RESUMO
Over the past decade and a half, there have been great advances in our understanding of how light is transduced into electrical signals by the retinal rod and cone photoreceptors in vertebrates. One essential feature of these sensory neurons is their ability to adapt to background illumination, which allows them to function over a broad range of light intensities. This adaptation appears to arise mostly from negative feedback on phototransduction that is mediated by calcium ions. Recent work has suggested that this feedback is fairly complex, and involves several pathways directed at different components of phototransduction. From direct measurements of these feedback pathways in rods, it is possible to evaluate their relative contributions to the overall sensitivity of the cell. At the same time, these feedback mechanisms, as currently known, appear to be sufficient for explaining the change in sensitivity of rods during adaptation to light.
Assuntos
Cálcio/fisiologia , Células Fotorreceptoras/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Vertebrados/fisiologia , Animais , HumanosRESUMO
Over the past two decades there has been an explosive growth in our understanding of phototransduction, leading to the development of a comprehensive scheme for the process. On the basis of this scheme the finer details of the process are being elucidated. Additional protein components and pathways have been identified, successful quantitative models of parts of the process have been developed, and a detailed understanding of the molecular basis of physiological function has begun to emerge. Here we summarize the most recent developments.
Assuntos
Células Fotorreceptoras Retinianas Cones/fisiologia , Segmento Externo da Célula Bastonete/fisiologia , Animais , Cálcio/metabolismo , GMP Cíclico/metabolismo , Retroalimentação , Humanos , Modelos Neurológicos , Doenças Retinianas/genética , Doenças Retinianas/fisiopatologia , Transdução de SinaisRESUMO
We have used the truncated outer segment preparation to measure rod cGMP-phosphodiesterase activity, as well as its modulation by Ca2+, in darkness and in light. The basal enzyme activity in darkness was approximately 0-3 s-1, and was largely independent of Ca2+ concentration from 10 nM to 10 microM. The steady state activity elicited by a step of light (lambda = 520 nm) was strongly enhanced by Ca2+, increasing from approximately 0.005 s-1/(h nu micron-2 s-1) at 10 nM Ca2+ to approximately 0.16 s-1/h nu micron-2 s-1) at 10 microM Ca2+. Based on these measurements, as well as previous measurements on the effects of Ca2+ on rod guanylate cyclase and the cGMP-gated channel, we have calculated the step response-intensity relation for the rod cell in steady state. This relation agrees reasonably well with the relation directly measured from intact rods. We have also evaluated the relative contributions from the three Ca2+ effects to rod sensitivity. At low background light intensities, the Ca2+ modulation of the guanylate cyclase appears to be the most important for sensitivity regulation. At higher light intensities, especially above half-saturation of the response, the Ca2+ modulation of the light-stimulated phosphodiesterase shows a progressively important influence on the light response; it also extends the Weber-Fechner behavior of the cell to higher intensities. The contribution of the Ca2+ modulation of the cGMP-gated channel is slight throughout.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , GMP Cíclico/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Eletrofisiologia , Ativação do Canal Iônico/fisiologia , Matemática , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Sensibilidade e Especificidade , Urodelos , Visão Ocular/fisiologiaRESUMO
cGMP mediates vertebrate phototransduction by directly gating cationic channels on the plasma membrane of the photoreceptor outer segment. This second messenger is produced by a guanylate cyclase and hydrolyzed by a light-activated cGMP-phosphodiesterase. Both of these enzyme activities are Ca2+ sensitive, the guanylate cyclase activity being inhibited and the light-activated phosphodiesterase being enhanced by Ca2+. Changes in these activities due to a light-induced decrease in intracellular Ca2+ are involved in the adaptation of photoreceptors to background light. We describe here experiments to characterize the guanylate cyclase activity and its modulation by Ca2+ using a truncated rod outer segment preparation, in order to evaluate the enzyme's role in light adaptation. The outer segment of a tiger salamander rod was drawn into a suction pipette to allow recording of membrane current, and the remainder of the cell was sheared off with a probe to allow internal dialysis. The cGMP-gated channels on the surface membrane were used to monitor conversion of GTP, supplied from the bath, into cGMP by the guanylate cyclase in the outer segment. At nominal 0 Ca2+, the cyclase activity had a Km of 250 microM MgGTP and a Vmax of 25 microM cGMP s-1 in the presence of 1.6 mM free Mg2+; in the presence of 0.5 mM free Mg2+, the Km was 310 microM MgGTP and the Vmax was 17 microM cGMP s-1. The stimulation by Mg2+ had an EC50 of 0.2 mM Mg2+ for MgGTP at 0.5 mM. Ca2+ inhibited the cyclase activity. In a K+ intracellular solution, with 0.5 mM free Mg2+ and 2.0 mM GTP, the cyclase activity was 13 microM cGMP s-1 at nominal 0 Ca2+; Ca2+ decreased this activity with a IC50 of approximately 90 nM and a Hill coefficient of approximately 2.0.
