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At least 973 records · Page 54Linked to original sources
PubMed1999

Time-resolved rhodopsin activation currents in a unicellular expression system.

The early receptor current (ERC) is the charge redistribution occurring in plasma membrane rhodopsin during light activation of photoreceptors. Both the molecular mechanism of the ERC and its relationship to rhodopsin conformational activation are unknown. To investigate whether the ERC could be a time-resolved assay of rhodopsin structure-function relationships, the distinct sensitivity of modern electrophysiological tools was employed to test for flash-activated ERC signals in cells stably expressing normal human rod opsin after regeneration with 11-cis-retinal. ERCs are similar in waveform and kinetics to those found in photoreceptors. The action spectrum of the major R(2) charge motion is consistent with a rhodopsin photopigment. The R(1) phase is not kinetically resolvable and the R(2) phase, which overlaps metarhodopsin-II formation, has a rapid risetime and complex multiexponential decay. These experiments demonstrate, for the first time, kinetically resolved electrical state transitions during activation of expressed visual pigment in a unicellular environment (single or fused giant cells) containing only 6 x 10(6)-8 x 10(7) molecules of rhodopsin. This method improves measurement sensitivity 7 to 8 orders of magnitude compared to other time-resolved techniques applied to rhodopsin to study the role particular amino acids play in conformational activation and the forces that govern those transitions.

Cell Fusion↗
PubMed1999

Simulation analysis of the retinal conformational equilibrium in dark-adapted bacteriorhodopsin.

In dark-adapted bacteriorhodopsin (bR) the retinal moiety populates two conformers: all-trans and (13,15)cis. Here we examine factors influencing the thermodynamic equilibrium and conformational transition between the two forms, using molecular mechanics and dynamics calculations. Adiabatic potential energy mapping indicates that whereas the twofold intrinsic torsional potentials of the C13==C14 and C15==N16 double bonds favor a sequential torsional pathway, the protein environment favors a concerted, bicycle-pedal mechanism. Which of these two pathways will actually occur in bR depends on the as yet unknown relative weight of the intrinsic and environmental effects. The free energy difference between the conformers was computed for wild-type and modified bR, using molecular dynamics simulation. In the wild-type protein the free energy of the (13,15)cis retinal form is calculated to be 1.1 kcal/mol lower than the all-trans retinal form, a value within approximately kBT of experiment. In contrast, in isolated retinal the free energy of the all-trans state is calculated to be 2.1 kcal/mol lower than (13,15)cis. The free energy differences are similar to the adiabatic potential energy differences in the various systems examined, consistent with an essentially enthalpic origin. The stabilization of the (13,15)cis form in bR relative to the isolated retinal molecule is found to originate from improved protein-protein interactions. Removing internal water molecules near the Schiff base strongly stabilizes the (13,15)cis form, whereas a double mutation that removes negative charges in the retinal pocket (Asp85 to Ala; Asp212 to Ala) has the opposite effect.

Bacteriorhodopsins↗
PubMed2001

Ternary complexes of liver alcohol dehydrogenase.

