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Photoreactions of cephalopod rhodopsin
CW2-6989/81/060935-07802.00/0 Pergamon Press Ltd
Vision Rrreurch Vol. 21. pp. 935 lo 941. 1981 Printed in Great Britain
PHOTOREACTIONS
OF CEPHALOPOD
RHODOPSIN
TAKAYUKINAITO, KAZUKONASHIMA-HAYAMA, KOHZOOHTSU’ and YUJI Ktro Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan and ‘Marine Laboratory, Faculty of Science, Okayama University, Ushimadocho, Okayama 701-43,Japan (Received 2 February 1980) Abstmet-In the cephalopod rhodopsin system, two new photoproducts from alkaline metarhodopsin were found. One was slowly converted to rhodopsin in the dark and called pseudo-rhodopsin. It could be photoconverted to the other, pseudo-metarhodopsin. This resulted in a photosteady state mixture in the
light. Absorbance spectra of both pigments depended reversibly on pH. Both pigments revealed weak, negatively signed circular dichroism corresponding to their absorbance spectra. Acid and alkaline metarhodopsin also had negatively signed circular dichroism corresponding to their respective absorbance spectra.
INTRODUCTION The visual pigment of cephalopod rhodopsin is located in the rhabdomeric membrane of photoreceptor cells. Considerable information concerning the photoreaction of rhodopsin and the ionic mechanism of the receptor potential has accumulated, but how the photoreaction is linked to permeability changes in the rhabdomeric membrane is still a matter of speculation (Hagins et al., 1962; Atzmon et al., 1978). It is necessary to investigate not only the events involved in the transformation of rhodopsin to its final photoproduct, metarhodopsin, but also the events in the reverse reaction, the photoregeneration process. Cephalopod metarhodopsin can take two forms according to pH (acid and alkaline metarhodopsin). Acid metarhodopsin is photoconverted preferentially to rhodopsin (Hubbard and St. George, 1958) via an intermediate, P3s0 having a relatively short life time at ordinary temperature and pH (Suzuki et al., 1973). On the other hand, alkaline metarhodopsin may be converted by light to rhodopsin and isorhodopsin in Loligo pealei (Hubbard and St. George, 1958) or to an unstable pigment other than isorhodopsin in Todurodes pacificus (Hara and Hara, 1966). In this study, it was found that the photoproduct of cephalopod rhodopsin illuminated with a light of &,,., = 4OOnm, was quite different from the photoproduct of rhodopsin illuminated with longer wavelength light (A > 560nm). This wavelength dependence in the photoreaction of cephalopod rhodopsin was attributed to the difference in the photoreaction of acid and alkaline metarhodopsin. A new relatively long-lived photoproduct of alkaline metarhodopsin was found to convert slowly to rhodopsin in the dark. METHODS Materials
The squid, Watasenia scintillans was mainly used in this study. Other species used were Todarodes paci935
jicuq Loligo japonica, Sepiella japonica, and Octopus minor. The eyes of these cephalopods were stored at
- 20°C. The extraction and purification of rhodopsin was carried out as described in a previous paper (Nashima et al., 1978). The rhabdomeric membranes were obtained by flotation on 400/, sucrose solution. Rhodopsin was extracted with 2% ~-1690 solution and purified by DEAE-cellulose and concanavalin A-Sepharose column chromatography. The AZsO/Amax values of the rhodopsin preparations obtained were between 2.8 and 3.0. In some experiments, the detergent digitonin was added to the rhodopsin in ~-1690 solution for three reasons. Firstly, the procedure enabled a comparison with the earlier observations by Hubbard and St. George (1958), and Hara and Hara (1966). Secondly, digitonin lowers the apparent pK of the retinal Schiff base in metarhodopsin and rhodopsin (Nashima and Kito, 1980). This makes it easier to investigate the pH dependence of metarhodopsin and the new product described later. Thirdly, the addition of digitonin decreases the rate of transition of the new product to rhodopsin and metarhodopsin, which proved useful in an analysis of the reactions observed. The pH dependence of metarhodopsin was measured by the methods described by Hubbard and St. George (1958) and Nashima and Kito (1980). The pH dependence of the new product was determined as follows. The rhodopsin solution was illuminated at pH 7.5 and 4°C for 10min with light obtained by passing the beam from a 500 W projection lamp through a Toshiba VV-40 filter which transmitted maximally at 400nm (half band width: 106mn). Immediately after cessation of the light, the pH of the solution was changed by addition of 1 N NaOH. The residual absorbance at 482 nm was determined at desired pHs. Retinal isomer compositions of rhodopsin, isorhodopsin, metarhodopsin and the new product were analyzed with a high performance liquid chromatograph (HPLC) system (Yanaco ~-2000) equipped with a silica gel column (Yanaco SA-1, 4 x 500mm (cf:
936
TAKAYUKI NAITO et al.
Wavelength inm)
/ 300
I
400
8
I
,
500
Wavelength fnm)
I
1
1
250 Vbdength
1
1
1
i
290
tnml
Fig. 1. Absorbance spectra and circular dichroism spectra of rhodopsin and metarhodopsin of the squid. ~~~us~t~iusci~ftj~~u~ls. Absorbance spectra (A) and circular dichroism spectra (B) and (C). Curve I : rhodopsin (i,,,: 482 nm) in 0.2% ~-1690, and 0.1 M KCI solution at pH 6.0. Curve 2: alkaline metarhodopsin (i.,,,: 380nm) produced by irradiating rhodopsin at pH I 1.6 and 4°C with orange light (E.> 560 nm) for 1min. Curve 3: acid metarhodopsin (i,,: 496nm) produced by readjusting the pH of the solution to 6.4. All the m~surements were carried out at 6°C (light path: IOmm) and curves in (B) and in (C) were averaged after summation of five and two measurements, respectively.
Rotmans and Kropf (1975)). The elution solvent was a mixture of hexane, diethylether (8%) and acetone (0.25%) which could separate 5 isomeric retinals. Pigments were denatured with 1% SDS solution, and then retinal was extracted into diethylether. The relative content of each isomer was determined after correcting for the difference in molar extinction of each isomer (Hubbard et al., 1971; DeGrip et al., 1976). Isorh~opsin was made by illuminating rhodopsin with red light (i Z=600nm) at liquid nitrogen temperature (Kito er al., 1961). Absorbance spectra were determined with a Union ShI-401 s~~rophotometer (scanning rate of lOnm/ set). The circular dichroism (CD) spectra were measured with the computer-controlled Jovin-Yvon Dichrograph Mark III-J (scanning rate of 1 rim/see). The temperature of the cell-holders was controlled by circulating water of a constant temperature. The detergent ~-1690 (lauryl ester of sucrose) was kindly synthesized by Ryoto Co. Ltd (Tokyo). Digito-
nin and mercapt~thanol Chemicals (Osaka).