Assuntos
Guanilato Ciclase/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Animais , Cálcio/farmacologia , Membrana Celular/enzimologia , GMP Cíclico/análise , GMP Cíclico/fisiologia , Eletrofisiologia , Guanilato Ciclase/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Magnésio/farmacologia , Matemática , Potássio/farmacologia , Células Fotorreceptoras Retinianas Bastonetes/química , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Urodelos , Visão Ocular/fisiologiaRESUMO
The basis of the duplex theory of vision is examined in view of the dazzling array of data on visual pigment sequences and the pigments they form, on the microspectrophotometry measurements of single photoreceptor cells, on the kinds of photoreceptor cascade enzymes, and on the electrophysiological properties of photoreceptors. The implications of the existence of five distinct visual pigment families are explored, especially with regard to what pigments are in what types of photoreceptors, if there are different phototransduction enzymes associated with different types of photoreceptors, and if there are electrophysiological differences between different types of cones.
Assuntos
Células Fotorreceptoras de Vertebrados , Adaptação Ocular , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , GMP Cíclico/metabolismo , Eletrofisiologia , Humanos , Dados de Sequência Molecular , Células Fotorreceptoras de Vertebrados/classificação , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/fisiologia , Pigmentos da Retina/química , Pigmentos da Retina/fisiologia , Homologia de Sequência de Aminoácidos , Visão Ocular/fisiologiaRESUMO
The resonance Raman spectrum of octopus bathorhodopsin in the fingerprint region and in the ethylenic-Schiff base region have been obtained at 80 K using the "pump-probe" technique as have its deuterated chromophore analogues at the C7D; C8D; C8,C7D2; C10D; C11D; C11, C12D2; C14D; C15D; C14, C15D2; and N16D positions. While these data are not sufficient to make definitive band assignments, many tentative assignments can be made. Because of the close spectral similarity between the octopus bathorhodopsin spectrum and that of bovine bathorhodopsin, we conclude that the essential configuration of octopus bathorhodopsin's chromophore is all-trans like. The data suggest that the Schiff base, C = N, configuration is trans (anti). The observed conformationally sensitive fingerprint bands show pronounced isotope shifts upon chromophore deuteration. The size of the shifts differ, in certain cases, from those found for bovine bathorhodopsin. Thus, the internal mode composition of the fingerprint bands differs somewhat from bovine bathorhodopsin, suggesting a somewhat different in situ chromophore conformation. An analysis of the NH bend frequency, the Schiff base C = N stretch frequency, and its shift upon Schiff base deuteration suggests that the hydrogen bonding between the protonated Schiff base with its protein binding pocket is weaker in octopus bathorhodopsin than in bovine bathorhodopsin but stronger than that found in bacteriorhodopsin's bR568 pigment.
Assuntos
Retinaldeído/metabolismo , Rodopsina/análogos & derivados , Animais , Deutério , Octopodiformes , Ligação Proteica , Conformação Proteica , Retinaldeído/química , Rodopsina/química , Rodopsina/metabolismo , Bases de Schiff , Análise Espectral Raman/métodosRESUMO
Previous resonance Raman spectroscopic studies of bovine and octopus rhodopsin and bathorhodopsin in the C-C stretch fingerprint region have shown drastically different spectral patterns, which suggest different chromophore-protein interactions. We have extended our resonance Raman studies of bovine and octopus pigments to the C=C stretch region in order to reveal a more detailed picture about the difference in retinal-protein interactions between these two pigments. The C=C stretch motions of the protonated retinal Schiff base are strongly coupled to form highly delocalized ethylenic modes located in the 1500 to 1650 cm-1 spectral region. In order to decouple these vibrations, a series of 11,12-D2-labeled retinals, with additional 13C labeling at C8, C10, C11 and C14, respectively, are used to determine the difference of specific C=C stretch modes between bovine and octopus pigments. Our results show that the C9=C10 and C13=C14 stretch mode are about 20 cm-1 lower in the Raman spectrum of octopus bathorhodopsin than in bovine bathorhodopsin, while the other C=C stretch modes in these two bathorhodopsins are similar. In contrast, only the C9=C10 stretch mode in octopus rhodopsin is about 10 cm-1 lower than in bovine rhodopsin, while other C=C stretches are similar.