Liver alcohol dehydrogenase (LADH; E.C. 1.1.1.1) provides an excellent system for probing the role of binding interactions with NAD(+) and alcohols as well as with NADH and the corresponding aldehydes. The enzyme catalyzes the transfer of hydride ion from an alcohol substrate to the NAD(+) cofactor, yielding the corresponding aldehyde and the reduced cofactor, NADH. The enzyme is also an excellent catalyst for the reverse reaction. X-ray crystallography has shown that the NAD(+) binds in an extended conformation with a distance of 15 A between the buried reacting carbon of the nicotinamide ring and the adenine ring near the surface of the horse liver enzyme. A major criticism of X-ray crystallographic studies of enzymes is that they do not provide dynamic information. Such data provide time-averaged and space-averaged models. Significantly, entries in the protein data bank contain both coordinates as well as temperature factors. However, enzyme function involves both dynamics and motion. The motions can be as large as a domain closure such as observed with liver alcohol dehydrogenase or as small as the vibrations of certain atoms in the active site where reactions take place. Ternary complexes produced during the reaction of the enzyme binary entity, E-NAD(+), with retinol (vitamin A alcohol) lead to retinal (vitamin A aldehyde) release and the enzyme binary entity E-NADH. Retinal is further metabolized via the E-NAD(+)-retinal ternary complex to retinoic acid (vitamin A acid). To unravel the mechanistic aspects of these transformations, the kinetics and energetics of interconversion between various ternary complexes are characterized. Proton transfers along hydrogen bond bridges and NADH hydride transfers along hydrophobic entities are considered in some detail. Secondary kinetic isotope effects with retinol are not particularly large with the wild-type form of alcohol dehydrogenase from horse liver. We analyze alcohol dehydrogenase catalysis through a re-examination of the reaction coordinates. The ground states of the binary and ternary complexes are shown to be related to the corresponding transition states through topology and free energy acting along the reaction path.

Alcohol Dehydrogenase↗
PubMed2001

Bioinorganic and bioorganic studies of liver alcohol dehydrogenase.

Liver alcohol dehydrogenase (E.C.1.1.1.1) is an NAD(+)/NADH dependent enzyme with a broad substrate specificity being active on an assortment of primary and secondary alcohols. It catalyzes the reversible oxidation of a wide variety of alcohols to the corresponding aldehydes and ketones as well as the oxidation of certain aldehydes to their related carboxylic acids. Although the bioinorganic and bioorganic aspects of the enzymatic mechanism, as well as the structures of various ternary complexes, have been extensively studied, the kinetic significance of certain intermediates has not been fully evaluated. Nevertheless, the availability of computer-assisted programs for kinetic simulation and molecular modeling make it possible to describe the biochemical mechanism more completely. Although the true physiological substrates of this zinc metalloenzyme are unknown, alcohol dehydrogenase effectively catalyzes not only the interconversion of all-trans-retinol and all-trans-retinal but also the oxidation of all-trans-retinal to the corresponding retinoic acid. Retinal and related vitamin A derivatives play fundamental roles in many physiological processes, most notably the vision process. Furthermore, retinoic acid is used in dermatology as well as in the prevention and treatment of different types of cancer. The enzyme-NAD(+)-retinol complex has an apparent pK(a) value of 7.2 and loses a proton rapidly. Proton inventory modeling suggests that the transition state for the hydride transfer step has a partial negative charge on the oxygen of retinoxide. Spectral evidence for an intermediate such as E-NAD(+)-retinoxide was obtained with enzyme that has cobalt(II) substituted for the active site zinc(II). Biophysical considerations of water in these biological processes coupled with the inverse solvent isotope effect lead to the conclusion that the zinc-bound alkoxide makes a strong hydrogen bond with the hydroxyl group of Ser48 and is thus activated for hydride transfer. Moderate pressure accelerates enzyme action indicative of a negative volume of activation. The data with retinol is discussed in terms of enzyme stability, mechanism, adaptation to extreme conditions, as well as water affinities of substrates and inhibitors. Our data concern all-trans, 9-cis, 11-cis, and 13-cis retinols as well as the corresponding retinals. In all cases the enzyme utilizes an approximately ordered mechanism for retinol-retinal interconversion and for retinal-retinoic acid transformation.

Alcohol Dehydrogenase↗
PubMed1983

Stimulation of cell proliferation by vitamin A derivatives on murine sarcoma virus-transformed mouse cells in serum-free culture.

The effect of retinoids (Rds) on cell proliferation was studied in serum-free culture condition, using non-transformed and transformed derivatives of BALB 3T3. Cell proliferation of an SV40-transformed line was inhibited significantly by Rd treatment. However, proliferation of two cell lines that were transformed by a Kirsten and Moloney strain of murine sarcoma virus (MSV) and produced growth factor into culture medium, was remarkably stimulated by Rds. Addition of serum masked both the inhibitory and stimulatory effects of Rds.