were purchased from Wako
RESULTS
Figure 1 shows the absorbance and CD spectra characteristic of squid rhodopsin and its thermally stable photoproducts, acid and alkaline metarhodopsin. In the region above 300 nm, both metarh~opsins have negative CD, corresponding to the absorbance spectra. The molar ellipticities, [@J of rhodopsin and acid and alkaline metarhodopsin at their respective x bands were 7.3 x IO4 at 470 nm (rh~opsin), - 1.2 x lo4 at 490 nm (acid metarhodopsin) and -2.1 x lo4 at 380 nm (alkaline metarhodopsin). In the region below 300 nm, the rhodopsin has small CD peaks or shoulders at 295, 2%5,279, 262 and 256nm, attributable to some aromatic residues and disulfide bonds of the protein. In the CD spectrum of acid metarhodopsin, peaks at 285 and 279 nm are less
Photoreactions
of cephalopod rhodopsin
Wavelength (nm -.----. 1
488 486 464 482 480
, 0
I I
I 2
1 3
I 4
;
j&Y-
931
sorbance around 380nm increased in curve 2 and 3 (Fig. 2A) and then gradually decreased (curves 5 and 7). On the other hand, the squid rhodopsin illuminated with the orange light at 4°C and pH 7.5, had already attained the photosteady state by 2Osec (Fig. 2B). Figure 3 shows the absorbance and CD spectra of the photosteady state mixtures. The photosteady state mixture (curve 2) produced by, the orange light contained almost an equal amount of rhodopsin and acid metarh~opsin as judged from the CD spectrum (curve 2), the mixture contained only a small amount of alkaline metarhodopsin at this pH to judge from the slightly higher absorbance at 380nm compared with seen for rhodopsin (curve 1). The photosteady state produced by the 400nm light (curve 3) appeared as if the squid rhodopsin were completely changed into acid metarh~opsin, having negatively signed CD in the visible. However, the I.,,,., of the . curve 3 was similar to that of rhodopsin (482 nm), at a considerably shorter wavelength than that of acid metarhodopsin (496nm). As will be shown later, the solution of Fig. 3, curve 3, in fact does not contain rhodopsin, nor acid nor alkaline metarh~opsin, and the absorbance is due to a new photoproduct obtained with the 4OOnm light (Pmx). In a parallel experiment, it was shown that the new product could be produced from squid isorhodopsin in a similar man-
Imin) Fig. 2. Photo~action of ~uruse~ia rhodopsin. (A) changes in absorbance spectrum of the rhodopsin illuminated with the 400nm light. Curve 1: Watasenia rhodopsin in 0.4% L-1690 and 0.05 M Tris-HCl (pH 7.5), curve 2: illuminated with the 400 nm light at 4°C for 1 set, curve 3: for 5 set, curve 4: for 15 set, curve 5: for 1min, curve 6: for 2 min, and curve 7: for 5 min. (B) Changes in the ,I,,,,, of the squid rhodoosin illuminated with the 4C@nm lkhht and the orange light (J. > 560nm). Open squares (U)~J.,,,,, of rhodopsin solution illuminated with the 4OOnm light, and with orange light open circles (o), illuminated (d z 560 nm). All the measurements were carried out at 10°C.
obvious. In the absorbance spectrum of alkaline metarhodopsin, a shoulder at 295 nm becomes more prominent and absorbance below 260nm was largely enhanced, possibly due to dissociation of some tyrosine residues at pH 11.6. An obvious CD peak at 290 nm is also characteristic of alkaline metarhodopsin, since the CD of rhodopsin made alkaline did not show the 290 nm peak. Thus these three pigments can be distinguished spectroscopically from one another. As is well known (Hubbard and St. George, 1958), in the squid rhodopsin system the photosteady state mixture above 0°C should contain a certain amount of rhodopsin, acid and alkaline metarhodopsin depending on the wavelength of illumination and the pH of the solution. As shown in Fig. 2A and B, in the initial stage of illuminating squid rhodopsin with the 4OOnm light at 4°C and pH 7.5, the J,,,,,, shifted from 482 nm (rhodopsin) to a longer wavelength (491 nm) and gradually returned to 482 nm after 5 min illumination (Photosteady state). At the same time, the ab-
A
0.6
Whwl6ftgth tnmf
”
-aooss wovd0ngm (nm)
Fig. 3. Photoproducts of Warosenia rhodoosin irradiated wiih orange light and a light of &,., = 4OO’nmat pH 7.5. (0.05 M Tris-HCll and 4°C. Absorbance snectra fAl and CD spectra (B). durve 1: rhodopsin in O.&, ~-l&b solution. Curve 2: preparation irradiated at 4°C with the orange light (J. z 560 nm) for 2 min. Curve 3: preparation irradiated with the 400 nm light for 10 min. Curves 2 and 3 are those of the photosteady-state made with respective light. All the measurements were carried out at 6°C.
TAKAYUKI NAITO et al.
938
0.6 L
o, 0.4
E a
0.2
Wovelength(nm)
%
Fig. 4. Dark reaction of Pmx of Wrctasenia rhodopsin. (A) Spectral changes of Pmx during dark incubation at 15°C. Absorbance spectra were measured every 5min after irradiation of the rhodopsin at 4°C and pH 7.5 (0.05 M Tris-HCI) for 10 min with the 400 nm light. The initial curve corresponds to curve 3 in Fig 3. (B) Circular dichroism spectra of Wutusenia rhodopsin and Pmx. Curve 1: Watuseniu rhodopsin. Curve 2: the rhodopsin solution illuminated with the 400 nm light for 10 mm at 4°C and pH 7.5. Curve 3: the solution incubated at 15°C for lOOmin in the dark.