Assuntos
Retina/química , Rodopsina/análogos & derivados , Animais , Carbono/química , Bovinos , Marcação por Isótopo , Octopodiformes , Rodopsina/química , Análise Espectral RamanAssuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Guanilato Ciclase/metabolismo , Segmento Externo da Célula Bastonete/enzimologia , Ambystoma , Animais , Bufo marinus , Cálcio/metabolismo , GMP Cíclico/metabolismo , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Cinética , Luz , Rana catesbeianaRESUMO
Since the 1930s, the spectrum of vertebrate rhodopsin has been considered to be independent of pH (Lythgoe, R.J. 1937. J. Physiol. 89:331-358; Wald, G. 1938. J. Gen. Physiol. 21:795-832). Here I report that the spectrum of bovine rhodopsin is pH dependent. At pHs greater than 9.0, there is a shift to shorter wavelengths of its 500-nm absorption band. This shift is accounted for by the existence of a high pH form of bovine rhodopsin, with absorption maximum at 494 nm and a slightly lower extinction coefficient. The high-pH form results from the low-pH form by the deprotonation of a single group with a pK of approximately 10.2 for rhodopsin in rod disk membranes in 4.0 M KCl. The shift is observed for sheep and chicken rhodopsins, but not for frog, toad, and octopus rhodopsins. This indicates a specific amino acid difference between these rhodopsins that is potentially relevant for the mechanism of color regulation.
Assuntos
Rodopsina/química , Animais , Bovinos , Concentração de Íons de Hidrogênio , Rodopsina/isolamento & purificação , Segmento Externo da Célula Bastonete/fisiologia , EspectrofotometriaRESUMO
Cyclic AMP (cAMP) is one of the intracellular messengers that mediate odorant signal transduction in vertebrate olfactory cilia. Therefore, the diffusion coefficient of cAMP in olfactory cilia is an important factor in the transduction of the odorous signal. We have employed the excised cilium preparation from the grass frog (Rana pipiens) to measure the cAMP diffusion coefficient. In this preparation an olfactory cilium is drawn into a patch pipette and a gigaseal is formed at the base of the cilium. Subsequently the cilium is excised, allowing bath cAMP to diffuse into the cilium and activate the cyclic nucleotide-gated channels on the plasma membrane. In order to estimate the cAMP diffusion coefficient, we analyzed the kinetics of the currents elicited by step changes in the bath cAMP concentration in the absence of cAMP hydrolysis. Under such conditions, the kinetics of the cAMP-activated currents has a simple dependence on the diffusion coefficient. From the analysis we have obtained a cAMP diffusion coefficient of 2.7 +/- 0.2. 10(-6) cm2 s-1 for frog olfactory cilia. This value is similar to the expected value in aqueous solution, suggesting that there are no significant diffusional barriers inside olfactory cilia. At cAMP concentrations higher than 5 microM, diffusion slowed considerably, suggesting the presence of buffering by immobile cAMP binding sites. A plausible physiological function of such buffering sites would be to prolong the response of the cell to strong stimuli.