Animals↗
PubMed1985

Localization of retinoid-binding proteins in developing rat retina.

Antibodies to cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRALBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antigens were localized on frozen sections of rat retina using indirect FITC immunofluorescence. In the retinal pigment epithelium of rats from postnatal day 1 (the day of birth) to postnatal day 32, specific staining with anti-CRBP was restricted to the cytoplasm; the nuclei were unstained. In the neurosensory retina, Muller cell endfeet were stained with anti-CRBP at all ages examined. No CRBP reactivity was found in the pigment epithelium of the ciliary body at any age examined. On postnatal days 14 and 32, in addition to Muller cell endfeet and radial processes, two fine laminae in the inner plexiform layer were faintly positive for CRBP. Anti-CRALBP stained the cytoplasm of the RPE and Muller cells in the adult rat. In developing rat retina, in addition to Muller cells and the retinal pigment epithelium, the ciliary body pigment epithelium and the outer epithelium of the iris were stained with anti-CRALBP in the first postnatal week. The intensity of staining of the ciliary body pigment epithelium decreased gradually from postnatal day 8 until postnatal day 14, at which point a clear line of demarcation was found between the positively stained retinal pigment epithelium and the adjacent, lightly stained, pigment epithelial cells of the ciliary body.

Aging↗
PubMed1987

Interaction of visual pigment with G-protein: effects of bleaching in native and reconstituted ROS preparations.

Native and reconstituted preparations of bullfrog rod outer segment (ROS) membranes were analysed for the binding of the alpha and beta subunits of G-protein. Following incubation at 22-28 degrees C under varying conditions of illumination and chemical treatment, preparations were chilled and extracted with hypotonic medium lacking-vs.-containing GTP (H and HG extracts, respectively). The extent of binding of G alpha,beta ('percentage G bound') was determined by measurement of the relative abundance of G alpha, beta in the H and HG extracts. The addition of G to unbleached, G-depleted membranes (D-ROS) yielded relatively little binding of the G (14 +/- 13%). G reintroduced at congruent to 1 min vs. congruent to 90 min after major bleaching yielded, respectively, levels of binding of 88 +/- 8% and 24 +/- 8%. Native G, extracted at congruent to 90 min post-bleach and added to freshly bleached D-ROS, exhibited binding exceeding that in the parent (bleached +90 min) sample. Supplementation of reconstituted suspensions with all-trans retinal, and subsequent incubation in darkness, yielded 30-35% G-binding; similar treatment with 11-cis retinal yielded binding in the range of 0-32%. Upon irradiation, suspensions previously supplemented with either isomer exhibited approximately 60% binding. Incubation with 11-cis retinal had no substantial effect on the condition of tight binding observed shortly after bleaching. The data are discussed in relation to previous studies of visual pigment bleaching and regeneration.

Animals↗
PubMed1988

A novel photopigment candidate in membranes of cultured chick pineal cells.

The photopigment of avian pineal which mediates light sensitivity was sought via its chromophore. Chick pineal cells in primary cultures were incubated overnight in the dark with [3H]retinol. Reduction of Schiff's bases with cyanoborohydride prior to SDS-PAGE revealed radioactivity bound to a 30 kDa component in pinealocyte membranes. All-trans-retinal, but not retinol or retinoic acid, incubated with pinealocyte homogenates prior to reduction, resulted in a loss of radioactivity from the 30 kDa region of the gel. The radioactivity was also displaced by NH2OH in the dark. Incubation of cultured cells or homogenates with retinoyl fluoride, an acylating agent specific for the retinal binding site of opsins, also displaced radioactivity from the protein. Furthermore, retinoyl fluoride, added to chick pineal cells in culture, blocked the suppressive effect of light on melatonin output by these cells. Taken together these results raise the possibility that the 30 kDa protein mediates photosensitivity in the chick pineal.

