythrocyte membrane phospholipid (EMP) fatty acid content. Measurement of the fatty acid composition of plasma phospholipid and EMP were performed in 29 UC patients, 20 CD patients, and 31 healthy controls. Anthropometric characteristics and data on dietary intake were also collected. We observed significantly lower lipid intake in UC and CD patients vs controls. The UC and CD patients had significantly higher levels of linoleic acid in their EMP than did controls. There were no significant differences in the levels of n-3 polyunsaturated fatty acids, but there were significantly higher levels of the n-6 in the EMP of UC and CD patients compared with controls. The significant differences persisted after the data were adjusted for potential confounders and lipid intake. Higher levels of linoleic acids and n-6 fatty acids, which are involved in production of proinflammatory mediators, were found in IBD patients compared with controls, thereby implicating n-6 fatty acids in the pathophysiology of the disease.

Changes of egg retinoids during the development of Xenopus laevis

The changes of egg retinoids during the development of Xenopus laevis were investigated by high-performance liquid chromatography (HPLC). All-trans retinal and 3-dehydroretinal are endogenous in the egg and are distributed to both the eyes and the ventral portion of the larval body. These retinals are converted to all-trans retinyl palmitate and 3-dehydroretinyl palmitate during the development up to stage 46. 11-cis retinal and 3-dehydroretinal can be detected after stage 40 in the eyes but not in the larval ventral portion. It is suggested that retinoids are transported from the larval ventral portion to the eyes after stage 41/42.

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.

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.

Formation of visual pigment chromophores during the development of Xenopus laevis

Retinoids in the eyes of Xenopus laevis at several developmental stages, were analyzed by high-performance liquid chromatography (HPLC). At stage 37/38, larval eyes contained mainly all-trans isomers of retinal, 3-dehydroretinal, retinyl ester and 3-dehydroretinyl ester. Ratios of all-trans 3-dehydroretinal to retinal and of all-trans 3-dehydroretinyl ester to retinyl ester were almost 1 at the stage. With the advance of development, the amounts of all-trans retinal and 3-dehydroretinal decreased; however, those of all-trans retinyl ester and 3-dehydroretinyl ester increased. The chromophores of visual pigments, 11-cis retinal and 3-dehydroretinal, were detected at stage 40 (total; 0.2 pmol/eye) and their amounts increased after that stage. The ratio of 11-cis 3-dehydroretinal to retinal was almost 1 at stages 40-42. The ratio became larger after stage 43 and was almost 19 at stage 46. The ratio of all-trans 3-dehydroretinyl ester to retinyl ester, also, increased after stage 42 and reached 11 at stage 46. The mechanism of 11-cis formation during development is discussed in relation to retinoid conversions in the eyes.

THE REGENERATION OF VISUAL PIGMENTS AND THE CHANGE OF ROD HYPERSENSITIVITY AFTER IRRADIATION BY BLEACHING LIGHT IN FROG RETINA

Abstract— The regeneration processes of visual pigments and the dark adaptation processes of rod photoreceptor after irradiation by bleaching light were studied by spectrophotometric, electroretinographic(ERG) methods and the measurement of early receptor potentials (ERPs) in bullfrog retina. After irradiation by bleaching light, rhodopsin in the isolated retina regenerated to an extent depending on the wavelength and intensity of the bleaching light as well as pH. Intense blue light and a weak alkaline environment (pH 7.5–9.5) favoured the regeneration. The regeneration of pigment in the green rods could not be detected in these experiments on the isolated retina. The regeneration of cone pigment was studied by measuring ERPs from both isolated retinas and retinas with pigment epithelium‐choroid complex separated from scleras, which are called PEC‐retinas. In the PEC‐retinas, cone pigment regenerated more rapidly and with better efficiency than in the isolated retinas.

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 changes in circular dichroism spectra of cattle rhodopsin and isorhodopsin.

When the disk membrane of rod outer segment is treated with detergents, the alpha-band CD of rhodopsin decreases and the gamma-band CD increases. This tendency of CD change is most prominent in the purified rhodopsin in cholic acid obtained by the ammonium sulfate fractionation of disk membranes, and the gamma-band CD is three times larger than the alpha-band CD. The beta-band CD of rhodopsin is only slightly influenced by detergents. The gamma-band of isorhodopsin shows two CD bands, one negative and one positive. Both in rhodopsin and isorhodopsin the gamma-band CD is lost by light irradiation. It is supposed that both chromophore retinal and aromatic amino acid residues of opsin are responsible for the gamma-band CD. When ammonium sulfate is added to the sonicated disk membranes suspended in cholic acid solution, the alpha-band CD of rhodopsin decreases to about a third and the gamma-band CD increases remarkably. The CD spectrum goes back to the original one on eliminating ammonium sulfate from the solution with dialysis. However, the purified rhodopsin recovers native CD spectrum on addition of lipids extracted from disk membranes. The retinal-opsin interaction that induces optical activity depends upon the property of a local environment formed by lipid and detergent.