When Pmx was left to stand at 15°C in the dark, the absorbance at 482 nm gradually decreased and the absorbance at 380nm rose, as shown in Fig. 4. The half time of this change was 30 min. After 100 min, the sign of CD was reversed, suggesting the appearance of rhodopsin (curve 3). After the incubation, addition of
hydroxyl~ine (5OmM) to the product shifted the 380 nm peak to 367 rnn, indicative of formation of the retinal oxime. The fact suggests that Pmx contained two fractions convertible to rhodopsin or free retinal in the dark, since rhodopsin and metarh~opsin are resistant to hydroxylamine. When Pmx was incubated in the presence of hydroxylamine, the absorbance peak at 482nm shifted to a longer wavelength. This cannot be due to oxime formation (see above) but NHsOH is also a mild reductant. This was checked by using mercaptoethanol as shown in Fig. 5. The 488nm product formed with the aid of 0.5% mer~ptoethanol contained a significant amount of metarhodopsin and the same amount of rhodopsin as in the case incubated in the absence of the reagent The absorbance spectrum of Pmx depended reversibly on pH in the same way as does the absorbance spectrum of metarhodopsin. The alkaline form of Pmx had a &,,,, of 380 nm, identical to that of alkaline metarhodopsin, possibly due to deprotonation of the retinal Schiff base of the product. As shown in Fig. 6, the apparent pK of the interconversion of the two forms of the product was higher (10.6) than that of metarhodopsin (7.9). The HPLC anaIysis of the chromophoric retinal indicates (Table 1) that almost an equal amount of all-trans and 11-cis retinal are contained in Pmx. The 13-cis retinal (loo/,) of the product may possibly be an artifact, due to isomer~tion during the extraction procedure. This isomer is always found when other pigments that are believed to be free from the isomer are analyzed with column separation, Acid metarh~opsins of cephalopods generally have a &,,,, longer than that of the parent rhodopsins. As demonstrated in Table 2, the &,,,, of Pmx of three species is largely independent of the hamalof the parent rhodopsin. The decay in Table 2, is estimated from the increase in absorbance at 380nm as in Fig. 4,
P
6
s 0
5
6
t
8
9
10
II
12
PH WavabnQth
t nm )
Fig. 5. Effect of mercaptoethanol on Pmx of Watasenia The rhodopsin solution illuminated with the 400 nm light at 4°C and pH 7.5 (0.05 M Tris-HCl) was incubated in the presence of 0.5% mercaptoethanol at 15°C and spectra were recorded every 5 min. Absorbance maximum shifted from the initial 482 nm to 488 nm.
scintikms.
Fig. 6. The pH dependence of Pmx and metarhodopsin of Warasenia scjnti~~u~s. Curve t : per cent of acid metarhodopsin at 4°C was plotted against pH. Curve 2: per cent of absorbance at 480nm of Pmx at 4°C was plotted against pH. The initial rhodopsin solution contained 0.2% digitonin, 0.1% ~-1690 and 0.1 M KCI. The pH of the rhodopsin solution ii~uminated with the 400nm tight was changed immediately after cessation of the light.
Photoreactions
of cephalopdd rhodopsin
Table 1. Retinal isomer composition of the photoproduct (Pmx) of Wataseniarhodopsin irradiated with light of &,,.. = 400 nm, comparing with those of rhodopsin, isorhodopsin and metarhodopsin
Rhodopsin Isorhodopsin Metarhodopsin
Pmx
Table 2. Absorbance maximum and decay time of photoproduct (Pmx) of squid rhodopsin illuminated with a light of &,.. = 400nm Decay of Pmx Absorbance maximum (nm) at 15°C Rhod acMR Pmx (min)
l-cis all-trans
13-cis
I I-cis
9-cis
4 9 8
76 5 5
1
0
12 0
1 0
19 13 87
10
46
I
0
43
Preparation of isorhodopsin, metarhodopsin and Pmx is described in Materials and Methods.
corresponding to the formation of retinal and not to the formation of rhodopsin or metarhodopsin. Squid Pmx released some retinal even in the presence of mercaptoethanol. However, Pmx in the cuttle fish (Sepiella) and octopus hardly released any retinal even in the absence of mercaptoethanol. In the cuttle fish, the conversion of Pmx to rhodopsin was complete within 1Omin at 10°C and pH 7.5, judging from the recovery of the CD intensity. The rate of the thermal change of the product to rhodopsin and metarhodop sin was species-specific. In the octopus, two components of Pmx were found that had different &,,., values, possibly due to the different chromophores (11-cis and all-truns retinal), although the two components of squid Pmx, also with different chromophores, were spectroscopically indistinguishable. As shown in Fig. 7, the &,,., of acid metarhodopsin of the octopus (52Onm, not illustrated) is far from that of the rhodopsin at 480mn (curve l), and Pmx has a J_. at 496 nm (curve 2). When Pmx was further illuminated with red light (A > 6OOmn) the &,,., altered to 474nm. This photoproduct can convert almost entirely to rhodopsin after incubation at 15°C in the dark as judged from absorbance and CD in the visible. Pmx can be obtained again if the preparation is exposed once more to 400nm illumination at 4°C for 1 min.
939
Species
Wataseniascintillans 482 Loligo japonica 496 Todarodespacificus 482
496 500 491
482 488 488
30 5 7
Rhod and acMR are rhodopsin and acid metarhodopsin. Pmx is photoproduct produced by irradiating rhodopsins in 0.4% ~-1690 solution with a light of I,,, = 4OOnm for 10min at pH 7.5 and 4°C. The half time of the spectral change (decay of Pmx) was determined by incubating at 15°C in the dark as shown in Fig. 4.
reaction of short lived intermediates in the process of rhodopsin + metarhodopsin is also improbable since the exhaustive illumination of squid rhodopsin by the 400nm light at liquid nitrogen temperature (Yoshizawa and Wald, 1964), dry ice temperature and around -40°C (Azuma et al., 1973, did not produce the Pmx observed in this study, and Pmx was formed even by quite weak light provided the pH was alkaline.
600
600
400
khf6kJWth
(nm)
DISCUSSION
Formation of the new photoproduct from metarhodopsin
alkaline
Illuminating rhodopsin or isorhodopsin with orange light yields a photosteady-state of rhodopsin and metarhodopsin. The 400 nm light irradiation produces a new product, Pmx. The light can be absorbed not only by rhodopsin and acid metarhodopsin but also by alkaline metarhodopsin. However below pH 5.5, little tir no Pmx is produced, so a wavelengthdependent photoreaction of rhodopsin and acid metarhodopsin forming the product seems improbable. On the contrary, the appearance of Pmx becomes more rapid at higher pH, implying a path way of alkaline metarhodopsin + Pmx. Another possibility that Pmx might be produced by photo-
L
I
Jo0
I
I 400 wd6ngrn
I 600
I
I
(nm)
Fig. 7. Photoreaction of octopus rhodopsin. Absorbance spectra (A) and CD spectra (B). Curve I : rhodopsin at pH 7.5 (0.05 M Tris-HCI) and 4°C in 0.1% digitonin and 0.3% ~-1690 solution. Curve 2: rhodopsin solution irradiated with the 4OOnm light for IOmin at 4°C. Curve 3: the solution of curve 2 was irradiated further with the red light (1 z 600 nm) for 1min at 4°C.
TAKAYUKI NAITO et
al.