Animals↗
PubMed1988

Substitution of porphyropsin for rhodopsin in mouse retina.

Weanling, male albino mice were placed on a vitamin A-free diet for three months to deplete their vitamin A stores. The vitamin A-deficient mice were injected intraperitoneally with all-trans 3-dehydroretinol. 3-dehydroretinol was rapidly incorporated into the liver as a fatty acid ester. The chromophore of visual pigment increased gradually and reached a normal level 13 days after the injection. 3-Dehydroretinal accounted for 95% of the total chromophore in the retina. The high proportion of 3-dehydroretinal was observed also in the long-term experiment which was continued for six weeks with the injection of 3-dehydroretinol once a week. When the animal was injected with a mixture of 3-dehydroretinol and retinol, the ratio of dehydroretinal/retinal in the retina was far lower than the ratio of dehydroretinol/retinol in the liver. These results indicate that 3-dehydroretinol is not converted to retinol in mouse and is used less efficiently than retinol for the chromophore of visual pigment. The synthesis of visual pigment was observed even when the animal was kept in complete darkness after the injection of all-trans 3-dehydroretinol. This fact indicates that light is not required for the production of 11-cis chromophore of visual pigment.

Animals↗
PubMed1988

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.

Animals↗
PubMed2000

Effects of mutation at a conserved N-glycosylation site in the bovine retinal cyclic nucleotide-gated ion channel.

Bovine retinal cyclic nucleotide-gated (CNG) ion channel contains an evolutionary conserved N-glycosylation site in the external loop between the fifth transmembrane segment and the pore-forming region. The effect of tunicamycin treatment and the site-specific mutation suggested that the channel is glycosylated when expressed in Xenopus oocytes. To test the role of glycosylation in this channel, N-glycosylation was abolished by mutation, and the detailed permeation and the gating characteristics of the mutant channel were investigated. The charge contribution turned out to be detectable, although the mutation of the N-glycosylation site did not affect expression and functionality of the CNG channel in oocytes.

Amino Acid Sequence↗
PubMed2002

Amphioxus homologs of Go-coupled rhodopsin and peropsin having 11-cis- and all-trans-retinals as their chromophores.

Because of low contents in the native organs and failure of the expression in cultured cells, the chromophore configurations of the pigments in Go-coupled opsin and peropsin groups in the opsin family are unknown. Here we have succeeded in expression of the amphioxus homologs of these groups in HEK293s cells and found that they can be regenerated with 11-cis- and all-trans-retinals, respectively. Light isomerized the chromophores of these opsins into the all-trans and 11-cis forms, respectively. The results strongly suggest that the physiological function of peropsin would be a retinal photoisomerase, while 11-cis configuration is necessary for the Go-coupled opsin groups.

Animals↗
PubMed2004

Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products.

Epidemiological studies testing the effect of beta-carotene in humans have found a relative risk for lung cancer in smokers supplemented with beta-carotene. We investigated the reactions of retinal and beta-apo-8'-carotenal, two beta-carotene oxidation products, with 2'-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N(2)-etheno-2'-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with beta-carotene or beta-carotene oxidation products, significantly increased levels of 1,N(2)-etheno-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of beta-carotene oxidation products.

Animals↗
PubMed1997

The cytosolic aldehyde dehydrogenase gene (Aldh1) is developmentally expressed in Leydig cells.

Cytosolic aldehyde dehydrogenase, ALDH1, participates in the oxidation of different aldehydes including that of all-trans retinal to retinoic acid. The accumulation of mouse Aldh1 transcripts is characterized by having different patterns in different tissues. This paper reports the greatest expression of Aldh1 in testis and liver. It was demonstrated that in testis, Aldh1 is specifically expressed in Leydig cells and is under developmental regulation. In vitro studies of cultured Leydig TM3 cells confirmed these results though such gene expression was found not to be mediated by LH regulation. Previous investigations have associated androgen receptors, and hence the androgen insensitivity syndrome in man, with the presence of ALDH1 in genital skin fibroblasts. However, this relationship was not established in a functional cell type, as is reported here for Leydig cells. These results could suggest a model for a molecular pathway from androgen receptor to retinoic acid biogenesis in Leydig cells via the mediation of ALDH.