Assuntos
Cílios/metabolismo , AMP Cíclico/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Animais , Sítios de Ligação , Fenômenos Biofísicos , Biofísica , Membrana Celular/metabolismo , Difusão , Técnicas In Vitro , Ativação do Canal Iônico , Potenciais da Membrana , Rana pipiens , Transdução de SinaisRESUMO
1. The outer segment of an isolated rod photoreceptor from the bullfrog retina was drawn into a pipette containing choline solution for recording membrane current. The rest of the cell was sheared off with a glass probe to allow internal dialysis of the outer segment with a bath potassium solution ('truncated rod outer segment' preparation). The potential between the inside and the outside of the pipette was held at 0 mV. 2. Application of bath cGMP, in the presence of 3-isobutyl-1-methylxanthine (IBMX), gave rise to an outward membrane current. At saturating cGMP concentrations, this current was insensitive to intracellular Ca2+ at concentrations between 0 and 10 microM. At subsaturating cGMP concentrations, however, this current was inhibited by intracellular Ca2+. This sensitivity to Ca2+ declined after dialysis with a low-Ca2+ solution, suggesting the involvement of a soluble factor. 3. At low (nominally 0) Ca2+, the half-maximal activation constant and Hill coefficient for the activation of the cGMP-gated current by cGMP were 27 microM and 2.0, respectively. At high (ca 10 microM) Ca2+, the corresponding values were 40 microM cGMP and 2.4. 4. The inhibition of the current by Ca2+ was characterized at 20 microM cGMP. Ca2+ inhibited the current by up to 60%, with half-maximal inhibition at 48 nM Ca2+ and a Hill coefficient of 1.6.
Assuntos
Cálcio/farmacologia , GMP Cíclico/farmacologia , Canais Iônicos/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Relação Dose-Resposta a Droga , Canais Iônicos/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Rana catesbeiana , Fatores de TempoRESUMO
Cyclic GMP (cGMP) is the intracellular messenger that mediates phototransduction in retinal rods. As photoisomerizations of rhodopsin molecules are local events, the longitudinal diffusion of cGMP in the rod outer segment should be a contributing factor to the response of the cell to light. We have employed the truncated rod outer segment preparation from bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) to measure the cGMP diffusion coefficient. In this preparation, the distal portion of a rod outer segment was drawn into a suction pipette for measuring membrane current, and the rest of the cell was then sheared off with a glass probe, allowing bath cGMP to diffuse into the outer segment and activate the cGMP-gated channels on the surface membrane. Addition and removal of bath cGMP were fast enough to produce effectively step changes in cGMP concentration at the open end of the outer segment. When cGMP hydrolysis is inhibited by isobutylmethylxanthine (IBMX), the equation for the diffusion of cGMP inside the truncated rod outer segment has a simple analytical solution, which we have used to analyze the rise and decay kinetics of the cGMP-elicited currents. From these measurements we have obtained a cGMP diffusion coefficient of approximately 70 x 10(-8) cm2 s-1 for bullfrog rods and approximately 60 x 10(-8) cm2 s-1 for tiger salamander rods. These values are six to seven times lower than the expected value in aqueous solution. The estimated diffusion coefficient is the same at high (20-1000 microM) and low (5-10 microM) concentrations of cGMP, suggesting no significant effect from buffering over these concentration ranges.
Assuntos
GMP Cíclico/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Ambystoma , Animais , Fenômenos Biofísicos , Biofísica , Difusão , Eletroquímica , Hidrólise , Técnicas In Vitro , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Transporte de Íons , Cinética , Potenciais da Membrana/efeitos dos fármacos , Modelos Biológicos , Fotoquímica , Rana catesbeiana , Segmento Externo da Célula Bastonete/efeitos dos fármacos , Segmento Externo da Célula Bastonete/efeitos da radiaçãoRESUMO
The chromophore of octopus rhodopsin is 11-cis retinal, linked via a protonated Schiff base to the protein backbone. Its stable photoproduct, metarhodopsin, has all-trans retinal as its chromphore. The Schiff base of acid metarhodopsin (lambda max = 510 nm) is protonated, whereas that of alkaline metarhodopsin (lambda max = 376 nm) is unprotonated. Metarhodopsin in photoreceptor membranes was titrated and the apparent pK of the Schiff base was measured at different ionic strengths. From these salt-dependent pKs the surface charge density of the octopus photoreceptor membranes and the intrinsic Schiff base pK of metarhodopsin were obtained. The surface charge density is sigma = -1.6 +/- 0.1 electronic charges per 1,000 A2. Comparison of the measured surface charge density with values from octopus rhodopsin model structures suggests that the measured value is for the extracellular surface and so the Schiff base in metarhodopsin is freely accessible to protons from the extracellular side of the membrane. The intrinsic Schiff base pK of metarhodopsin is 8.44 +/- 0.12, whereas that of rhodopsin is found to be 10.65 +/- 0.10 in 4.0 M KCl. These pK values are significantly higher than the pK value around 7.0 for a retinal Schiff base in a polar solvent; we suggest that a plausible mechanism to increase the pK of the retinal pigments is the preorganization of their chromophore-binding sites. The preorganized site stabilizes the protonated Schiff base with respect to the unprotonated one. The difference in the pK for the octopus rhodopsin compared with metarhodopsin is attributed to the relative freedom of the latter's chromophore-binding site to rearrange itself after deprotonation of the Schiff base.
