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Biochemistry Feb 2007Metabolism of vitamin A, all-trans-retinol, leads to the formation of 11-cis-retinaldehyde, the visual chromophore, and all-trans-retinoic acid, which is involved in the...
Metabolism of vitamin A, all-trans-retinol, leads to the formation of 11-cis-retinaldehyde, the visual chromophore, and all-trans-retinoic acid, which is involved in the regulation of gene expression through the retinoic acid receptor. Enzymes and binding proteins involved in retinoid metabolism are highly conserved across species. We previously described a novel mammalian enzyme that saturates the 13-14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol, which then follows the same metabolic fate as that of all-trans-retinol. Specifically, all-trans-13,14-dihydroretinol is transiently oxidized to all-trans-13,14-dihydroretinoic acid before being oxidized further by Cyp26 enzymes. Here, we report the identification of two putative RetSat homologues in zebrafish, one of which, zebrafish RetSat A (zRetSat A), also had retinol saturase activity, whereas zebrafish RetSat B (zRetSat B) was inactive under similar conditions. Unlike mouse RetSat (mRetSat), zRetSat A had an altered bond specificity saturating either the 13-14 or 7-8 double bonds of all-trans-retinol to produce either all-trans-13,14-dihydroretinol or all-trans-7,8-dihydroretinol, respectively. zRetSat A also saturated the 13-14 or 7-8 double bonds of all-trans-3,4-didehydroretinol (vitamin A2), a second endogenous form of vitamin A in zebrafish. The dual enzymatic activity of zRetSat A displays a newly acquired specificity for the 13-14 double bond retained in higher vertebrates and also the evolutionarily preserved activity of bacterial phytoene desaturases and plant carotenoid isomerases. Expression of zRetSat A was restricted to the liver and intestine of hatchlings and adult zebrafish, whereas zRetSat B was expressed in the same tissues but at earlier developmental stages. Exogenous all-trans-retinol, all-trans-13,14-dihydroretinol, or all-trans-7,8-dihydroretinol led to the strong induction of the expression of the retinoic acid-metabolizing enzyme, Cyp26A1, arguing for an active signaling function of dihydroretinoid metabolites in zebrafish. These findings point to a conserved function but altered specificity of RetSat in vertebrates, leading to the generation of various dihydroretinoid compounds, some of which could have signaling functions.
Topics: Amino Acid Sequence; Animals; Catalysis; Molecular Sequence Data; Organ Specificity; Oxidoreductases Acting on CH-CH Group Donors; Stereoisomerism; Substrate Specificity; Vitamin A; Zebrafish; Zebrafish Proteins
PubMed: 17253779
DOI: 10.1021/bi062147u -
Investigative Ophthalmology & Visual... Nov 2002In the photic visual cycle, retinal G protein-coupled receptor (RGR) isomerizes all-trans retinal to 11-cis retinal in the retinal pigment epithelium (RPE) after...
PURPOSE
In the photic visual cycle, retinal G protein-coupled receptor (RGR) isomerizes all-trans retinal to 11-cis retinal in the retinal pigment epithelium (RPE) after illumination. It is unclear, however, how all-trans retinal, the substrate for RGR, is generated in the RPE, because no all-trans retinol dehydrogenase (atRDH) has been identified in the RPE. This study was conducted to identify the atRDH that generates all-trans retinal in the RPE.
METHODS
The full-length cDNA encoding a novel atRDH, RDH10, was cloned by PCR based on an expressed sequence tag (EST). Cellular localization was determined at the mRNA level by Northern blot analysis, RT-PCR, and in situ hybridization and at the protein level by immunohistochemistry with an antibody specific to RDH10. The activity was measured by an RDH activity assay with recombinant RDH10 expressed in COS cells.
RESULTS
The full-length RDH10 was cloned from the human, cow, and mouse. These cDNAs encode a protein of 341 amino acids and have significant sequence homology with other short-chain dehydrogenases/reductases (SDRs). The human RDH10 shares 100% and 98.6% amino acid sequence identity with the bovine and mouse proteins, respectively, suggesting a highly conserved sequence during evolution. RDH10 is predominantly expressed in the microsomal fraction of the RPE. Human RDH10 expressed in COS cells oxidized all-trans retinol to all-trans retinal. RDH10 displayed substrate specificity for all-trans retinol and preferred nicotinamide adenine dinucleotide phosphate (NADP) as the cofactor.
CONCLUSIONS
RDH10 is a novel retinol oxidase expressed in the RPE. This enzyme can generate all-trans retinal from all-trans retinol and may play an important role in the photic visual cycle.
Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Animals; Blotting, Northern; COS Cells; Cattle; Cloning, Molecular; Expressed Sequence Tags; Humans; Immunoenzyme Techniques; In Situ Hybridization; Mice; Molecular Sequence Data; Pigment Epithelium of Eye; RNA, Messenger; Retinaldehyde; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Transfection; Vitamin A
PubMed: 12407145
DOI: No ID Found -
Biotechnology Letters Apr 2015A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min(-1) mg(-1), a K m of 1.8 μM,...
A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min(-1) mg(-1), a K m of 1.8 μM, and a k cat of 1.7 min(-1) for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD (+) as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l(-1), and 2,200 mg all-trans-retinal l(-1) in the presence of 5% (v/v) methanol, 1% (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l(-1) after 3 h, with a conversion yield of 27.3% (w/w) and a productivity of 200 mg l(-1) h(-1). This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH.
Topics: Alcanivoraceae; Alcohol Dehydrogenase; Amino Acid Sequence; Coenzymes; Enzyme Stability; Hydrogen-Ion Concentration; Hydroquinones; Kinetics; Methanol; Molecular Sequence Data; Molecular Weight; NAD; Phylogeny; Protein Multimerization; Recombinant Proteins; Sequence Homology, Amino Acid; Temperature; Vitamin A
PubMed: 25481533
DOI: 10.1007/s10529-014-1740-x -
Methods in Molecular Biology (Clifton,... 2010The mobility of all-trans-retinol makes a crucial contribution to the rate of the reactions in which it participates. This is even more so because of its low aqueous...
The mobility of all-trans-retinol makes a crucial contribution to the rate of the reactions in which it participates. This is even more so because of its low aqueous solubility, which makes the presence of carrier proteins and the spatial arrangement of cellular membranes especially relevant. In rod photoreceptor outer segments, all-trans-retinol is generated after light exposure from the reduction of all-trans-retinal that is released from bleached rhodopsin. The mobility of all-trans-retinol in rod outer segments was measured with fluorescence recovery after photobleaching (FRAP), using two-photon excitation of its fluorescence. The values of the lateral and axial diffusion coefficients indicate that most of the all-trans-retinol in rod outer segments move unrestricted and without being aided by carriers.
Topics: Animals; Anura; Cell Separation; Diffusion; Fluorescence Recovery After Photobleaching; Movement; Photons; Rod Cell Outer Segment; Vitamin A
PubMed: 20552425
DOI: 10.1007/978-1-60327-325-1_6 -
Biochimica Et Biophysica Acta Oct 1987As reported previously squamous cell differentiation of rabbit tracheal epithelial (RTE) cells in culture is a multi-step process. This program of differentiation is...
As reported previously squamous cell differentiation of rabbit tracheal epithelial (RTE) cells in culture is a multi-step process. This program of differentiation is inhibited by retinoic acid and retinol; retinoic acid is about 100 times more effective than retinol. To examine the metabolism of these agents in this in vitro model system, RTE cells were grown in the presence of all-trans-[3H]retinol or all-trans-[3H]retinoic acid and their metabolites analyzed by high-pressure liquid chromatography. RTE cells converted most of the retinol to retinyl esters, predominantly retinyl palmitate. A small fraction was metabolized to polar compounds, one of which coeluted with retinoic acid. After methylation this compound eluted as 13-cis-methyl retinoate and as all-trans-methyl retinoate. Conversion to 13-cis-retinol was also observed. All-trans-retinoic acid was rapidly taken up by RTE cells and converted to more polar (peak 1) and less polar (peak 3) metabolites. A proportion of all-trans-[3H]retinoic acid was metabolized to 13-cis-[3H]retinoic acid. These metabolic reactions appeared to be constitutive and were not induced by pretreatment with retinoic acid. The peak 1 metabolites were rapidly secreted into the medium whereas the peak 3 metabolites were retained by the cells and were not detected in the medium. Alkaline hydrolysis of the metabolites in peak 3 yielded retinoic acid, indicating the formation of retinoyl derivatives. Our results establish that RTE cells can convert all-trans-retinol to 13-cis-retinol and retinoic acid. RTE can metabolize all-trans-retinoic acid to 13-cis-retinoic acid and to an unidentified ester of retinoic acid.
Topics: Animals; Cell Differentiation; Cells, Cultured; Chromatography, High Pressure Liquid; Epithelium; Rabbits; Stereoisomerism; Trachea; Tretinoin; Vitamin A
PubMed: 3663700
DOI: 10.1016/0005-2760(87)90240-2 -
Investigative Ophthalmology & Visual... Jun 2007To investigate the effect of light stimulation on lipid droplets (LDs) and LD proteins in the retinal pigment epithelium (RPE).
PURPOSE
To investigate the effect of light stimulation on lipid droplets (LDs) and LD proteins in the retinal pigment epithelium (RPE).
