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PubMed2005

Defining the retinoid binding site in the rod cyclic nucleotide-gated channel.

Rod vision is initiated when 11-cis-retinal, bound within rhodopsin, absorbs a photon and isomerizes to all-trans-retinal (ATR). This triggers an enzyme cascade that lowers cGMP, thereby closing cyclic nucleotide-gated (CNG) channels. ATR then dissociates from rhodopsin, with bright light releasing millimolar levels of ATR. We have recently shown that ATR is a potent closed-state inhibitor of the rod CNG channel, and that it requires access to the cytosolic face of the channel (McCabe, S.L., D.M. Pelosi, M. Tetreault, A. Miri, W. Nguitragool, P. Kovithvathanaphong, R. Mahajan, and A.L. Zimmerman. 2004. J. Gen. Physiol. 123:521-531). However, the details of the interaction between the channel and ATR have not been resolved. Here, we explore the nature of this interaction by taking advantage of specific retinoids and retinoid analogues, namely, beta-ionone, all-trans-C15 aldehyde, all-trans-C17 aldehyde, all-trans-C22 aldehyde, all-trans-retinol, all-trans-retinoic acid, and all-trans-retinylidene-n-butylamine. These retinoids differ in polyene chain length, chemical functionality, and charge. Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid-channel interaction. We propose that the cytoplasmic face of the channel contains a retinoid binding site. This binding site likely contains a hydrophobic region that allows the ionone ring and polyene tail to sit in an optimal position to promote interaction of the terminal functional group with residues approximately 15 A away from the ionone ring. Based on our functional data with retinoids possessing either a positive or a negative charge, we speculate that these amino acid residues may be polar and/or aromatic.

Animals↗
PubMed2006

Visual cycle: Dependence of retinol production and removal on photoproduct decay and cell morphology.

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50-70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10-40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.

Ambystoma↗
PubMed2006

Cyclic nucleotide-gated ion channels in rod photoreceptors are protected from retinoid inhibition.

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.

1-Methyl-3-isobutylxanthine↗
PubMed2006

Turning cones off: the role of the 9-methyl group of retinal in red cones.

Our ability to see in bright light depends critically on the rapid rate at which cone photoreceptors detect and adapt to changes in illumination. This is achieved, in part, by their rapid response termination. In this study, we investigate the hypothesis that this rapid termination of the response in red cones is dependent on interactions between the 9-methyl group of retinal and red cone opsin, which are required for timely metarhodopsin (Meta) II decay. We used single-cell electrical recordings of flash responses to assess the kinetics of response termination and to calculate guanylyl cyclase (GC) rates in salamander red cones containing native visual pigment as well as visual pigment regenerated with 11-cis 9-demethyl retinal, an analogue of retinal in which the 9-methyl group is missing. After exposure to bright light that photoactivated more than approximately 0.2% of the pigment, red cones containing the analogue pigment had a slower recovery of both flash response amplitudes and GC rates (up to 10 times slower at high bleaches) than red cones containing 11-cis retinal. This finding is consistent with previously published biochemical data demonstrating that red cone opsin regenerated in vitro with 11-cis 9-demethyl retinal exhibited prolonged activation as a result of slowed Meta II decay. Our results suggest that two different mechanisms regulate the recovery of responsiveness in red cones after exposure to light. We propose a model in which the response recovery in red cones can be regulated (particularly at high light intensities) by the Meta II decay rate if that rate has been inhibited. In red cones, the interaction of the 9-methyl group of retinal with opsin promotes efficient Meta II decay and, thus, the rapid rate of recovery.

1-Methyl-3-isobutylxanthine↗
PubMed1952

The role of sulfhydryl groups in the bleaching and synthesis of rhodopsin.

