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Biochimica et Biophysica Acta, 404 (1975) 3 0 0 - - 3 0 8 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m -- Printed in The Netherlands

BBA 27737

ACCESSIBILITY OF THE IODOPSIN CHROMOPHORE*

H I R O Y U K I MATSUMOTO, FUMIO T O K U N A G A and T O R U YOSHIZAWA

Department of Biophysics, Faculty of Science, Kyoto University, Kyoto (Japan) (Received F e b r u a r y 5th, 1975)

Summary Iodopsin can replace its chromophore {ll-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH4 bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes ll-c/s retinal from iodopsin in the dark. These facts suggest that the linkage of the chromophore to opsin in the iodopsin molecule {presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.

Introduction Iodopsin is a cone visual pigment that has been extracted from chicken retinas [1,2]. It has the same chromophore, ll-cis retinal, as the rod visual pigment, rhodopsin. The absorption maximum of iodopsin (approx. 562 nm) lies at a longer wavelength than that of chicken rhodopsin (approx. 509 nm). This difference must have its origin in the different properties of their protein moieties or opsins. The protein moieties of iodopsin and rhodopsin are called photopsin and scotopsin, respectively [2]. Iodopsin differs from rhodopsin not only in this spectral property, but also in its biochemical and photochemical behaviour. Iodopsin is unstable to such reagents as alum or hydroxylamine and to alkaline and acidic conditions under which rohodopsin is stable [2]. The velocity constant of regeneration of iodopsin at 10°C and pH 6.5 from l l ¢ i s retinal and photopsin is about 500 times greater than that of rhodopsin [2]. Though the photochemical behaviour of iodopsin at liquid-nitrogen temperature resembles that of rhodopsin, there is a striking difference in the dark * AH the figures in this p a p e r w e r e p r e s e n t e d at the m e e t i n g o f the R e s e a r c h G r o u p o n ' M o l e c u l a r M e c h a n i s m o f P h o t o r e c e p t i o n ' at T o k y o , O c t o b e r , 1 9 7 3 (in J a p a n e s e ) , and at t h e 1st S Y m p o s i u m o f the Japanese C h a p t e r o f t h e I n t e r n a t i o n a l C o m m i t t e e for E y e R e s e a r c h at Osaka, October, 1973

(in English).

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reaction between bathoiodopsin and bathorhodopsin (formerly called prelumiiodopsin and pre-lumirhodopsin); on warming in the dark, bathorhodopsin is converted to the second intermediate, lumirhodopsin, while bathoiodopsin reverts to iodopsin [3,4]. Another difference between the iodopsin and rhodopsin systems is that cattle and chicken rhodopsin axe converted to hypsorhodopsin by irradiation at liquid-helium temperature [5,6], but iodopsin does not form a hypso-product. All these differences must be attributed to the differences in the interactions of ll-c/s retinal with photopsin and scotopsin. Becently, it has been shown that in cattle rhodopsin the aldehyde group of ll-c/s retinal is bound to the e-amino group of a lysine residue by a Schiff-base linkage [7--9], but the linkage between ll-cis retinal and photopsin has not been examined. Because of their spectroscopic and chemical properties, it is reasonable to assume that iodopsin has a similar Schiff-base linkage as rhodopsin. About 20 years ago, Wald et al. [2] reported that the addition of hydroxylamine to a preparation of iodopsin bleached it without denaturing the photopsin; when ll-c/s retinal was added to the bleached preparation, iodopsin was regenerated. We confirmed the observations and assumed that the hydroxylamine may react with ll-cis retinal, if iodopsin dissociates into photopsin and ll-c/s retinal, and/or with Schiff-base linkage directly (transimination mechanism) [10], resulting in the formation of retinal-oxime in any case. Thus, the accessibility of the chromophores of iodopsin and rhodopsin to some reagents was examined. Materials and Methods Through the kindness of a local poulterer, Toripin Co. Ltd, we obtained about fifty chicken heads within several hours after slaughtering, which was carried out under artificial light. The heads were thrown into a dark ice box and brought to the laboratory. All further procedures were carried out under dim deep red light near 0 ° C. After enucleation of the eyes from the heads and then removal of the retina, outer segments were prepared by the method of Wald et al. [2]. Further purification of outer segments was performed by sucrose density-gradient centrifugation. A linear sucrose density gradient of specific gravity from 1.13 to 1.17 was prepared in a 30 ml centrifuge tube using sucrose solutions in M/15 phosphate buffer (pH 6.5). The crude preparation of rod and cone outer segments in M/15 phosphate buffer (pH 6.5) was placed on top of the gradient. After centrifuging (Hitachi 55P) for 1 h at 40 000 X g and 4°C in a swinging rotor (SW-25), the preparation was divided into 2 ml fractions by removing samples from the bottom of the tube. The absorption spectrum of each fraction was measured in a recording spectrophotometer (Hitachi EPS-3T) by the opal glass method [11]. After the visual pigments in the fractions were completely bleached by orange light at wavelengths longer than 510 nm, the absorption spectra were measured again. Difference spectra before and after bleaching were calculated. The contents of rhodopsin and iodopsin were determined on the basis of the difference spectra of iodopsin and rhodopsin reported by Wald et al. [2]. We found that cone and rod outer segments have the same specific gravity of 1.14, so that one cannot separate them by