METHODS
Dark-adapted mouse eyes were exposed to intense flashes of light, and ARPE-19 cells were treated with all-trans-retinol. The two specimens were labeled with BODIPY493/503 for LDs and with antibodies for three LD proteins: adipocyte differentiation-related protein (ADRP), TIP47, and Rab18. The labeling intensity in fluorescence microscopy was quantified by image analysis. Localization of mutated TIP47 was also examined. Immunoelectron microscopy was performed for ADRP in mouse RPE. Expression of TIP47 in ARPE-19 cells was knocked down by RNA interference (RNAi), and its effect on retinyl ester storage was measured by HPLC.
RESULTS
Both flashes of light on mouse eyes and all-trans-retinol on ARPE-19 cells caused rapid translocation of TIP47 from the cytosol to LDs, whereas ADRP distributed constitutively in LDs. The density of LDs did not show visible changes by any treatment. The localization of TIP47 to LDs was abolished when either the amino-terminal or the carboxyl-terminal half of the molecule was deleted, but was enhanced by a short deletion in the carboxyl terminus. Manipulation of TIP47 expression by RNAi or cDNA transfection did not affect the retinyl ester amounts in ARPE-19 cells significantly.
CONCLUSIONS
All-trans-retinol generated by photobleaching in the retina induces rapid translocation of TIP47 to LDs in the RPE.
Topics: Adipocytes; Animals; Blotting, Western; Cells, Cultured; Chromatography, High Pressure Liquid; Cytosol; DNA-Binding Proteins; Dark Adaptation; Humans; Intracellular Signaling Peptides and Proteins; Lipid Metabolism; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Microscopy, Immunoelectron; Perilipin-2; Perilipin-3; Photic Stimulation; Pigment Epithelium of Eye; Pregnancy Proteins; Protein Transport; RNA Interference; Rhodopsin; Vesicular Transport Proteins; Vitamin A; rab GTP-Binding Proteins
PubMed: 17525222
DOI: 10.1167/iovs.06-0768 -
Journal of Biochemistry May 1998It is known that exogenous 11-cis-retinol inhibits the recovery of photosensitivity of bleached rod outer segments (ROS) and 11-cis-retinol exists in the...
It is known that exogenous 11-cis-retinol inhibits the recovery of photosensitivity of bleached rod outer segments (ROS) and 11-cis-retinol exists in the interphotorecepter matrix. We examined the conversion of 11-cis-retinol with bovine ROS. ROS was incubated with 11-cis-retinol under dim red light. Retinoids were extracted from the reaction mixture with hexane and analyzed by HPLC coupled with a fluorescence spectrophotometer. Isomerization of 11-cis-retinol to all-trans-retinol was observed in the presence of ROS. This isomerization was not suppressed by heat treatment and did not have stereospecificity. In addition, we incubated purified rhodopsin and phospholipids extracted from ROS with 11-cis-retinol. Rhodopsin was found to isomerize 11-cis-retinol to all-trans-retinol as well as ROS, but phospholipids did not. In contrast, the phospholipids inhibited the isomerization of 11-cis-retinol to all-trans-retinol by the purified rhodopsin. Commercially available phospholipids, especially phosphatidylserine, also inhibited the isomerization. Our results suggest that rhodopsin has activity for the isomerization of 11-cis-retinol to all-trans-retinol and may play an important role in the detoxification of 11-cis-retinol in the ROS.
Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Hot Temperature; In Vitro Techniques; Light; Phospholipids; Rhodopsin; Rod Cell Outer Segment; Spectrometry, Fluorescence; Stereoisomerism; Vitamin A
PubMed: 9562631
DOI: 10.1093/oxfordjournals.jbchem.a022030 -
Archives of Biochemistry and Biophysics Nov 1993A kinetic quantification of the lipoperoxyl radical-scavenging activity of all-trans-retinol has been carried out in homogeneous solution, when radicals were produced...