The condensation of retinene(1) with opsin to form rhodopsin is optimal at pH about 6, a pH which favors the condensation of retinene(1) with sulfhydryl rather than with amino groups. The synthesis of rhodopsin, though unaffected by the less powerful sulfhydryl reagents, monoiodoacetic acid and its amide, is inhibited completely by p-chloromercuribenzoate (PCMB). This inhibition is reversed in part by the addition of glutathione. PCMB does not attack rhodopsin itself, nor does it react with retinene(1). Its action in this system is confined to the -SH groups of opsin. Under some conditions the synthesis of rhodopsin is aided by the presence of such a sulfhydryl compound as glutathione, which helps to keep the -SH groups of opsin free and reduced. By means of the amperometric silver titration of Kolthoff and Harris, it is shown that sulfhydryl groups are liberated in the bleaching of rhodopsin, two such groups for each retinene(1) molecule that appears. This is true equally of rhodopsin from the retinas of cattle, frogs) and squid. The exposure of new sulfhydryl groups adds an important element to the growing evidence that relates the bleaching of rhodopsin to protein denaturation. The place of sulfhydryl groups in the structure of rhodopsin is still uncertain. They may be concerned directly in binding the chromophore to opsin; or alternatively they may furnish hydrogen atoms for some reductive change by which the chromophore is formed from retinene(1). In the amperometric silver titration, the bleaching of rhodopsin yields directly an electrical variation. This phenomenon may have some fundamental connection with the role of rhodopsin in visual excitation, and may provide a model of the excitation process in general.

Animals↗
PubMed1952

Cis-trans isomers of vitamin A and retinene in the rhodopsin system.

Vitamin A and retinene, the carotenoid precursors of rhodopsin, occur in a variety of molecular shapes, cis-trans isomers of one another. For the synthesis of rhodopsin a specific cis isomer of vitamin A is needed. Ordinary crystalline vitamin A, as also the commercial synthetic product, both primarily all-trans, are ineffective. The main site of isomer specificity is the coupling of retinene with opsin. It is this reaction that requires a specific cis isomer of retinene. The oxidation of vitamin A to retinene by the alcohol dehydrogenase-cozymase system displays only a low degree of isomer specificity. Five isomers of retinene have been isolated in crystalline condition: all-trans; three apparently mono-cis forms, neoretinenes a and b and isoretinene a; and one apparently di-cis isomer, isoretinene b. Neoretinenes a and b were first isolated in our laboratory, and isoretinenes a and b in the Organic Research Laboratory of Distillation Products Industries. Each of these substances is converted to an equilibrium mixture of stereoisomers on simple exposure to light. For this reaction, light is required which retinene can absorb; i.e., blue, violet, or ultraviolet light. Yellow, orange, or red light has little effect. The single geometrical isomers of retinene must therefore be protected from low wave length radiation if their isomerization is to be avoided. By incubation with opsin in the dark, the capacity of each of the retinene isomers to synthesize rhodopsin was examined. All-trans retinene and neoretinene a are inactive. Neoretinene b yields rhodopsin indistinguishable from that extracted from the dark-adapted retina (lambda(max.) 500 mmicro). Isoretinene a yields a similar light-sensitive pigment, isorhodopsin, the absorption spectrum of which is displaced toward shorter wave lengths (lambda(max.) 487 mmicro). Isoretinene b appears to be inactive, but isomerizes preferentially to isoretinene a, which in the presence of opsin is removed to form isorhodopsin before the isomerization can go further. The synthesis of rhodopsin in solution follows the course of a bimolecular reaction, as though one molecule of neoretinene b combines with one of opsin. The synthesis of isorhodopsin displays similar kinetics. The bleaching of rhodopsin, whether by chemical means or by exposure to yellow or orange (i.e., non-isomerizing) light, yields primarily or exclusively all-trans retinene. The same appears to be true of isorhodopsin. The process of bleaching is therefore intrinsically irreversible. The all-trans retinene which results must be isomerized to active configurations before rhodopsin or isorhodopsin can be regenerated. A cycle of isomerization is therefore an integral part of the rhodopsin system. The all-trans retinene which emerges from the bleaching of rhodopsin must be isomerized to neoretinene b before it can go back; or if first reduced to all-trans vitamin A, this must be isomerized to neovitamin Ab before it can regenerate rhodopsin. The retina obtains new supplies of the neo-b isomer: (a) by the isomerization of all-trans retinene in the eye by blue or violet light; (b) by exchanging all-trans vitamin A for new neovitamin Ab from the blood circulation; and (c) the eye tissues may contain enzymes which catalyze the isomerization of retinene and vitamin A in situ. When the all-trans retinene which results from bleaching rhodopsin in orange or yellow light is exposed to blue or violet light, its isomerization is accompanied by a fall in extinction and a shift of absorption spectrum about 5 mmicro toward shorter wave lengths. This is a second photochemical step in the bleaching of rhodopsin. It converts the inactive, all-trans isomer of retinene into a mixture of isomers, from which mixtures of rhodopsin and isorhodopsin can be regenerated. Isorhodopsin, however, is an artefact. There is no evidence that it occurs in the retina; nor has isovitamin Aa or b yet been identified in vivo. In rhodopsin and isorhodopsin, the prosthetic groups appear to retain the cis configurations characteristic of their retinene precursors. In accord with this view, the beta-bands in the absorption spectra of both pigments appear to be cis peaks. The conversion to the all-trans configuration occurs during the process of bleaching. The possibility is discussed that rhodopsin may represent a halochromic complex of a retinyl ion with opsin. The increased resonance associated with the ionic state of retinene might then be responsible both for the color of rhodopsin and for the tendency of retinene to assume the all-trans configuration on its release from the complex. A distinction must be made between the immediate precursor of rhodopsin, neovitamin Ab, and the vitamin A which must be fed in order that rhodopsin be synthesized in vivo. Since vitamin A isomerizes in the body, it is probable that any geometrical isomer can fulfill all the nutritional needs for this vitamin.