PMR to rhodopsin and metarhodopsin may be accompanied by a further change in protein structure, possibly involving the rearrangement of sullhydryl links as suggested in the thermal change of Pmx as illustrated in Fig. 5. Other remarks
PR _=
pm light
._
--
.-
(4)
--- - -
Fig. 8. Photoreaction of cephalopod rhodopsin at normal temperature and pH. R, acMR, alMR, PR and PMR are rhodopsin, acid and alkaline metarhodopsin, pseudorhodopsin and pseudo-metarhodopsin, respectively. Species-specificity of Pmx
Hubbard and St. George (1958) and Hubbard and Kropf (1958) found that the photoproduct of alkaline metarhodopsin had a J_, shorter than that of the parent rhodopsin (I.&go pealei). Hara and Hara (1966) reported that the photoproduct of alkaline metarhodopsin was unstable and had a &,a, almost similar to that of the parent rhodopsin (Todarodes pactjicus). These observations were reconfirmed, as shown in Table 2. In those earlier experiments, the illumination was carried out in alkaline conditions and this made it difficult to understand the nature of the products because of their instability and pH dependence as in Figs 4 and 6. Two components of Pmx and photoreaction of cephalopod rhodopsin Pmx contains two isomeric chromophores and hence should be considered as two, one of which converts to rhodopsin and the other of which releases retinal or can be converted to metarhodopsin with the aid of mercaptoethanol. The component produced by illuminating octopus Pmx with red light may be called pseudo-rhodopsin; it has 11-cis retinal as its chromophore but a negative CD in the visible. This is converted by light to pseudo-metarhodopsin which has all-trans retinal as the chromophore. Thus the photoreaction of cephalopod rhodopsin at normal temperatures can be illustrated as shown in Fig. 8. The photoreaction (1) of acid metarhodopsin to rhodopsin involves an intermediate, PBS0 which is long-lived at an alkaline pH (Suzuki et al., 1973). Under the condition (pH 7 and lYC), the P,,, should have too short life time as compared with Pmx. The photoreaction (3) of alkaline metarhodopsin to pseudo-rhgopsin (PR) presumably involves the isomerization of the chromophore, accompanying the rise of pK of the retinal Schiff base due to rearrangement of protein structure. Further photoreaction (4) of PR to PMR also involves the isomerization of the chromophore. The dark reaction (5) of PR and
The apparent pK of the interconversion of metarhodopsin is considerably affected by the phospholipid and detergents used for dispersing cephalopod rhodopsin and also by the salt concentration (Kito and Nashima, 1980). The apparent pK of the interconversion of acid and alkaline metarhodopsin in rhabdomeric membranes was higher (above 9) than in digitonin solution (about 7), but the titration curve deviated from the theoretical titration curve of a single ionizable group of the same pK. In the rhabdomerit membrane, only a small amount of alkaline metarhodopsin would be formed and if the wavelength of light is such that light is absorbed by alkaline metarhodopsin, the photoproducts (PR and PMR) will accumulate. It is of interest to examine the formation of those products in the arthropod rhodopsin system, since a photoproduct with a relatively long life time is assumed to cause the so-called PDA (prolonged depolarizing afterpotential) in the photoreceptors (Nolte and Brown, 1972; Hillman et Al., 1972). Previously, the CD characteristic of cephalopod metarhodopsin could not be detected in the region above 300 nm (Kito et al., 1968). In digitonin solution, even rhodopsin tended to release protons from the retinal Schiff base at an alkaline pH and to form alkaline rhodopsin (Nashima and Kito, 1980), and this had a positive CD around 380nm and could convert to rhodopsin at a neutral pH. Possibly, this substance disturbed to detect the weak negative CD of metarhodopsin. Recently Tsuda (1979) reported values of CD for octopus metarhodopsin in the visible that were a little different from the data of this paper. Taking into account of the formation of alkaline rhodopsin, the discrepancy would be explained. At present all the intermediates from cephalopod rhodopsin to metarhodopsin have shown a negative CD in the visible (Azuma et al., 1975; Shichida et al., 1978), differing from the vertebrate rhodopsin system. The explanation of CD data in rhodopsin system is presently difficult to understand; however, as CD is very sensitive to the conformation or situation of the chromophore in question, the information might be useful as in this study. Another point to be mentioned is that the recovery of CD in the visible during the conversion of PR to rhodopsin occurred independently from the recovery of CD in the UV region. This is also being investigated together with the UV difference spectrum of PR and rhodopsin. Acknowledgements-The authors wish to express their thanks to Professor T. Hara, Professor Y. Tsukahara and Professor B. Minke for their valuable discussions.
Photoreactions
of cephalopod rhodopsin
REFERENCES Atzmon Z., Hillman P. and Hochstein S. (1978) Visual response in barnacle photoreceptors is not initiated by transitions to and from metarhodopsin. Nature 274, 74-76. Azuma K., Azuma M. and Suzuki T. (1975) Circular dichroism of cephalopod rhodopsin and its intermediates in the bleaching and photoregeneration process. Biochim. biophys.
Acta 393, 520-530.
DeGrip W. J., Liu R. S. H., Ramamurthy V. and Asato A. (1976) Rhodopsin analogues from highly hindered 7-cis isomers of retinal. Nature 262, 416-418. Hagins W. A., Zonana H. V. and Adams R. G. (1962) Local membrane current in the outer segments of squid photoreceptors. Nature 194, 844-847. Hara T. and Hara R. (1966) Electrical conductance of rhodopsin solutions. Proceedings of the 4th ISCERG symposium Jap. J. Ophthal., Suppl. 10, 22-28. Hillman P., Hochstein S. and Minke B. (1972) A visual pigment with two physiologically active stable states. Science 175, i 4861488. Hubbard R. and St. George R. C. C. (1958) The rhodopsin system of the squid. J. gen. Physiol. 41, 501-528. Hubbard R. and Xropf A. (1958) The action of light on rhodopsin. Proc. Natl. Acad. Sci. U.S.A. 44. 13&139. Hubbard R., Brown P. K. and Bownds D. (1971) Methodology of vitamin A and visual pigments. Meth. Enzym. MC, 615-653. Kito Y., Ishigami M. and Yoshizawa T. (1961) On the labile intermediate of rhodopsin as demonstrated by low temperature illumination. Biochim. biophys. Acta 48, 287-298.
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Kito Y., Azuma M. and Maeda Y. (1968) Circular dichroism of squid rhodopsin. Biochim. biophys. Acta 154, 352-359.
Kito I’. and Nashima K. (1980) Retinal-opsin linkage of cephalopod rhodopsin. Photochem. Photobiol. 32, 443-445.
Nashima K., Mitsudo M. and Kito Y. (1978) Studies on cephalopod rhodopsin: Fatty acid esters of sucrose as effective detergents. Biochim. biophys. Acta 536, 78-87. Nashima K. and Kito Y. (1980) Effect of phospholipid and detergent on Retinal Schiff base in cephalopod rhodopsin and metarhodopsin. Biochim. biophys. Actu. 626, 39&396.
Nolte J. and Brown J. E. (1972) Ultraviolet-induced sensitivity to visible light in ultraviolet receptors of Limulus. J. gen. Physiol. 59, 186200. Rotmans J. P. and Kropf A. (1975) The analysis of retinal isomers by high speed liquid chromatography. Vision Res. 15, 1301-1302. Shichida Y., Tokunaga F. and Yoshizawa T. (1978) Circular dichroism of squid rhodopsin and its intermediates. Biochim.
biovhvs.
