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1.
Recent studies of the human, mouse and bovine genes for 11-cis-retinol dehydrogenase (11cRDH) and human and mouse 9-cis-retinol dehydrogenase (9cRDH) suggest that they are homologs of the same enzyme. This conclusion is inconsistent with earlier literature indicating that 11cRDH is expressed solely in the eye and does not utilize 9-cis-retinol as a substrate. We have compared directly the kinetic properties of recombinant human and mouse 9cRDH with those of bovine 11cRDH for 9-cis- and 11-cis-retinol and investigated the inhibitory properties of 13-cis-retinoic acid on each of these enzymes. Human and mouse 9cRDH and bovine 11cRDH have very similar kinetic properties towards 9-cis- and 11-cis-retinol oxidation and they respond identically to 13-cis-retinoic acid inhibition. Our biochemical data are consistent with the conclusion that 9cRDH and 11cRDH are the same enzyme. 相似文献
2.
9-cis-retinoids: biosynthesis of 9-cis-retinoic acid 总被引:5,自引:0,他引:5
3.
Cis-retinol dehydrogenase: 9-cis-retinol metabolism and its effect on proliferation of human MCF7 breast cancer cells 总被引:3,自引:0,他引:3
9-Cis-retinoic acid (RA) suppresses cancer cell proliferation via binding and activation of nuclear receptors, retinoid X receptors (RXRs). In vivo, 9-cis-RA is formed through oxidation of 9-cis-retinol by cis-retinol dehydrogenase (cRDH), an enzyme that we characterized previously. Since 9-cis-RA is a potent inhibitor of breast cancer cell proliferation, we hypothesized that overexpression of cRDH in breast cancer cells would result in increased production of 9-cis-RA, which in turn would suppress cell proliferation. To investigate this hypothesis, MCF7 human breast carcinoma cells were transduced with cRDH cDNA (LRDHSN/MCF7), and the growth kinetics and retinoid profiles of cells were examined following treatment with 9-cis-retinol. LRDHSN/MCF7 cells showed a marked reduction in cell numbers (60-80%) upon treatment with 9-cis-retinol compared to vehicle alone. Within 24 h of treatment, approximately 75% of the 9-cis-retinol was taken up and metabolized by LRDHSN/MCF7 cells. Despite the rapid uptake and oxidation of 9-cis-retinol to 9-cis-retinal, 9-cis-RA was not formed in these cells. We detect at least one novel metabolite formed from both 9-cis-retinol and 9-cis-retinal that may play a role in inhibition of MCF7 cell proliferation. Our studies demonstrate that 9-cis-retinol in combination with cRDH inhibits breast cancer cell proliferation by production of retinol metabolites other than RA. 相似文献
4.
Sílvia Martras Rosana Alvarez Susana E Martínez Dámaso Torres Oriol Gallego Gregg Duester Jaume Farrés Angel R de Lera Xavier Parés 《European journal of biochemistry》2004,271(9):1660-1670
Studies in knockout mice support the involvement of alcohol dehydrogenases ADH1 and ADH4 in retinoid metabolism, although kinetics with retinoids are not known for the mouse enzymes. Moreover, a role of alcohol dehydrogenase (ADH) in the eye retinoid interconversions cannot be ascertained due to the lack of information on the kinetics with 11-cis-retinoids. We report here the kinetics of human ADH1B1, ADH1B2, ADH4, and mouse ADH1 and ADH4 with all-trans-, 7-cis-, 9-cis-, 11-cis- and 13-cis-isomers of retinol and retinal. These retinoids are substrates for all enzymes tested, except the 13-cis isomers which are not used by ADH1. In general, human and mouse ADH4 exhibit similar activity, higher than that of ADH1, while mouse ADH1 is more efficient than the homologous human enzymes. All tested ADHs use 11-cis-retinoids efficiently. ADH4 shows much higher k(cat)/K(m) values for 11-cis-retinol oxidation than for 11-cis-retinal reduction, a unique property among mammalian ADHs for any alcohol/aldehyde substrate pair. Docking simulations and the kinetic properties of the human ADH4 M141L mutant demonstrated that residue 141, in the middle region of the active site, is essential for such ADH4 specificity. The distinct kinetics of ADH4 with 11-cis-retinol, its wide specificity with retinol isomers and its immunolocalization in several retinal cell layers, including pigment epithelium, support a role of this enzyme in the various retinol oxidations that occur in the retina. Cytosolic ADH4 activity may complement the isomer-specific microsomal enzymes involved in photopigment regeneration and retinoic acid synthesis. 相似文献
5.