Assuntos
Células Fotorreceptoras/fisiologia , Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/fisiologia , Concentração de Íons de Hidrogênio , Cinética , Matemática , Microvilosidades/fisiologia , Modelos Moleculares , Modelos Teóricos , Dados de Sequência Molecular , Octopodiformes , Conformação Proteica , Rodopsina/análogos & derivados , Bases de Schiff , Espectrofotometria , Propriedades de SuperfícieRESUMO
When light is absorbed within the outer segment of a vertebrate photoreceptor, the conformation of the photopigment rhodopsin is altered to produce an activated photoproduct called metarhodopsin II or Rh(*). Rh(*) initiates a transduction cascade similar to that for metabotropic synaptic receptors and many hormones; the Rh(*) activates a heterotrimeric G protein, which in turn stimulates an effector enzyme, a cyclic nucleotide phosphodiesterase. The phosphodiesterase then hydrolyzes cGMP, and the decrease in the concentration of free cGMP reduces the probability of opening of channels in the outer segment plasma membrane, producing the electrical response of the cell. Photoreceptor transduction can be modulated by changes in the mean light level. This process, called light adaptation (or background adaptation), maintains the working range of the transduction cascade within a physiologically useful region of light intensities. There is increasing evidence that the second messenger responsible for the modulation of the transduction cascade during background adaptation is primarily, if not exclusively, Ca(2+), whose intracellular free concentration is decreased by illumination. The change in free Ca(2+) is believed to have a variety of effects on the transduction mechanism, including modulation of the rate of the guanylyl cyclase and rhodopsin kinase, alteration of the gain of the transduction cascade, and regulation of the affinity of the outer segment channels for cGMP. The sensitivity of the photoreceptor is also reduced by previous exposure to light bright enough to bleach a substantial fraction of the photopigment in the outer segment. This form of desensitization, called bleaching adaptation (the recovery from which is known as dark adaptation), seems largely to be due to an activation of the transduction cascade by some form of bleached pigment. The bleached pigment appears to activate the G protein transducin directly, although with a gain less than Rh(*). The resulting decrease in intracellular Ca(2+) then modulates the transduction cascade, by a mechanism very similar to the one responsible for altering sensitivity during background adaptation.
Assuntos
Adaptação Ocular/fisiologia , Células Fotorreceptoras de Vertebrados/metabolismo , Animais , Cálcio/metabolismo , Adaptação à Escuridão/fisiologia , Adaptação à Escuridão/efeitos da radiação , Humanos , Luz , Fotoquímica , Rodopsina/metabolismo , Rodopsina/efeitos da radiação , Transdução de Sinais/fisiologia , Transdução de Sinais/efeitos da radiação , Vertebrados , Visão Ocular/fisiologia , Visão Ocular/efeitos da radiaçãoRESUMO
Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectra were obtained from living toad retinae and toad retinal extracts at 4 degrees C. Several phosphorus metabolites--nucleoside di- and triphosphates (NTP), phosphocreatine, phosphodiesters, inorganic phosphate, and phosphomonoesters--were identified from the spectra of whole retinae. The intracellular pH was determined to be 7.27 +/- 0.06 at 4 degrees C and the intracellular MgNTP/NTP ratio was at least 0.77. These results are consistent with those reported by other techniques, and they show that 31P-NMR spectroscopy can be used for noninvasively and quantitatively studying the metabolism of living toad retinae, and for monitoring its changes over time.