Aldehyde Dehydrogenase↗
PubMed1997

Titration kinetics of Asp-85 in bacteriorhodopsin: exclusion of the retinal pocket as the color-controlling cation binding site.

The spectrum (the purple blue transition) and function of the light-driven proton pump bacteriorhodopsin are determined by the state of protonation of the Asp-85 residue located in the vicinity of the retinal chromophore. The titration of Asp-85 is controlled by the binding/unbinding of one or two divalent metal cations (Ca2+ or Mg2+). The location of such metal binding site(s) is approached by studying the kinetics of the cation-induced titration of Asp-85 using metal ions and large molecular cations, such as quaternary ammonium ions, R4N+ (R = Et, Pr, a divalent 'bolaform ion' [Et3N+-(CH2)4-N+Et3] and the 1:3 molecular complex formed between Fe2+ and 1,10-phenanthroline (OP). The basic multi-component kinetic features of the titration, extending from 10(-2) to 10(4) s, are unaffected by the charge and size of the cation. This indicates that cation binding to bR triggers the blue --> purple titration in a fast step, which is not rate-determining. In view of the size of the cations involved, these observations indicate that the cation binding site is in an exposed location on, or close to, the membrane surface. This excludes previous models, which placed the color-controlling Ca2+ ion in the retinal binding pocket.

Aspartic Acid↗
PubMed1998

Photoreceptor rhodopsin: structural and conformational study of its chromophore 11-cis retinal in oriented membranes by deuterium solid state NMR.

Rhodopsin is the retinal photoreceptor responsible for visual signal transduction. To determine the orientation and conformation of retinal within the binding pocket of this membrane bound receptor, an ab initio solid state 2H NMR approach was used. Bovine rhodopsin containing 11-cis retinal, specifically deuterated at its methyl groups at the C19 or C20 position, was uniaxially oriented in DMPC bilayers. Integrity of the membranes and quality of alignment were monitored by 31P NMR. Analysis of the obtained 2H NMR spectra provided angles for the individual labelled chemical bond vectors leading to an overall picture for the three dimensional structure of the polyene chain of the chromophore in the protein binding pocket around the Schiff base attachment site.

Animals↗
PubMed1998

Induction of the mitogen-activated protein kinase phosphatase MKP3 by nerve growth factor in differentiating PC12.

In PC12 sympathetic neurons activation and nuclear translocation of ERK family MAP kinases plays an essential role in processes underlying nerve growth factor (NGF)-dependent differentiation. We have recently cloned MKP-3 as a novel dual specificity phosphatase displaying selectivity towards inactivation of the ERK1 and ERK2 MAP kinases. Here we report that in PC12 cells, MKP-3 undergoes powerful and specific up-regulation by NGF while a number of mitogens and cellular stresses are ineffective. NGF-stimulated MKP-3 expression appears after 1 h, is maximal at 3 h, and is sustained for 5 days. This coincides with a critical period of neurite outgrowth and terminal differentiation. Consistent with a role mediating inhibition of PC12 cell MAP kinases, NGF-stimulated ERK2 activation was suppressed considerably following pretreatment with fibroblast growth factor and 9-cis-retinal, two additional differentiation factors found to induce powerfully MKP-3 expression. Given the clear cytosolic localization of MKP3 in PC12 cells and sympathetic neurons, these results suggest a critical role for inactivating ERK MAP kinases in non-nuclear compartments during essential stages of NGF-mediated PC12 differentiation.

Animals↗