A kinetic quantification of the lipoperoxyl radical-scavenging activity of all-trans-retinol has been carried out in homogeneous solution, when radicals were produced from the oxidation of methyl linoleate in methanol, initiated by the lipid-soluble 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) as well as in a soybean phosphatidylcholine membrane model, in which peroxidation was induced either by AMVN or the hydrophylic 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH). The physical microenvironment contributes to the determination of antioxidant efficiency of all-trans-retinol. In homogeneous solution the kinetic constant kinh is 3.5 x 10(5) M-1 s-1 and appears of the same order of magnitude as the inhibition constant measured for alpha-tocopherol under the same experimental conditions. Nevertheless, despite its very high chemical reactivity toward lipoperoxyl radicals, the overall antioxidant efficiency of all-trans-retinol in this system appears quite limited, since the evaluated stoichiometric factor is 0.21. When the polyenoic chain of all-trans-retinol is incorporated into a phosphatidylcholine lipid bilayer, the antioxidant efficiency depends on the site of peroxyl-radical production. The highest lipoperoxyl radical-scavenging activity is measured when radicals are generated by AHVN inside the bilayer multilamellar liposomes. Under these conditions, the relative antioxidant efficiency is similar to that of alpha-tocopherol, and the stoichiometric factor is 3.1. When radicals are generated by AAPH in the aqueous phase of an unilamellar liposomal system, the antioxidant effectiveness of all-trans-retinol appears reduced and lower than that measured with equivalent amounts of alpha-tocopherol. Synergistic antioxidant effects between all-trans-retinol and alpha-tocopherol are observed when both antioxidants are simultaneously incorporated into unilamellar liposomes in which peroxidation is induced by AAPH. This suggests that all-trans-retinol may interact with tocopheroxyl radicals, thereby regenerating alpha-tocopherol. This interaction, which may be related to molecular features and to the relative location of the antioxidants in the bilayer, could provide an effective antioxidant system that may be of great importance in vivo.
Topics: Amidines; Azo Compounds; Chromatography, High Pressure Liquid; Free Radical Scavengers; Free Radicals; Kinetics; Lipid Bilayers; Lipid Peroxidation; Liposomes; Nitriles; Oxidants; Phosphatidylcholines; Solutions; Tritium; Vitamin A
PubMed: 8239660
DOI: 10.1006/abbi.1993.1581 -
Biochemical Pharmacology Mar 1987All-trans-retinoic acid formation from topically applied retinol has been demonstrated in the skin of skh/hr1 (hairless) mice. The all-trans-retinoic acid was identified... (Comparative Study)
Comparative Study
All-trans-retinoic acid formation from topically applied retinol has been demonstrated in the skin of skh/hr1 (hairless) mice. The all-trans-retinoic acid was identified on the basis of its chromatographic properties on HPLC at various pH values, its photoisomerization to reaction products identical to those formed from authentic all-trans-retinoic acid, and its co-chromatography with methyl retinoate after methylation with diazomethane. Topically applied retinol is about 2-fold less potent at inducing epidermal hyperplasia and 7-fold less potent at inhibiting the induction of epidermal ornithine decarboxylase by phorbol esters than all-trans-retinoic acid in this strain of mice. To elucidate the possible role all-trans-retinoic acid formation from retinol may have in these pharmacological activities, the epidermal and dermal all-trans-retinoic acid levels were compared in mice treated topically with retinol or [11-3H]-all-trans-retinoic acid. The levels of all-trans-retinoic acid found after retinol treatment were several orders of magnitude lower than those found after [11-3H]-all-trans-retinoic acid treatment, and they were insufficient to account for the difference in potencies between all-trans-retinoic acid and retinol. Retinol was eliminated from the epidermis at a rate similar to that of all-trans-retinoic acid after topical administration, but the initial tissue levels achieved were lower. These results suggest that the lower potencies of retinol may simply reflect lower tissue uptake.
Topics: Animals; Chromatography, High Pressure Liquid; Enzyme Induction; Epidermis; Female; Half-Life; Hydrogen-Ion Concentration; Hyperplasia; Mice; Mice, Hairless; Ornithine Decarboxylase; Skin; Spectrophotometry; Tretinoin; Vitamin A
PubMed: 3566789
DOI: 10.1016/0006-2952(87)90185-7 -
Neuroreport Jun 2008Embryonic cerebrospinal fluid (E-CSF) is involved in the regulation of survival, proliferation and neurogenesis of neuroectodermal progenitor cells, as well as in the...
Embryonic cerebrospinal fluid (E-CSF) is involved in the regulation of survival, proliferation and neurogenesis of neuroectodermal progenitor cells, as well as in the control of mesencephalic gene expression in collaboration with the isthmic organizer. Recently, we showed the presence of retinol-binding protein (RBP) within the E-CSF proteome. RBP is an all-trans retinol carrier, a molecule that can be metabolized into retinoic acid, a morphogen involved in central nervous system (CNS) morphogenesis and patterning. Here we demonstrate the presence of all-trans retinol within the E-CSF and analyse the dynamics of RBP and all-trans retinol within this fluid, as well as the expression of retinoic acid-synthesizing enzymes during early CNS development. Our results suggest a relationship between the dynamics of these molecules and the early events of CNS patterning.
Topics: Animals; Body Patterning; Central Nervous System; Chick Embryo; Chromatography, High Pressure Liquid; Gene Expression Regulation, Developmental; Mass Spectrometry; Retinol-Binding Proteins; Vitamin A
PubMed: 18520998
DOI: 10.1097/WNR.0b013e3283021c94