Purification and low temperature spectroscopy of gecko visual pigments green and blue.

We purified two kinds of visual pigments, gecko green and gecko blue, from retinas of Tokay geckos (Gekko gekko) by two steps of column chromatography, and investigated their photobleaching processes by means of low temperature spectroscopy. Absorption maxima of gecko green and blue solubilized in a mixture of 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS) and phosphatidylcholine were 522 and 465 nm, respectively, which are close to those observed in the photoreceptor cells. Low temperature spectroscopy identified six intermediates in the photobleaching process of gecko green; batho (lambda max = 569 nm), BL (lambda max = 519 nm), lumi (507 nm), meta I (approximately 486 nm), meta II (approximately 384 nm), and meta III intermediates (approximately 500 nm). In contrast to the high similarity in amino acid sequence between gecko green and iodopsin [Kojima, D., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6841-6845], the batho-green did not revert thermally to original gecko green but converts to the next intermediate. The photobleaching process of gecko blue was investigated by low temperature spectroscopy, and three intermediates, meta I (lambda max = approximately 470 nm), meta II (lambda max = approximately 370 nm) and meta III (lambda max = approximately 475 nm), were identified. A comparative study on the thermal behavior of meta intermediates revealed that the thermal stability of meta II intermediate of both of the gecko visual pigments is lower than that of metarhodopsin II. The result supports the idea that both the gecko visual pigments are cone-type ones.

The eyespot of Euglena gracilis: a microspectrophotometric study.

The eyespots in cells of streptomycin-bleached strains and of dark-grown cultures of Euglena gracilis, were examined by means of fluorescence microscopy and microspectrophotometry. When viewed with light in the region of 380-500 nm, the stigma appeared as a dark spot. Adjacent to this was a second spot, not seen with white light, but which was seen to fluoresce when excited with radiation at 370 +/- 20 nm. This fluorescence proved to be polarized in contrast to other fluorescing bodies in the cell. The absorption curves, obtained by microspectrophotometry of individual eyespots, were found to consist of two spectral maxima, an A-band in the blue and a B-band in the green. Unlike the A-band, the B-band provided evidence of originating from an anisotropic structure. Relating these data to literature findings, we conclude that the B-band is the absorbance of a pigment in the quasi-crystalline paraflagellar body and the A-band perhaps a pigment in the orange-red stigma. The spectrum of the B-band does not appear to be that of a flavoprotein or of a free carotenoid but its resemblance to the spectrum of rhodopsin is significant in relation to published data for the Chlamydomonas eyespot that suggests the presence of a rhodopsin-like pigment as the photosensitive system responsible for phototaxis in this alga.

The eyespot of Chlamydomonas reinhardtii: a comparative microspectrophotometric study.

The eyespot of Chlamydomonas reinhardtii is believed to utilize a rhodopsin-like pigment in its responses to light. This paper examines its eyespot by means of microspectrophotometry with the finding of an absorption spectrum with two bands, an A-band in the blue, and a B-band in the green. This spectrum is identical to that previously recorded from the eyespot of Euglena gracilis. As with Euglena the B-band was found to have dichroic character and its spectrum was similar to the absorption curve of rhodopsin. This A-B-spectrum was always recorded from a single granule in each cell. It is concluded that both E. gracilis and C. reinhardtii may utilize a rhodopsin-like pigment as the photopigment associated with the eyespot response to light. In both these algae a few particles in each cell were found whose spectra consisted of two other bands, C and D, blue- and red-shifted, respectively, relative to the eyespot A-B-bands. There is some reason to believe that the C-D-granules may also be involved in certain light-controlled activities of the cells.

The gecko visual pigment: the anion hypsochromic effect.

The 521-pigment in the retina of the Tokay gecko (Gekko gekko) readily responds to particular physical and chemical changes in its environment. When solubilized in chloride deficient state the addition of Class I anions (Cl-, Br-) induces a bathochromic shift of the absorption spectrum. Class II anions (NO3-, IO3-, N3-, OCN-, SCN-, SeCN-, N(CN)2-), which exhibit ambidental properties, cause an hypsochromic shift. Class III anions (F-, I-, NO2-, CN-, AsO3-, SO2(4-), S2O2(3-) have no spectral effect on the 521-pigment. Cations appear to have no influence on the pigment absorption and Class I anions prevent or reverse the hypsochromic shift caused by Class II anions. It is suggested that the spectral displacements reflect specific changes in the opsin conformation, which alter the immediate (dipolar) environment of the retinal chromophore. The protein conformation seems to promote excited-state processes most in the native 521-pigment state and least in the presence of Class II anions. This in turn suggests that the photosensitivity of the 521-pigment is controlled by the excited rather than by the ground-state properties of the pigment.