Dark Adaptation↗
PubMed1953

Geometrical isomers of retinene.

Five crystalline retinenes have been isolated, which have every appearance of being cis-trans isomers of one another. They are all-trans retinene; three apparently mono-cis isomers: neoretinenes a and b and isoretinene a; and isoretinene b, an apparently di-cis isomer. The absorption spectra of these substances display the relations expected of cis-trans isomers. The main absorption band is displaced 5.5 to 7 mmicro toward shorter wave lengths for each presumptive cis linkage. Some of the presumptive cis isomers also display a cis peak at 255 to 260 mmicro. All five substances yield an identical blue product on mixing with antimony chloride. All of them are converted by light to what appears to be an identical mixture of stereoisomers. Heat isomerizes them very slowly; only neoretinene b exhibits large changes on heating at 70 degrees C. for 3 hours. The various isomers have been extensively interconverted by gentle procedures, and all of them have been converted to all-trans retinene. The present theory of cis-trans isomerism in carotenoids predicts the existence of four stable isomers of retinene. Instead we seem to have five-specifically three mono-cis forms where two are expected. There is no doubt that all these substances are closely related isomers of one another. The only point in question is whether they differ in part by something other than cis-trans configuration. One possibility, as yet little supported by evidence, is that isomerization between beta- and alpha-ionone rings may be involved. If, however, as seems more likely, all these substances are geometrical isomers of one another, some modification is needed in the present theory of configurational relationships in this class of compounds.

Hot Temperature↗
PubMed1953

The molar extinction of rhodopsin.

The molar extinction of rhodopsin is 40,600 cm.(2) per mole equivalent of retinene; i.e., this is the extinction of a solution of rhodopsin which is produced by, or yields on bleaching, a molar solution of retinene. The molar extinctions of all-trans retinene and all-trans retinene oxime have also been determined in ethyl alcohol and aqueous digitonin solutions. On the assumption that each chromophoric group of rhodopsin is made from a single molecule of retinene, it is concluded that the primary photochemical conversion of rhodopsin to lumi-rhodopsin has a quantum efficiency of 1; though the over-all bleaching of rhodopsin in solution to retinene and opsin may have a quantum efficiency as low as one-half. On bleaching cattle rhodopsin, about two sulfhydryl groups appear for each molecule of retinene liberated. In frog rhodopsin the -SH:retinene ratio appears to be higher, 5:2 or perhaps even 3:1. Some of this sulfhydryl appears to have been engaged in binding retinene to opsin; some may have been exposed as the result of changes in opsin which accompany bleaching, comparable with protein denaturation.

Animals↗
PubMed1955

Iodopsin.