Acta 504. 413430.
Suzuki T., Sugadara M., Azuma K., Azuma M., Saimi Y. and Kito Y. (1973) Studies on cephalopod rhodopsin: Conformational changes in chromophore and protein during the photoregeneration process. Biochim. biophys. Acta 333, 149-l 60.
Tsuda M. (1979) Optical activity of octopus metarhodopsins. Biochim. biophys. Acta 578, 372-380. Yoshizawa T. and Wald G. (1964) Transformations of squid rhodopsin at low temperatures. Nature 201, 340-345.
Vision Rrreurch Vol. 21. pp. 935 lo 941. 1981 Printed in Great Britain
PHOTOREACTIONS
OF CEPHALOPOD
RHODOPSIN
TAKAYUKINAITO, KAZUKONASHIMA-HAYAMA, KOHZOOHTSU’ and YUJI Ktro Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan and ‘Marine Laboratory, Faculty of Science, Okayama University, Ushimadocho, Okayama 701-43,Japan (Received 2 February 1980) Abstmet-In the cephalopod rhodopsin system, two new photoproducts from alkaline metarhodopsin were found. One was slowly converted to rhodopsin in the dark and called pseudo-rhodopsin. It could be photoconverted to the other, pseudo-metarhodopsin. This resulted in a photosteady state mixture in the
light. Absorbance spectra of both pigments depended reversibly on pH. Both pigments revealed weak, negatively signed circular dichroism corresponding to their absorbance spectra. Acid and alkaline metarhodopsin also had negatively signed circular dichroism corresponding to their respective absorbance spectra.
INTRODUCTION The visual pigment of cephalopod rhodopsin is located in the rhabdomeric membrane of photoreceptor cells. Considerable information concerning the photoreaction of rhodopsin and the ionic mechanism of the receptor potential has accumulated, but how the photoreaction is linked to permeability changes in the rhabdomeric membrane is still a matter of speculation (Hagins et al., 1962; Atzmon et al., 1978). It is necessary to investigate not only the events involved in the transformation of rhodopsin to its final photoproduct, metarhodopsin, but also the events in the reverse reaction, the photoregeneration process. Cephalopod metarhodopsin can take two forms according to pH (acid and alkaline metarhodopsin). Acid metarhodopsin is photoconverted preferentially to rhodopsin (Hubbard and St. George, 1958) via an intermediate, P3s0 having a relatively short life time at ordinary temperature and pH (Suzuki et al., 1973). On the other hand, alkaline metarhodopsin may be converted by light to rhodopsin and isorhodopsin in Loligo pealei (Hubbard and St. George, 1958) or to an unstable pigment other than isorhodopsin in Todurodes pacificus (Hara and Hara, 1966). In this study, it was found that the photoproduct of cephalopod rhodopsin illuminated with a light of &,,., = 4OOnm, was quite different from the photoproduct of rhodopsin illuminated with longer wavelength light (A > 560nm). This wavelength dependence in the photoreaction of cephalopod rhodopsin was attributed to the difference in the photoreaction of acid and alkaline metarhodopsin. A new relatively long-lived photoproduct of alkaline metarhodopsin was found to convert slowly to rhodopsin in the dark. METHODS Materials
The squid, Watasenia scintillans was mainly used in this study. Other species used were Todarodes paci935
jicuq Loligo japonica, Sepiella japonica, and Octopus minor. The eyes of these cephalopods were stored at
- 20°C. The extraction and purification of rhodopsin was carried out as described in a previous paper (Nashima et al., 1978). The rhabdomeric membranes were obtained by flotation on 400/, sucrose solution. Rhodopsin was extracted with 2% ~-1690 solution and purified by DEAE-cellulose and concanavalin A-Sepharose column chromatography. The AZsO/Amax values of the rhodopsin preparations obtained were between 2.8 and 3.0. In some experiments, the detergent digitonin was added to the rhodopsin in ~-1690 solution for three reasons. Firstly, the procedure enabled a comparison with the earlier observations by Hubbard and St. George (1958), and Hara and Hara (1966). Secondly, digitonin lowers the apparent pK of the retinal Schiff base in metarhodopsin and rhodopsin (Nashima and Kito, 1980). This makes it easier to investigate the pH dependence of metarhodopsin and the new product described later. Thirdly, the addition of digitonin decreases the rate of transition of the new product to rhodopsin and metarhodopsin, which proved useful in an analysis of the reactions observed. The pH dependence of metarhodopsin was measured by the methods described by Hubbard and St. George (1958) and Nashima and Kito (1980). The pH dependence of the new product was determined as follows. The rhodopsin solution was illuminated at pH 7.5 and 4°C for 10min with light obtained by passing the beam from a 500 W projection lamp through a Toshiba VV-40 filter which transmitted maximally at 400nm (half band width: 106mn). Immediately after cessation of the light, the pH of the solution was changed by addition of 1 N NaOH. The residual absorbance at 482 nm was determined at desired pHs. Retinal isomer compositions of rhodopsin, isorhodopsin, metarhodopsin and the new product were analyzed with a high performance liquid chromatograph (HPLC) system (Yanaco ~-2000) equipped with a silica gel column (Yanaco SA-1, 4 x 500mm (cf:
936
TAKAYUKI NAITO et al.
Wavelength inm)
/ 300
I
400
8
I
,
500
Wavelength fnm)
I
1
1
250 Vbdength
1
1
1
i
290
tnml
Fig. 1. Absorbance spectra and circular dichroism spectra of rhodopsin and metarhodopsin of the squid. ~~~us~t~iusci~ftj~~u~ls. Absorbance spectra (A) and circular dichroism spectra (B) and (C). Curve I : rhodopsin (i,,,: 482 nm) in 0.2% ~-1690, and 0.1 M KCI solution at pH 6.0. Curve 2: alkaline metarhodopsin (i.,,,: 380nm) produced by irradiating rhodopsin at pH I 1.6 and 4°C with orange light (E.> 560 nm) for 1min. Curve 3: acid metarhodopsin (i,,: 496nm) produced by readjusting the pH of the solution to 6.4. All the m~surements were carried out at 6°C (light path: IOmm) and curves in (B) and in (C) were averaged after summation of five and two measurements, respectively.