Adaptations of visual pigments to the photic environment of the deep sea.

This is a summary of studies that bear on the problems of the adaptation of visual pigments to the photic environment of the deep sea. The results suggest that the spectral absorption of these retinal pigments is shifted toward the blue in order to match the dim, blue-green downwelling light and/or the bioluminescence of organisms that are critical to the life of the species. Through such a spectral match, greater visual sensitivity is achieved for life in the special photic condition of their habitat. This adaptation has been found for chimaerid fishes, for elasmobranchs, for teleosts, for mammals, and for certain crustaceans and cephalopods. The most convincing evidence for such an adaptive match has been found in teleosts that have red-emitting photophores. In these fishes a photopigment with absorbance shifted toward the red has been found by extraction and microspectrophotometry. A few exceptions to this idea of an adaptive match have appeared in the literature, the cone pigments, especially, being examples of such offset pigments. The malacosteid fishes have been shown to have a red-shifted retinal pigment with 11-cis-3-dehydroretinal as the chromophore and some invertebrates have also adopted this molecule to adjust the spectral absorption to the photic environment or to the bioluminescence. These studies are beginning to reveal that visual biochemistry is basically the same in vertebrates and invertebrates and that the visual pigment protein arose early in phylogeny and has been retained, with appropraite modifications, to the present.

The visual pigment cyanide effect.

The visual pigment of the Tokay gecko (Gekko gekko) with its in situ absorption maximum at 521 nm has its spectral position at 500 to 505 nm when chloride-deficient digitonin is used for the extraction. In this case the addition of chloride or bromide to the extract restores the maximum to 521 nm. This property, characteristic of gecko pigments in general, does not occur with any of the rhodopsins that have been tested. Simple salts of cyanide, a pseudohalogenoid with an ionic radius close to those of chloride and bromide and/or its hydrolysis product attacks both this gecko pigment and rhodopsins in the dark. This is seen as a slow thermal loss of photopigment if (sodium) cyanide is present at concentrations above 40 mM for the gecko pigment and 150 mM for the rhodopsins of the midshipman (Porichthys notatus) and of the frog (Rana pipiens). In all cases the loss of the photopigment is accompanied by the appearance of a spectral product with maximum absorption at about 340 nm. Cyanide addition has no effect on the photosensitivity of the native pigments and neither does it alter, as do chloride, bromide and other anions, the spectral absorbance curve. The spectral product at 340 nm also appears when the visual pigments are photolyzed in the presence of cyanide salts below the threshold concentrations given above. Incubation of digitonin-solubilized all-trans-retinal with (sodium) cyanide leads to a reaction product with absorption spectrum similar to that obtained with visual pigments under comparable conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

The Gecko visual pigment: the dark exchange reaction and the effects of anions.

A dark reaction is known to occur in retinal extracts of the gecko (Gekko gekko), in which the natural 11-cis-chromophore of the 521-pigment is apparently replaced by adding 9-cis-retinal to form the 9-cis-photopigment. With chloride-deficient extracts the reaction involves some 70% of the 521-pigment. Anions like nitrate, azide, thiocyanate and cyanate that shift the spectrum toward the blue do not affect this 70% exchange. Anions like fluoride, iodide and sulfate likewise do not alter this magnitude of reaction. In contrast, chloride and bromide that induce a bathochromic spectral shift lead to a decrease in this dark replacement of the 11-cis chromophore. This protection is similar to the action of these two anions in antagonizing the pigment loss by NH2OH and by temperature, both occurring in the dark. Apparently, chloride and bromide alter the opsin conformation so as to stabilize and/or protect the Schiff's base linkage but nitrate, azide, thiocyanate and cyanate act at a different opsin site or by a different mechanism.

The spectral properties and photosensitivities of analogue photopigments regenerated with 10- and 14-substituted retinal analogues.