The iodopsin system found in the cones of the chicken retina is identical with the rhodopsin system in its carotenoids. It differs only in the protein-the opsin -with which carotenoid combines. The cone protein may be called photopsin to distinguish it from the scotopsins of the rods. Iodopsin bleaches in the light to a mixture of photopsin and all-trans retinene. The latter is reduced by alcohol dehydrogenase and cozymase to all-trans vitamin A(1). Iodopsin is resynthesized from photopsin and a cis isomer of vitamin A, neovitamin Ab or the corresponding neoretinene b, the same isomer that forms rhodopsin. The synthesis of iodopsin from photopsin and neoretinene b is a spontaneous reaction. A second cis retinene, isoretinene a, forms iso-iodopsin (lambda(max) 510 mmicro). The bleaching of iodopsin in moderate light is a first-order reaction (Bliss). The synthesis of iodopsin from neoretinene b and opsin is second-order, like that of rhodopsin, but is very much more rapid. At 10 degrees C. the velocity constant for iodopsin synthesis is 527 times that for rhodopsin synthesis. Whereas rhodopsin is reasonably stable in solution from pH 4-9, iodopsin is stable only at pH 5-7, and decays rapidly at more acid or alkaline reactions. The sulfhydryl poison, p-chloromercuribenzoate, blocks the synthesis of iodopsin, as of rhodopsin. It also bleaches iodopsin in concentrations which do not attack rhodopsin. Hydroxylamine also bleaches iodopsin, yet does not poison its synthesis. Hydroxylamine acts by competing with the opsins for retinene. It competes successfully with chicken, cattle, or frog scotopsin, and hence blocks rhodopsin synthesis; but it is less efficient than photopsin in trapping retinene, and hence does not block iodopsin synthesis. Though iodopsin has not yet been prepared in pure form, its absorption spectrum has been computed by two independent procedures. This exhibits an alpha-band with lambda(max) 562 mmicro, a minimum at about 435 mmicro, and a small beta-band in the near ultraviolet at about 370 mmicro. The low concentration of iodopsin in the cones explains to a first approximation their high threshold, and hence their status as organs of daylight vision. The relatively rapid synthesis of iodopsin compared with rhodopsin parallels the relatively rapid dark adaptation of cones compared with rods. A theoretical relation is derived which links the logarithm of the visual sensitivity with the concentration of visual pigment in the rods and cones. Plotted in these terms, the course of rod and cone dark adaptation resembles closely the synthesis of rhodopsin and iodopsin in solution. The spectral sensitivities of rod and cone vision, and hence the Purkinje phenomenon, have their source in the absorption spectra of rhodopsin and iodopsin. In the chicken, for which only rough spectral sensitivity measurements are available, this relation can be demonstrated only approximately. In the pigeon the scotopic sensitivity matches the spectrum of rhodopsin; but the photopic sensitivity is displaced toward the red, largely or wholly through the filtering action of the colored oil globules in the pigeon cones. In cats, guinea pigs, snakes, and frogs, in which no such colored ocular structures intervene, the scotopic and photopic sensitivities match quantitatively the absorption spectra of rhodopsin and iodopsin. In man the scotopic sensitivity matches the absorption spectrum of rhodopsin; but the photopic sensitivity, when not distorted by the yellow pigmentations of the lens and macula lutea, lies at shorter wave lengths than iodopsin. This discrepancy is expected, for the human photopic sensitivity represents a composite of at least three classes of cone concerned with color vision.

Animals↗
PubMed1956

The stability of rhodopsin and opsin; effects of pH and aging.

THE STABILITY OF CATTLE RHODOPSIN AND OF ITS PROTEIN MOIETY OPSIN TOWARD ACIDS AND ALKALIES AND ON AGING WAS DETERMINED BY TWO CRITERIA: maintenance of absorption spectrum, and capacity to regenerate after exposure to light. On storage at 3 degrees C. at pH near neutrality, the absorption spectrum in the visible region may remain unchanged for as long as 6 months; but the regenerability progressively declines, at very different rates in different preparations. The cause of this decline has not been determined. It may involve denaturation at sites other than the retinene-protein bond, which by the evidence of the absorption spectrum remains intact. Cattle rhodopsin maintains its absorption spectrum at any pH from 3.9-9.6 for at least an hour at 25-27 degrees C. To both sides of this pH range the pigment bleaches, the extinction falling to half in 1 hour at pH 3.3 and 10.5. The exposure of rhodopsin to light greatly increases the vulnerability of the product (opsin) to acids and bases. Opsin rapidly loses its capacity to regenerate rhodopsin to both sides of the range of pH 5.5-7.0. Half the regenerability is lost within 45 seconds at pH 3.4 and 9.1; and within 1 hour at pH 5 and 8.