Assuntos
Retina/análise , Animais , Bufo marinus , Liofilização , Concentração de Íons de Hidrogênio , Magnésio/análise , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética , Isótopos de FósforoRESUMO
Cyclic GMP (cGMP) is the intracellular messenger mediating phototransduction in retinal rods, with its longitudinal diffusion in the rod outer segment (ROS) likely to be a factor in determining light sensitivity. From the kinetics of cGMP-activated currents in the truncated ROS of the salamander (Ambystoma tigrinum), the cGMP diffusion coefficient was previously estimated to be approximately 60 x 10(-8) cm2 s-1. On the other hand, fluorescence measurements in intact salamander ROS using 8-(fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate (Fl-cGMP) led to a diffusion coefficient for this compound of 1 x 10(-8) cm2 s-1; after corrections for differences in size and in binding to cellular components between cGMP and Fl-cGMP, this gave an upper limit of 11 x 10(-8) cm2 s-1 for the cGMP diffusion coefficient. To properly compare the two sets of measurements, we have examined the diffusion of Fl-cGMP in the truncated ROS. From the kinetics of Fl-cGMP-activated currents, we have obtained a diffusion coefficient of 3 x 10(-8) cm2 s-1 for this analog; the cGMP diffusion coefficient measured from the same truncated ROSs was approximately 80 x 10(-8) cm2 s-1. Thus, a factor of 27 appears appropriate for correcting differences in size and intracellular binding between cGMP and Fl-cGMP. Application of this correction factor to the Fl-cGMP diffusion coefficient measurements by Olson and Pugh (1993) gives a cGMP diffusion coefficient of approximately 30 x 10(-8) cm2 s-1, in reasonable agreement with the value measured from the truncated ROS.
Assuntos
Ambystoma/metabolismo , GMP Cíclico/análogos & derivados , Fluoresceínas/farmacocinética , Segmento Externo da Célula Bastonete/metabolismo , Animais , Transporte Biológico Ativo , Fenômenos Biofísicos , Biofísica , GMP Cíclico/metabolismo , GMP Cíclico/farmacocinética , Difusão , Técnicas In Vitro , CinéticaRESUMO
We consider the problem of color regulation in visual pigments for both bovine rhodopsin (lambda max = 500 nm) and octopus rhodopsin (lambda max = 475 nm). Both pigments have 11-cis-retinal (lambda max = 379 nm, in ethanol) as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 +/- 3000 M-1 cm-1 at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.
Assuntos
Proteínas do Olho/metabolismo , Pigmentos da Retina/metabolismo , Retinaldeído/metabolismo , Retinoides/metabolismo , Rodopsina/metabolismo , Animais , Bovinos , Cinética , Octopodiformes , Retinaldeído/análogos & derivados , Retinaldeído/síntese química , Segmento Externo da Célula Bastonete/metabolismo , Opsinas de Bastonetes , Espectrofotometria/métodos , Relação Estrutura-AtividadeRESUMO
Resonance Raman spectra of the hydrogen out-of-plane (HOOP) vibrational modes in the retinal chromophore of octopus bathorhodopsin with deuterium label(s) along the polyene chain have been obtained. In clear contrast with bovine bathorhodopsin's HOOP modes, there are only two major HOOP bands at 887 and 940 cm-1 for octopus bathorhodopsin. On the basis of their isotopic shifts upon deuterium labeling, we have assigned the band at 887 cm-1 to C10H and C14H HOOP modes, and the band at 940 cm-1 to C11H = C12H Au-like HOOP mode. Except for a 26 cm-1 downward shift, the C11H = C12H Au-like wag appears to be little disturbed in octopus bathorhodopsin from the chromophore in solution since its changes upon deuterium labeling are close to those found in solution model-compound studies. We found also that the C10H and C14H HOOP wags are also similar to those in the model-compound studies. However, we have found that the interaction between the C7H and C8H HOOP internal coordinates of the chromophore in octopus bathorhodopsin is different from that of the chromophore in solution. The intensity of the C11H = C12H and the other HOOP modes suggests that the chromophore of octopus bathorhodopsin is somewhat torsionally distorted from a planar trans geometry. Importantly, a twist about C11 = C12 double bond is inferred. Such a twist breaks the local symmetry, resulting in the observation of the normally Raman-forbidden C11H = C12H Au-like HOOP mode. The twisted nature of the chromophore, semiquantitatively discussed here, likely affects the lambda max of the chromophore and its enthalpy.(ABSTRACT TRUNCATED AT 250 WORDS)