Analogues of 11-cis- and 9-cis-retinal with substitutions at positions 10 and 14 were used to regenerate analogue photopigments with two opsins: that of the transmuted (cone-like) 521-pigment of Gekko gekko and that of the rhodopsin of Porichthys notatus. The spectral absorbances and photosensitivities of the regenerated photopigments were determined and compared, first, between the two systems of analogue photopigments, and second, in the responses to the two opsins. Unlike the 10-fluoropigments, the comparable 14-compounds were significantly red-shifted by 19-30 nm and their sensitivity to light was similar to that of the parent 11-cis- and 9-cis-pigments. These were the results for both analogue pigments. In contrast, the 10-pigments were spectrally located close to the wavelengths of the parent compounds and the photosensitivity was significantly reduced, especially in the case of the 9-cis-analogues. Evidence was obtained for a steric hindrance effect at position 14, for no regeneration was obtained when methyl or ethyl groups were at this carbon. In the 10-substituted retinals, steric hindrance was noted only for the gecko; only the fluorosubstituted, but not the chloro-, the methyl- or the ethyl-substituted, retinals reacted. With the fish opsin, pigments were regenerated with all but the ethyl-substituted retinal. The gecko opsin appears to have a more restricted binding site. Another feature of the gecko was related to the chloride bathochromic and hyperchromic effects, in which the 521-pigment prepared in a chloride-deficient state has a blue-shifted spectrum compared with the spectrum obtained after the addition of chloride, and its extinction is raised by the addition of chloride to give a mean ratio of 1.23 for the two extinctions, one with, the other without, added chloride. The 11-cis-10-F-analogue pigment gave both chloride effects and the hyperchromic ratio was the same as that recorded for the native visual pigment. In contrast, the pigment formed with 11-cis-14-F-retinal gave a hyperchromic ratio significantly greater than 1.23. A similar contrast in the responses to chloride was obtained with the analogue photopigments regenerated with the 9-cis-10-F- and 9-cis-14-F-chromophores. This difference between the two systems is interpreted as the result of a specific configurational feature of the gecko opsin when in the chloride-deficient state that is relevant to the binding of the retinal analogue.(ABSTRACT TRUNCATED AT 400 WORDS)

The gecko visual pigment: the chromophore dark exchange reaction.

This study confirms the occurrence of a dark-exchange reaction in the extracted 521-pigment of the Tokay gecko (G. gekko). The present study involved the exchange, in the dark, of the natural 11-cis-chromophore by the 9-cis-10-F-retinal analog. This analog is able to combine with the 521-opsin to regenerate a photopigment at 492 nm. In addition to this shift in absorbance from 521 to 492 nm, the analog photopigment has a photosensitivity some 2.4% that of the native 521-system in the chloride-sufficient state. These two properties of the regenerated analog pigment have simplified the demonstration of a dark exchange of chromophores. At 15 degrees C the 9-cis-10-F-analog replaces the 11-cis-chromophore by at least 30% (density-wise) in about 15 hr. This exchange occurs with the system in the chloride-deficient state. The presence of chloride during the period in the dark significantly reduces the magnitude of the exchange. Apparently, the protein has a more open structure at the chromophoric binding site, allowing this interchange of chromophores. The addition of chloride induces a conformational change at this site, 'burying' the Schiff base and reducing the exchange reaction. The biological implication of this mobile property of the gecko opsin is that it is similar to the behavior of the cone pigment iodopsin but is unlike that of rhodopsins. This supports the idea that the gecko visual cells, despite their appearance as rods, are phylogenetically related to ancestral photopic receptors.

Patterns of pigmentation in the eye lens of the deep-sea hatchetfish, Argyropelecus affinis Garman.

The present study is a morphological, biochemical and spectrophotometric characterization of the eye lens pigmentation in 45 specimens (11-88 mm in standard length) of the deep-sea hatchetfish, Argyropelecus affinis (Stomiiformes: Sternoptychidae). For comparison, we also examined available lenses of other members of the family Sternoptychidae, including three other species of the genus Argyropelecus, and two species of the genus Sternoptyx. Lens pigmentation was observed in all specimens of Argyropelecus spp. larger than about 36 mm in standard length, but was absent in all Argyropelecus spp. individuals less than 36 mm. However, lens pigmentation was not observed in Sternoptyx specimens of any size. Detailed studies of A. affinis indicated that at 36 mm the nascent lens fiber cells, which are continually laid down over preexisting, unpigmented cells, begin incorporating pigment, and the pigment concentration increases steadily as pigmented cells are added during lens growth. Spectrophotometric and biochemical data suggested that the pigment is a carotenoprotein complex, the carotenoid-like chromophore being strongly associated with a specific soluble lens protein, alpha crystallin. While the lens coloration in these fishes is age-related, analyses of the retinal visual pigment revealed no concomitant age-related change in the peak wavelength of retinal sensitivity in these fishes. Our data on the spectral absorbance of the lens and visual pigment of these fishes suggest that the lens pigmentation acts as a short-wave filter to improve acuity of the visual system.