Rotmans and Kropf (1975)). The elution solvent was a mixture of hexane, diethylether (8%) and acetone (0.25%) which could separate 5 isomeric retinals. Pigments were denatured with 1% SDS solution, and then retinal was extracted into diethylether. The relative content of each isomer was determined after correcting for the difference in molar extinction of each isomer (Hubbard et al., 1971; DeGrip et al., 1976). Isorh~opsin was made by illuminating rhodopsin with red light (i Z=600nm) at liquid nitrogen temperature (Kito er al., 1961). Absorbance spectra were determined with a Union ShI-401 s~~rophotometer (scanning rate of lOnm/ set). The circular dichroism (CD) spectra were measured with the computer-controlled Jovin-Yvon Dichrograph Mark III-J (scanning rate of 1 rim/see). The temperature of the cell-holders was controlled by circulating water of a constant temperature. The detergent ~-1690 (lauryl ester of sucrose) was kindly synthesized by Ryoto Co. Ltd (Tokyo). Digito-
nin and mercapt~thanol Chemicals (Osaka).
were purchased from Wako
RESULTS
Figure 1 shows the absorbance and CD spectra characteristic of squid rhodopsin and its thermally stable photoproducts, acid and alkaline metarhodopsin. In the region above 300 nm, both metarh~opsins have negative CD, corresponding to the absorbance spectra. The molar ellipticities, [@J of rhodopsin and acid and alkaline metarhodopsin at their respective x bands were 7.3 x IO4 at 470 nm (rh~opsin), - 1.2 x lo4 at 490 nm (acid metarhodopsin) and -2.1 x lo4 at 380 nm (alkaline metarhodopsin). In the region below 300 nm, the rhodopsin has small CD peaks or shoulders at 295, 2%5,279, 262 and 256nm, attributable to some aromatic residues and disulfide bonds of the protein. In the CD spectrum of acid metarhodopsin, peaks at 285 and 279 nm are less
Photoreactions
of cephalopod rhodopsin
Wavelength (nm -.----. 1
488 486 464 482 480
, 0
I I
I 2
1 3
I 4
;
j&Y-
931
sorbance around 380nm increased in curve 2 and 3 (Fig. 2A) and then gradually decreased (curves 5 and 7). On the other hand, the squid rhodopsin illuminated with the orange light at 4°C and pH 7.5, had already attained the photosteady state by 2Osec (Fig. 2B). Figure 3 shows the absorbance and CD spectra of the photosteady state mixtures. The photosteady state mixture (curve 2) produced by, the orange light contained almost an equal amount of rhodopsin and acid metarh~opsin as judged from the CD spectrum (curve 2), the mixture contained only a small amount of alkaline metarhodopsin at this pH to judge from the slightly higher absorbance at 380nm compared with seen for rhodopsin (curve 1). The photosteady state produced by the 400nm light (curve 3) appeared as if the squid rhodopsin were completely changed into acid metarh~opsin, having negatively signed CD in the visible. However, the I.,,,., of the . curve 3 was similar to that of rhodopsin (482 nm), at a considerably shorter wavelength than that of acid metarhodopsin (496nm). As will be shown later, the solution of Fig. 3, curve 3, in fact does not contain rhodopsin, nor acid nor alkaline metarh~opsin, and the absorbance is due to a new photoproduct obtained with the 4OOnm light (Pmx). In a parallel experiment, it was shown that the new product could be produced from squid isorhodopsin in a similar man-
Imin) Fig. 2. Photo~action of ~uruse~ia rhodopsin. (A) changes in absorbance spectrum of the rhodopsin illuminated with the 400nm light. Curve 1: Watasenia rhodopsin in 0.4% L-1690 and 0.05 M Tris-HCl (pH 7.5), curve 2: illuminated with the 400 nm light at 4°C for 1 set, curve 3: for 5 set, curve 4: for 15 set, curve 5: for 1min, curve 6: for 2 min, and curve 7: for 5 min. (B) Changes in the ,I,,,,, of the squid rhodoosin illuminated with the 4C@nm lkhht and the orange light (J. > 560nm). Open squares (U)~J.,,,,, of rhodopsin solution illuminated with the 4OOnm light, and with orange light open circles (o), illuminated (d z 560 nm). All the measurements were carried out at 10°C.
obvious. In the absorbance spectrum of alkaline metarhodopsin, a shoulder at 295 nm becomes more prominent and absorbance below 260nm was largely enhanced, possibly due to dissociation of some tyrosine residues at pH 11.6. An obvious CD peak at 290 nm is also characteristic of alkaline metarhodopsin, since the CD of rhodopsin made alkaline did not show the 290 nm peak. Thus these three pigments can be distinguished spectroscopically from one another. As is well known (Hubbard and St. George, 1958), in the squid rhodopsin system the photosteady state mixture above 0°C should contain a certain amount of rhodopsin, acid and alkaline metarhodopsin depending on the wavelength of illumination and the pH of the solution. As shown in Fig. 2A and B, in the initial stage of illuminating squid rhodopsin with the 4OOnm light at 4°C and pH 7.5, the J,,,,,, shifted from 482 nm (rhodopsin) to a longer wavelength (491 nm) and gradually returned to 482 nm after 5 min illumination (Photosteady state). At the same time, the ab-
A
0.6
Whwl6ftgth tnmf
”
-aooss wovd0ngm (nm)
Fig. 3. Photoproducts of Warosenia rhodoosin irradiated wiih orange light and a light of &,., = 4OO’nmat pH 7.5. (0.05 M Tris-HCll and 4°C. Absorbance snectra fAl and CD spectra (B). durve 1: rhodopsin in O.&, ~-l&b solution. Curve 2: preparation irradiated at 4°C with the orange light (J. z 560 nm) for 2 min. Curve 3: preparation irradiated with the 400 nm light for 10 min. Curves 2 and 3 are those of the photosteady-state made with respective light. All the measurements were carried out at 6°C.
TAKAYUKI NAITO et al.
938
0.6 L
o, 0.4
E a
0.2
Wovelength(nm)
%
Fig. 4. Dark reaction of Pmx of Wrctasenia rhodopsin. (A) Spectral changes of Pmx during dark incubation at 15°C. Absorbance spectra were measured every 5min after irradiation of the rhodopsin at 4°C and pH 7.5 (0.05 M Tris-HCI) for 10 min with the 400 nm light. The initial curve corresponds to curve 3 in Fig 3. (B) Circular dichroism spectra of Wutusenia rhodopsin and Pmx. Curve 1: Watuseniu rhodopsin. Curve 2: the rhodopsin solution illuminated with the 400 nm light for 10 mm at 4°C and pH 7.5. Curve 3: the solution incubated at 15°C for lOOmin in the dark.