This report describes the isolation of a heretofore uncharacterized aldehyde dehydrogenase (ALDH) with retinal dehydrogenase activity from rat kidney and the cloning and expression of a cDNA that encodes its human ortholog, the previously unknown ALDH12. The human ALDH12 cDNA predicts a 487-residue protein with the 23 invariant amino acids, four conserved regions, cofactor binding motif (G(209)XGX(3)G), and active site cysteine residue (Cys(287)) that typify members of the ALDH superfamily. ALDH12 seems at least as efficient (V(m)/K(m)) in converting 9-cis-retinal into the retinoid X receptor ligand 9-cis-retinoic acid as two previously identified ALDHs with 9-cis-retinal dehydrogenase activity, rat retinal dehydrogenase (RALDH) 1 and RALDH2. ALDH12, however, has approximately 40-fold higher activity with 9-cis- retinal than with all-trans-retinal, whereas RALDH1 and RALDH2 have equivalent and approximately 4-fold less efficiencies for 9-cis-retinal versus all-trans-retinal, respectively. Therefore, ALDH12 is the first known ALDH to show a preference for 9-cis-retinal relative to all-trans-retinal. Evidence consistent with the possibility that ALDH12 could function in a pathway of 9-cis-retinoic acid biosynthesis in vivo includes biosynthesis of 9-cis-retinoic acid from 9-cis-retinol in cells co-transfected with cDNAs encoding ALDH12 and the 9-cis-retinol/androgen dehydrogenase, cis-retinoid/androgen dehydrogenase type 1. Intense ALDH12 mRNA expression in adult and fetal liver and kidney, two organs that reportedly have relatively high concentrations of 9-cis-retinol, reinforces this notion. 相似文献
6.
Shang E Lai K Packer AI Paik J Blaner WS de Morais Vieira M Gouras P Wolgemuth DJ 《Journal of lipid research》2002,43(4):590-597
It has been proposed that cis-retinol dehydrogenase (cRDH) acts within the body to catalyze the oxidation of 9-cis-retinol, an oxidative step needed for 9-cis-retinoic acid synthesis, the oxidation of 11-cis-retinol [an oxidative step needed for 11-cis-retinal (visual chromophore) synthesis], and 3 alpha-hydroxysteroid transformations. To assess in vivo the physiological importance of each of these proposed actions of cRDH, we generated cRDH-deficient (cRDH-/-) mice. The cRDH-/- mice reproduce normally and appear to be normal. However, the mutant mice do have a mild visual phenotype of impaired dark adaptation. This phenotype is evidenced by electroretinagram analysis of the mice and by biochemical measures of eye levels of retinoid intermediates during recovery from an intense photobleach. Although it is thought that cRDH is expressed in the eye almost solely in retinal pigment epithelial cells, we detected cRDH expression in other retinal cells, including ganglion cells, amacrine cells, horizontal cells, and the inner segments of the rod photoreceptor cells. Aside from the eye, there are no marked differences in retinoid levels in other tissues throughout the body for cRDH-/- compared with cRDH+/+ mice. Moreover, we did not detect any non-visual phenotypic changes for cRDH-/- mice, suggesting that these mice do not have problems in metabolizing 3 alpha-hydroxysteroids.Thus, cRDH may act essentially in the visual cycle but is redundant for catalyzing 9-cis-retinoic acid formation and 3 alpha-hydroxysteroid metabolism. 相似文献
7.
Retinoic acid is generated by a two-step mechanism. First, retinol is converted into retinal by a retinol dehydrogenase, and, subsequently, retinoic acid is formed by a retinal dehydrogenase. In vitro, several enzymes are suggested to act in this metabolic pathway. However, little is known regarding their capacity to contribute to retinoic acid biosynthesis in vivo. We have developed a versatile cell reporter system to analyze the role of several of these enzymes in 9-cis-retinoic acid biosynthesis in vivo. Using a Gal4-retinoid X receptor fusion protein-based luciferase reporter assay, the formation of 9-cis-retinoic acid from 9-cis-retinol was measured in cells transfected with expression plasmids encoding different combinations of retinol and retinal dehydrogenases. The results suggested that efficient formation of 9-cis-retinoic acid required co-expression of retinol and retinal dehydrogenases. Interestingly, the cytosolic alcohol dehydrogenase 4 failed to efficiently catalyze 9-cis-retinol oxidation. A structure-activity analysis showed that mutants of two retinol dehydrogenases, devoid of the carboxyl-terminal cytoplasmic tails, displayed greatly reduced enzymatic activities in vivo, but were active in vitro. The cytoplasmic tails mediate efficient endoplasmic reticulum localization of the enzymes, suggesting that the unique milieu in the endoplasmic reticulum compartment is necessary for in vivo activity of microsomal retinol dehydrogenases. 相似文献
8.