Study of the shape of the binding site of bovine opsin using 10-substituted retinal isomers.

The 9-cis, 11-cis, 13-cis, and all-trans isomers of 10-fluoro-, 10-chloro-, 10-methyl-, and 10-ethylretinals have been prepared and characterized. Results of their interaction with bovine opsin are reported. The data have been analyzed in terms of the conformational properties of the retinal isomers and their steric compatibility with the binding site as defined by the two-dimensional map disclosed earlier. The need to expand the active zone and previously undetected restrictions in the third dimension are noted.

Photosensitivity of 10-substituted visual pigment analogues: detection of a specific secondary opsin-retinal interaction.

The photosensitivities of the bovine rhodopsin and gecko pigment 521 analogues regenerated from C-10-substituted analogues of 11-cis- and 9-cis-retinals were determined by two different methods. A similar reactivity trend was noted for both pigment systems as revealed in the photosensitivity of the gecko pigments and relative quantum yields of the bovine analogues. The 10-fluoro-11-cis photopigments had a photosensitivity less than, but approaching, that of the native (11-cis) visual pigment while the 10-fluoro-9-cis photopigments had a much lower photosensitivity than the parent 9-cis regenerated pigment. The results are interpreted in terms of recently described models of rhodopsin architecture and of the primary molecular reaction of visual pigments to light. The unusually low photoreactivity of the 10-fluoro-9-cis pigment molecule is viewed as the result of a regiospecific hydrogen-bonding interaction of the electronegative fluorine atom to the opsin.

The visual pigment sensitivity hypothesis: further evidence from fishes of varying habitats.

Visual pigments were extracted from the retinas of 8 species of marine teleosts and 4 species of elasmobranchs and a comparison was made of the pigment properties from these fishes, some inhabiting surface waters, others from the mesopelagic zone, and a few migrating vertically between these two environments. An association was found between the spectral position of the absorbance curve and the habitat depth or habitat behavior, with the blue-shifted chrysopsins being the pigments of the twilight zone fishes and the rhodopsins with fishes living near the surface. The retina of the swell shark (Cephaloscyllium ventriosum) yielded extracts with two photopigments; one, a rhodopsin at 498 nm; the second, a chrysopsin at 478 nm. This fish has been reported to practice seasonal vertical migrations between the surface and the mesopelagic waters. In addition to the spectral absorbance, several properties of these visual pigments were examined, including the meta-III product of photic bleaching, regeneration with added 11-cis and 9-cis retinals, and the chromophoric photosensitivity. The chrysopsin properties were found to be fundamentally similar to those of typical vertebrate rhodopsins. Correlating the spectral data with the habitat and habitat behavior of our fishes gives us confidence in the idea that the scotopic pigments have evolved as adaptations to those aspects of their color environment that are critical to the survival of the species.

Some properties of solubilized human rhodopsin.

This investigation involved an examination of some properties of solubilized human rhodopsin. In confirmation of previous work, the spectral maximum was found to be at 493 nm at temperatures 5-10 degrees C below 37.5 degrees C. An increase in temperature to 37.5 degrees C produced only a minor shift of 2-4 nm toward the blue. The opsin displayed the classic and typical stereospecificity of vertebrate visual pigments, regenerating a pigment at 493 nm with 11-cis retinal and an isopigment at 483 nm with 9-cis retinal. No regeneration occurred with either all-trans or 13-cis retinal. The chromophoric photosensitivity of human rhodopsin and of its 11-cis regenerated pigment was found to be the same at 13.2 X 10(-17) cm2; that of the isopigment, at 4.5 X 10(-17) cm2. The long-lived photoproduct of human rhodopsin at 475 nm (metarhodopsin-III) was found to be especially interesting because of its protracted growth following a brief (20 sec) light exposure of the pigment and because of its long decay time even at 27 degrees C and higher. This property (growth and decay of metarhodopsin-III) was studied at temperatures ranging from 1.9 to 37.5 degrees C. Though NH2OH (4.6 X 10(-3) M) was found to speed the decay of metarhodopsin-III, it did not prevent its presence during decay for minutes after the 20-sec bleach. It is clear that the human metarhodopsin-III is indeed a long-lived intermediate of bleaching and evidence from the literature, which is cited, suggests that this product persists for significant periods of time in the retinas of mammals, including that of man. This fact suggests the possible physiological role of metarhodopsin-III in some aspects of vertebrate vision.