When Pmx was left to stand at 15°C in the dark, the absorbance at 482 nm gradually decreased and the absorbance at 380nm rose, as shown in Fig. 4. The half time of this change was 30 min. After 100 min, the sign of CD was reversed, suggesting the appearance of rhodopsin (curve 3). After the incubation, addition of
hydroxyl~ine (5OmM) to the product shifted the 380 nm peak to 367 rnn, indicative of formation of the retinal oxime. The fact suggests that Pmx contained two fractions convertible to rhodopsin or free retinal in the dark, since rhodopsin and metarh~opsin are resistant to hydroxylamine. When Pmx was incubated in the presence of hydroxylamine, the absorbance peak at 482nm shifted to a longer wavelength. This cannot be due to oxime formation (see above) but NHsOH is also a mild reductant. This was checked by using mercaptoethanol as shown in Fig. 5. The 488nm product formed with the aid of 0.5% mer~ptoethanol contained a significant amount of metarhodopsin and the same amount of rhodopsin as in the case incubated in the absence of the reagent The absorbance spectrum of Pmx depended reversibly on pH in the same way as does the absorbance spectrum of metarhodopsin. The alkaline form of Pmx had a &,,,, of 380 nm, identical to that of alkaline metarhodopsin, possibly due to deprotonation of the retinal Schiff base of the product. As shown in Fig. 6, the apparent pK of the interconversion of the two forms of the product was higher (10.6) than that of metarhodopsin (7.9). The HPLC anaIysis of the chromophoric retinal indicates (Table 1) that almost an equal amount of all-trans and 11-cis retinal are contained in Pmx. The 13-cis retinal (loo/,) of the product may possibly be an artifact, due to isomer~tion during the extraction procedure. This isomer is always found when other pigments that are believed to be free from the isomer are analyzed with column separation, Acid metarh~opsins of cephalopods generally have a &,,,, longer than that of the parent rhodopsins. As demonstrated in Table 2, the &,,,, of Pmx of three species is largely independent of the hamalof the parent rhodopsin. The decay in Table 2, is estimated from the increase in absorbance at 380nm as in Fig. 4,
P
6
s 0
5
6
t
8
9
10
II
12
PH WavabnQth
t nm )
Fig. 5. Effect of mercaptoethanol on Pmx of Watasenia The rhodopsin solution illuminated with the 400 nm light at 4°C and pH 7.5 (0.05 M Tris-HCl) was incubated in the presence of 0.5% mercaptoethanol at 15°C and spectra were recorded every 5 min. Absorbance maximum shifted from the initial 482 nm to 488 nm.
scintikms.
Fig. 6. The pH dependence of Pmx and metarhodopsin of Warasenia scjnti~~u~s. Curve t : per cent of acid metarhodopsin at 4°C was plotted against pH. Curve 2: per cent of absorbance at 480nm of Pmx at 4°C was plotted against pH. The initial rhodopsin solution contained 0.2% digitonin, 0.1% ~-1690 and 0.1 M KCI. The pH of the rhodopsin solution ii~uminated with the 400nm tight was changed immediately after cessation of the light.
Photoreactions
of cephalopdd rhodopsin
Table 1. Retinal isomer composition of the photoproduct (Pmx) of Wataseniarhodopsin irradiated with light of &,,.. = 400 nm, comparing with those of rhodopsin, isorhodopsin and metarhodopsin
Rhodopsin Isorhodopsin Metarhodopsin
Pmx
Table 2. Absorbance maximum and decay time of photoproduct (Pmx) of squid rhodopsin illuminated with a light of &,.. = 400nm Decay of Pmx Absorbance maximum (nm) at 15°C Rhod acMR Pmx (min)
l-cis all-trans
13-cis
I I-cis
9-cis
4 9 8
76 5 5
1
0
12 0
1 0
19 13 87
10
46
I
0
43
Preparation of isorhodopsin, metarhodopsin and Pmx is described in Materials and Methods.
corresponding to the formation of retinal and not to the formation of rhodopsin or metarhodopsin. Squid Pmx released some retinal even in the presence of mercaptoethanol. However, Pmx in the cuttle fish (Sepiella) and octopus hardly released any retinal even in the absence of mercaptoethanol. In the cuttle fish, the conversion of Pmx to rhodopsin was complete within 1Omin at 10°C and pH 7.5, judging from the recovery of the CD intensity. The rate of the thermal change of the product to rhodopsin and metarhodop sin was species-specific. In the octopus, two components of Pmx were found that had different &,,., values, possibly due to the different chromophores (11-cis and all-truns retinal), although the two components of squid Pmx, also with different chromophores, were spectroscopically indistinguishable. As shown in Fig. 7, the &,,., of acid metarhodopsin of the octopus (52Onm, not illustrated) is far from that of the rhodopsin at 480mn (curve l), and Pmx has a J_. at 496 nm (curve 2). When Pmx was further illuminated with red light (A > 6OOmn) the &,,., altered to 474nm. This photoproduct can convert almost entirely to rhodopsin after incubation at 15°C in the dark as judged from absorbance and CD in the visible. Pmx can be obtained again if the preparation is exposed once more to 400nm illumination at 4°C for 1 min.
939
Species
Wataseniascintillans 482 Loligo japonica 496 Todarodespacificus 482
496 500 491
482 488 488
30 5 7
Rhod and acMR are rhodopsin and acid metarhodopsin. Pmx is photoproduct produced by irradiating rhodopsins in 0.4% ~-1690 solution with a light of I,,, = 4OOnm for 10min at pH 7.5 and 4°C. The half time of the spectral change (decay of Pmx) was determined by incubating at 15°C in the dark as shown in Fig. 4.
reaction of short lived intermediates in the process of rhodopsin + metarhodopsin is also improbable since the exhaustive illumination of squid rhodopsin by the 400nm light at liquid nitrogen temperature (Yoshizawa and Wald, 1964), dry ice temperature and around -40°C (Azuma et al., 1973, did not produce the Pmx observed in this study, and Pmx was formed even by quite weak light provided the pH was alkaline.
600
600
400
khf6kJWth
(nm)
DISCUSSION
Formation of the new photoproduct from metarhodopsin
alkaline
Illuminating rhodopsin or isorhodopsin with orange light yields a photosteady-state of rhodopsin and metarhodopsin. The 400 nm light irradiation produces a new product, Pmx. The light can be absorbed not only by rhodopsin and acid metarhodopsin but also by alkaline metarhodopsin. However below pH 5.5, little tir no Pmx is produced, so a wavelengthdependent photoreaction of rhodopsin and acid metarhodopsin forming the product seems improbable. On the contrary, the appearance of Pmx becomes more rapid at higher pH, implying a path way of alkaline metarhodopsin + Pmx. Another possibility that Pmx might be produced by photo-
L
I
Jo0
I
I 400 wd6ngrn
I 600
I
I
(nm)
Fig. 7. Photoreaction of octopus rhodopsin. Absorbance spectra (A) and CD spectra (B). Curve I : rhodopsin at pH 7.5 (0.05 M Tris-HCI) and 4°C in 0.1% digitonin and 0.3% ~-1690 solution. Curve 2: rhodopsin solution irradiated with the 4OOnm light for IOmin at 4°C. Curve 3: the solution of curve 2 was irradiated further with the red light (1 z 600 nm) for 1min at 4°C.
TAKAYUKI NAITO et
al.