Animals↗
PubMed1956

Retinene isomerase.

Rhodopsin is formed by the condensation of opsin with a cis isomer of retinene, called neo-b. The bleaching of rhodopsin releases all-trans retinene which must be isomerized back to neo-b in order for rhodopsin to regenerate. Both retinene isomers are in equilibrium with the corresponding isomers of vitamin A, through the alcohol dehydrogenase system. An enzyme is found in cattle retinas and frog pigment layers which catalyzes the interconversion of all-trans and neo-b retinene. We call it "retinene isomerase." It is soluble in neutral phosphate buffer, and precipitates between 20 and 35 per cent saturation with ammonium sulfate. In the dark, the isomerase converts all-trans and neo-b retinene to an equilibrium mixture of 5 parts neo-b and 95 parts all-trans. With opsin present to trap neo-b, the isomerase catalyzes the synthesis of rhodopsin from all-trans retinene. This reaction, however, is too slow to account for dark adaptation. Retinene is isomerized by light, but too slowly to supply the retina with neo-b. In aqueous solution the pseudoequilibrium mixture contains about 15 per cent neo-b. When all-trans retinene is irradiated in the presence of isomerase, the rate of formation of neo-b is increased about 5 times, and the pseudoequilibrium shifted so that the mixture now contains about 32 per cent neo-b. The isomerase is specific for all-trans and neo-b retinene. It does not act on two other cis isomers of retinene, nor on all-trans or neo-b vitamin A. The role of the isomerase in vision appears to be as follows: in the light, as rhodopsin is bleached to opsin and all-trans retinene, the latter is in part converted to the neo-b isomer and stored in the pigment epithelium as neo-b vitamin A. During dark adaptation, the dominant process is the trapping by opsin of neo-b retinene supplied from stores of neo-b vitamin A, and the slow isomerase-catalyzed "dark" conversion of all-trans to neo-b retinene.

Animals↗
PubMed1957

The vitamin A of a euphausiid crustacean.

The vitamin A of the euphausiid crustacean, Meganyctiphanes norvegica, consists almost wholly of the hindered cis isomer, neo-b (11-cis). In this animal vitamin A is concentrated almost entirely in the eyes; and its properties so closely resemble those of pure neo-b vitamin A as not in themselves to indicate that any other isomer is present. However, Fisher et al. (1955 b) have isolated a small fraction from this material which may be neo-c vitamin A (11, 13-dicis). The neo-b isomer was identified by its absolute absorption spectrum, the changes of absorption spectrum on isomerization, oxidation to neo-b retinene, and synthesis from the latter of rhodopsin. This identification is also in good accord with new, revised bioassays of Meganyctiphanes vitamin A by Plack et al. (1956).

Animals↗
PubMed1958

The rhodopsin system of the squid.

Squid rhodopsin (lambda(max) 493 mmicro)-like vertebrate rhodopsins-contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (lambda(max) 500 mmicro) near neutrality, yellow (lambda(max) 380 mmicro) in alkaline solution. The two forms-acid and alkaline metarhodopsin-are interconverted according to the equation, Alkaline metarhodopsin + H(+) right harpoon over left harpoonacid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.

Animals↗
PubMed1958

The thermal stability of rhodopsin and opsin.

Rhodopsin, the red photosensitive pigment of rod vision, is composed of a specific cis isomer of retinene, neo-b (11-cis), joined as chromophore to a colorless protein, opsin. We have investigated the thermal denaturation of cattle rhodopsin and opsin in aqueous digitonin solution, and in isolated rod outer limbs. Both rhodopsin and opsin are more stable in rods than in solution. In solution as well as in rods, moreover, rhodopsin is considerably more stable than opsin. The chromophore therefore protects opsin against denaturation. This is true whether rhodopsin is extracted from dark-adapted retinas, or synthesized in vitro from neo-b retinene and opsin. Excess neo-b retinene does not protect rhodopsin against denaturation. The protection involves the specific relationship between the chromophore and opsin. Similar, though somewhat less, protection is afforded opsin by the stereoisomeric iso-a (9-cis) chromophore in isorhodopsin. The Arrhenius activation energies (E(a)) and entropies of activation (DeltaSdouble dagger) are much greater for thermal denaturation of rhodopsin and isorhodopsin than of opsin. Furthermore, these values differ considerably for rhodopsins from different species -frog, squid, cattle-presumably due to species differences in the opsins. Heat or light bleaches rhodopsin by different mechanisms, yielding different products. Light stereoisomerizes the retinene chromophore; heat denatures the opsin. Photochemical bleaching therefore yields all-trans retinene and native opsin; thermal bleaching, neo-b retinene and denatured opsin.