302 density-gradient centrifugation, though this procedure is useful for removing some impurities from the outer segments. The purified outer segments were washed several times with M/15 phosphate buffer (pH 6.5) to remove t h e sucrose. They were then lyophilized and repeatedly washed with chilled light petroleum (boiling point 30--50°C) to extract lipids. The light petroleum was evaporated at room temperature and the visual pigments were extracted with 1.5% digitonin in M/15 phosphate buffer (pH 6.5). The outer segments were kept in digitonin for 1 h at 4°C and then centrifuged for 20 min at 10 000 × g. The supernatant was used for the experiment. The final preparation contained a b o u t 45% iodopsin and 55% rhodopsin estimated by the absorbance at each ~m ax, 562 nm and 509 nm, respectively. The yield of iodopsin was a b o u t 0.004 absorbance units per retinal per ml at ~knl a x °

Results

Effect o f horse liver alcohol dehydrogenase on iodopsin If iodopsin was partly dissociated to l l ¢ i s retinal and photopsin, the addition of horse liver alcohol dehydrogenase, which reduces l l-cis retinal to retinol in the presence of NADH [ 1 2 ] , should lead to bleaching. Our experiment showed that a preparation of alcohol dehydrogenase that reduces 0.69 nmol ll-cis retinal to retinol per rain in 1.5% digitonin (25°C, pH 6.5) did n o t bleach iodopsin (As 62, 0.08; volume, 0.37 ml). This indicates that there exists no free l l - c / s retinal to be reduced by alcohol dehydrogenase in the iodopsin preparation. Formation o f isoiodopsin from iodopsin in the dark If one still assumes a dissociation of iodopsin into ll-cis retinal and photopsin, the ll-cis retinal molecule must lie in a cleft of the photopsin that is not accessible to alcohol dehydrogenase. A low molecular weight reagent such as hydroxylamine, however, might be able to enter the cleft and react with the free retinal to form retinal-oxime. To test whether other small molecules have also accessibility to the chromophore, an excess of 9-cis retinal was added to the iodopsin preparation, in order to convert it to isoiodopsin in the dark by the replacement of the l l - c / s retinal c h r o m o p h o r e by 9-cis one. A sample of the solubilized visual pigment preparation, containing iodopsin and rhodopsin, was divided into t w o parts. One part was irradiated with deep red light containing wavelengths longer than 660 nm in order to bleach only iodopsin. This sample was used as a reference when the absorbances of the other part were measured. Thus, one can directly record the difference spectrum of iodopsin before and after bleaching as shown in Fig. l a (curve 1). The absorption spectrum of curve 1 shows a slightly greater )tin ax {572 nm) than that of iodopsin (562 nm) because of the sloping base-line. When 9-c/s retinal was added to the preparation shown in curve 1, it changed to curve 2. Prolonged incubation in the dark successively changed curve 2 to curves 3--5, and finally to curve 6. As shown in Fig. l b , the difference spectrum between curve 6 and curve 2 (---) reasonably agrees with the difference spectrum

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Accessibility of the iodopsin chromophore.

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH4 bleaches iodopsin in th...
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