PMR to rhodopsin and metarhodopsin may be accompanied by a further change in protein structure, possibly involving the rearrangement of sullhydryl links as suggested in the thermal change of Pmx as illustrated in Fig. 5. Other remarks
PR _=
pm light
._
--
.-
(4)
--- - -
Fig. 8. Photoreaction of cephalopod rhodopsin at normal temperature and pH. R, acMR, alMR, PR and PMR are rhodopsin, acid and alkaline metarhodopsin, pseudorhodopsin and pseudo-metarhodopsin, respectively. Species-specificity of Pmx
Hubbard and St. George (1958) and Hubbard and Kropf (1958) found that the photoproduct of alkaline metarhodopsin had a J_, shorter than that of the parent rhodopsin (I.&go pealei). Hara and Hara (1966) reported that the photoproduct of alkaline metarhodopsin was unstable and had a &,a, almost similar to that of the parent rhodopsin (Todarodes pactjicus). These observations were reconfirmed, as shown in Table 2. In those earlier experiments, the illumination was carried out in alkaline conditions and this made it difficult to understand the nature of the products because of their instability and pH dependence as in Figs 4 and 6. Two components of Pmx and photoreaction of cephalopod rhodopsin Pmx contains two isomeric chromophores and hence should be considered as two, one of which converts to rhodopsin and the other of which releases retinal or can be converted to metarhodopsin with the aid of mercaptoethanol. The component produced by illuminating octopus Pmx with red light may be called pseudo-rhodopsin; it has 11-cis retinal as its chromophore but a negative CD in the visible. This is converted by light to pseudo-metarhodopsin which has all-trans retinal as the chromophore. Thus the photoreaction of cephalopod rhodopsin at normal temperatures can be illustrated as shown in Fig. 8. The photoreaction (1) of acid metarhodopsin to rhodopsin involves an intermediate, PBS0 which is long-lived at an alkaline pH (Suzuki et al., 1973). Under the condition (pH 7 and lYC), the P,,, should have too short life time as compared with Pmx. The photoreaction (3) of alkaline metarhodopsin to pseudo-rhgopsin (PR) presumably involves the isomerization of the chromophore, accompanying the rise of pK of the retinal Schiff base due to rearrangement of protein structure. Further photoreaction (4) of PR to PMR also involves the isomerization of the chromophore. The dark reaction (5) of PR and
The apparent pK of the interconversion of metarhodopsin is considerably affected by the phospholipid and detergents used for dispersing cephalopod rhodopsin and also by the salt concentration (Kito and Nashima, 1980). The apparent pK of the interconversion of acid and alkaline metarhodopsin in rhabdomeric membranes was higher (above 9) than in digitonin solution (about 7), but the titration curve deviated from the theoretical titration curve of a single ionizable group of the same pK. In the rhabdomerit membrane, only a small amount of alkaline metarhodopsin would be formed and if the wavelength of light is such that light is absorbed by alkaline metarhodopsin, the photoproducts (PR and PMR) will accumulate. It is of interest to examine the formation of those products in the arthropod rhodopsin system, since a photoproduct with a relatively long life time is assumed to cause the so-called PDA (prolonged depolarizing afterpotential) in the photoreceptors (Nolte and Brown, 1972; Hillman et Al., 1972). Previously, the CD characteristic of cephalopod metarhodopsin could not be detected in the region above 300 nm (Kito et al., 1968). In digitonin solution, even rhodopsin tended to release protons from the retinal Schiff base at an alkaline pH and to form alkaline rhodopsin (Nashima and Kito, 1980), and this had a positive CD around 380nm and could convert to rhodopsin at a neutral pH. Possibly, this substance disturbed to detect the weak negative CD of metarhodopsin. Recently Tsuda (1979) reported values of CD for octopus metarhodopsin in the visible that were a little different from the data of this paper. Taking into account of the formation of alkaline rhodopsin, the discrepancy would be explained. At present all the intermediates from cephalopod rhodopsin to metarhodopsin have shown a negative CD in the visible (Azuma et al., 1975; Shichida et al., 1978), differing from the vertebrate rhodopsin system. The explanation of CD data in rhodopsin system is presently difficult to understand; however, as CD is very sensitive to the conformation or situation of the chromophore in question, the information might be useful as in this study. Another point to be mentioned is that the recovery of CD in the visible during the conversion of PR to rhodopsin occurred independently from the recovery of CD in the UV region. This is also being investigated together with the UV difference spectrum of PR and rhodopsin. Acknowledgements-The authors wish to express their thanks to Professor T. Hara, Professor Y. Tsukahara and Professor B. Minke for their valuable discussions.
Photoreactions
of cephalopod rhodopsin
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Acta 393, 520-530.
DeGrip W. J., Liu R. S. H., Ramamurthy V. and Asato A. (1976) Rhodopsin analogues from highly hindered 7-cis isomers of retinal. Nature 262, 416-418. Hagins W. A., Zonana H. V. and Adams R. G. (1962) Local membrane current in the outer segments of squid photoreceptors. Nature 194, 844-847. Hara T. and Hara R. (1966) Electrical conductance of rhodopsin solutions. Proceedings of the 4th ISCERG symposium Jap. J. Ophthal., Suppl. 10, 22-28. Hillman P., Hochstein S. and Minke B. (1972) A visual pigment with two physiologically active stable states. Science 175, i 4861488. Hubbard R. and St. George R. C. C. (1958) The rhodopsin system of the squid. J. gen. Physiol. 41, 501-528. Hubbard R. and Xropf A. (1958) The action of light on rhodopsin. Proc. Natl. Acad. Sci. U.S.A. 44. 13&139. Hubbard R., Brown P. K. and Bownds D. (1971) Methodology of vitamin A and visual pigments. Meth. Enzym. MC, 615-653. Kito Y., Ishigami M. and Yoshizawa T. (1961) On the labile intermediate of rhodopsin as demonstrated by low temperature illumination. Biochim. biophys. Acta 48, 287-298.
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Nashima K., Mitsudo M. and Kito Y. (1978) Studies on cephalopod rhodopsin: Fatty acid esters of sucrose as effective detergents. Biochim. biophys. Acta 536, 78-87. Nashima K. and Kito Y. (1980) Effect of phospholipid and detergent on Retinal Schiff base in cephalopod rhodopsin and metarhodopsin. Biochim. biophys. Actu. 626, 39&396.
Nolte J. and Brown J. E. (1972) Ultraviolet-induced sensitivity to visible light in ultraviolet receptors of Limulus. J. gen. Physiol. 59, 186200. Rotmans J. P. and Kropf A. (1975) The analysis of retinal isomers by high speed liquid chromatography. Vision Res. 15, 1301-1302. Shichida Y., Tokunaga F. and Yoshizawa T. (1978) Circular dichroism of squid rhodopsin and its intermediates. Biochim.
biovhvs.
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Tsuda M. (1979) Optical activity of octopus metarhodopsins. Biochim. biophys. Acta 578, 372-380. Yoshizawa T. and Wald G. (1964) Transformations of squid rhodopsin at low temperatures. Nature 201, 340-345.