Animals↗
PubMed1963

THE PHOTOSENSITIVE RETINAL PIGMENT SYSTEM OF GEKKO GEKKO.

Retinal extracts of Gekko gekko were found to contain two retinene(1) photopigments, one with maximum absorption at about 521 mmicro, the second with a maximum in the region of 478 mmicro. These pigments were assayed by the method of partial bleaching and their spectral characteristics studied by examining their difference spectra. The 478 mmicro pigment was present in the extracts as 8 per cent of the total photopigment concentration. The two pigment systems were shown to be biochemically independent and to have different properties. Unlike the 521 mmicro pigment, for example, the 478 mmicro pigment was found to resist the action of NH(2)OH and, within the cells, to be unaffected by sucrose solutions. These solutions destroyed or altered the 521 system so that extracts of sucrose-treated retinae were found to contain significantly less 521 photopigment. In digitonin solution the 521 pigment was unaffected by sucrose treatment. Both pigments were extracted from separated, washed outer segments and so are considered to be visual pigments. This dual system accounts for the spectral sensitivity of this gecko as determined by Denton. A search was made, but no evidence was secured for the presence of a photopigment absorbing at longer wavelengths. Electoretinographic data suggest, however, that an elevated sensitivity at longer wavelengths occurs in some geckos so that a continued search is justified for a third photopigment.

Animals↗
PubMed1963

TAUTOMERIC FORMS OF METARHODOPSIN.

Light isomerizes the chromophore of rhodopsin, 11-cis retinal (formerly retinene), to the all-trans configuration. This introduces a succession of unstable intermediates-pre-lumirhodopsin, lumirhodopsin, metarhodopsin -in which all-trans retinal is still attached to the chromophoric site on opsin. Finally, retinal is hydrolyzed from opsin. The present experiments show that metarhodopsin exists in two tautomeric forms, metarhodopsins I and II, with lambda(max) 478 and 380 mmicro. Metarhodopsin I appears first, then enters into equilibrium with metarhodopsin II. In this equilibrium, the proportion of metarhodopsin II is favored by higher temperature or pH, neutral salts, and glycerol. The change from metarhodopsin I to II involves the binding of a proton by a group with pK 6.4 (imidazole?), and a large increase of entropy. Metarhodopsin II has been confused earlier with the final mixture of all-trans retinal and opsin (lambda(max) 387 mmicro), which it resembles in spectrum. These two products are, however, readily distinguished experimentally.

Animals↗
PubMed1963

CHANGES IN ELECTRICAL CONDUCTANCE OF RHODOPSIN ON PHOTOLYSIS.

The change in electrical conductance of rhodopsin solutions was studied with flash-photolysis techniques. The whole pattern of the conductance change on illumination consists of three different processes: (I) the initial decrease, (II) the increase, and (III) the slow decrease, which are in decreasing order of reaction rate. The processes I, II, and III can be most distinctly recognized on flash illumination of acid, slightly acid, and alkaline rhodopsins, respectively. On the other hand, the bleaching of rhodopsin also shows at least three successive phases of different rates, but none of them corresponds in reaction rate to any of the processes of the conductance change. The conductance change may be related to conformational changes of opsin following photoisomerization of retinene, being due to hydrogen or hydroxyl ions and some other inorganic electrolytes. The amount of the change, especially the initial decrease, is proportional to the amount of rhodopsin bleached, even when the photochemical back reaction towards rhodopsin and isorhodopsin occurs in the chromophore depending on the intensity of illumination. Of the three processes, the slow decrease is most severely affected by aging, but the initial decrease and increase are slightly affected. These two processes promptly caused by illumination are connected closely to the conformational changes during the conversion of rhodopsin to metarhodopsin, and perhaps to the initial stage of excitation of rod cells.

Chemical Phenomena↗