The gecko visual pigment: its photosensitivity and the effects of chloride and nitrate ions.

By use of the method of photometric curves, the photosensitivity of the major and ion-sensitive pigment of Gekko gekko has been determined and compared with that of rhodopsins of the frog (Rana pipiens) and of the fish (Porichthys notatus). In the presence of Cl- (or Br-), the gecko pigment has the same photosensitivity as the other A1 rod pigments, but unlike these, the addition of NH2OH does not lead to a Dartnall effect, i.e. an enhancement in the measured rate of photic bleaching. This is because the gecko pigment has no meta-III intermediate. In the Cl- -deficient state the gecko pigment has a photosensitivity 0.8 times that of the Cl- -provided system. The increase in photosensitivity brought on by Cl- is quantitatively accounted for by the Cl- -induced hyperchromic effect. The addition of NH2OH to the system without added Cl- leads to a small increase in measured rate of photic bleaching with an apparent 13% increment in photosensitivity. This is not a classical Dartnall effect for here again no meta-III is involved. The possibility is raised of an additional, yet undiscovered, action of NH2OH on the opsin moiety. Nitrate ions (NO3-) are known to produce an increase in extinction coefficient similar to that of Cl- and a hypochromic shift in the spectral absorbance. Despite the hyperchromic action, NO3- produces a reduction in the measured rate of photic bleaching, an effect explained by the appearance of a meta-III type intermediate absorbing at about 470 nm. While Cl- is able to antagonize the NO3- -induced hypochromic shift, it is unable to reverse the NO3- -induction of meta-III. This, along with other differences in responses of the gecko pigment to these two ions, suggests that Cl- and NO3- act at two different sites and produce unique conformational changes in the protein molecule.

The gecko visual pigment: a pH indicator with a salt effect.

1. Unlike rhodopsin, the extracted 521-pigment of the Tokay gecko (Gekko gekko) is pH-sensitive and changes its spectral absorbance in the pH range of 4.5-7.3. The colour change is reversible and pH can be employed to adjust the spectral maximum anywhere between 490 nm and its native location at 521 nm.2. The hypsochromic shift with increasing acidity is opposite to that expected for the protonation of the Schiff base nitrogen and suggests an action on the secondary system of interacting charges that have long been postulated to adjust vertebrate visual pigment colour within the visible spectrum.3. Chloride ions modulate this pH effect in a systematic and significant manner. For the pigment extracted in the chloride-deficient state the colour change occurs in the pH range of 6.0-7.0, the midpoint being close to 6.5, suggesting the possible participation of the imidazole group of histidine as the functional moiety. With added NaCl the colour shifts to the region below pH 6.2.4. The modulating action of chloride is postulated to be a conformational change of the opsin leading to a shift of the secondary interacting site from one functional group to another or else to a change in pK of a single group due to the conformational alteration of the electrostatics of the system.5. At pH values between 7.5 and 9.0 a different mechanism becomes apparent. In this region a decrease occurs in the photopigment density as well as a shift in absorbance toward the blue. This alkaline effect is readily reversed either by adding NaCl or else by lowering the pH. Along with the other protective effects of chloride these ions serve to reduce or prevent this alkaline loss in density.6. Associated with this reversible photopigment loss is a reversible appearance of a product with a maximum at about 366 nm. The spectrum of this product is like that produced by the addition of 11-cis retinal to the extract. Acidification of the alkaline preparation leads to a restitution of the photopigment as well as to a reduction of the 366-product.7. Addition of hydroxylamine to the alkaline extract in appropriate concentration inhibits the restitution of pigment-521 with acid or NaCl, but adding 11-cis retinal to the system leads to restoration of the photopigment after acidification. All the evidence suggests that product-366 is either free 11-cis retinal or else held to the opsin in a form that does not alter its spectral absorbance. The alkaline effect is therefore a disruption of the aldimine bond of the visual pigment.8. In many respects the gecko 521-pigment behaves like the chicken cone pigment, iodopsin, suggesting that an investigation of the latter in terms of pH may be a worthy project for future study.9. With its ability to change colour with pH, with chloride, with nitrate, etc. the extractable gecko pigment offers possibilities for the investigation of mechanisms responsible for adjusting visual pigment absorbance throughout the visible spectrum. The techniques of circular dichroism, Raman spectroscopy, infra-red spectroscopy, etc. may find here a suitable material for these studies.