Characterization of the oxidative 3alpha-hydroxysteroid dehydrogenase activity of human recombinant 11-cis-retinol dehydrogenase. 总被引:1,自引:0,他引:1
11-cis-Retinol dehydrogenase catalyzes the oxidation of cis-retinols, a rate-limiting step in the biosynthesis of 9-cis-retinoic acid. It is also active toward 3alpha-hydroxysteroids, and thus might be involved in steroid metabolism. To better understand the role of this enzyme, we produced stable transfectants expressing 11-cis-retinol dehydrogenase in human embryonic kidney 293 cells. In vitro enzymatic assays have demonstrated that, with an appropriate exogenous cofactor, the enzyme catalyzes the interconversion of 5alpha-androstane-3alpha,17beta-diol and dihydrotestosterone and that of androsterone and androstanedione. However, using intact transfected cells, we found that the enzyme catalyzes reactions only in the oxidative direction. Thus, it is possible that 5alpha-androstane-3alpha,17beta-diol (an inactive androgen) can be converted into dihydrotestosterone, the most potent androgen, by the action of 11-cis-retinol dehydrogenase. This reaction could constitute a non-classical pathway of production of active androgens in the peripheral tissues. We also showed that all-trans-, 9-cis- and 13-cis-retinol inhibit the oxidative 3alpha-hydroxysteroid steroid activity of 11-cis-retinol dehydrogenase with similar K(i) values. Since all-trans-retinol is a precursor of cis-retinols, its inhibitory effect on the activity suggests that it could play an important role in modulating the formation of 9-cis-retinoic acid. In addition, we examined the effect of several known enzyme modulators, namely carbenoxolone, phenylarsine oxide and phosphatidylcholine, on 11-cis-retinol dehydrogenase activity. Taken together, our results suggest that, in humans, this enzyme might play a role in the biosynthesis of both 9-cis-retinoic acid and dihydrotestosterone. 相似文献
9.
Metabolism of all-trans-retinol and all-trans-retinoic acid in rabbit tracheal epithelial cells in culture 总被引:1,自引:0,他引:1
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. 相似文献
10.
Vogel S Mendelsohn CL Mertz JR Piantedosi R Waldburger C Gottesman ME Blaner WS 《The Journal of biological chemistry》2001,276(2):1353-1360
Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol. Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family. In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse. Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm. Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III. 相似文献
11.
Karlsson T Vahlquist A Kedishvili N Törmä H 《Biochemical and biophysical research communications》2003,303(1):273-278
Retinol dehydrogenase-4 (RoDH-4) converts retinol and 13-cis-retinol to corresponding aldehydes in human liver and skin in the presence of NAD(+). RoDH-4 also converts 3 alpha-androstanediol and androsterone into dihydrotestosterone and androstanedione, which may stimulate sebum secretion. This oxidative 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) activity of RoDH-4 is competitively inhibited by retinol and 13-cis-retinol. Here, we further examine the substrate specificity of RoDH-4 and the inhibition of its 3 alpha-HSD activity by retinoids. Recombinant RoDH-4 oxidized 3,4-didehydroretinol-a major form of vitamin A in the skin-to its corresponding aldehyde. 13-cis-retinoic acid (isotretinoin), 3,4-didehydroretinoic acid, and 3,4-didehydroretinol, but not all-trans-retinoic acid or the synthetic retinoids acitretin and adapalene, were potent competitive inhibitors of the oxidative 3 alpha-HSD activity of RoDH-4, i.e., reduced the formation of dihydrotestosterone and androstandione in vitro. Extrapolated to the in vivo situation, this effect might explain the unique sebosuppressive effect of isotretinoin when treating acne. 相似文献
12.
Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina 总被引:2,自引:0,他引:2
11-cis-Retinaldehyde bound to cellular retinaldehyde-binding protein (CRALBP) is unaffected in bovine eyecup preparations by illumination that bleaches approximately 70% of the rhodopsin. Illumination of retinal homogenates to which CRALBP X [3H]11-cis-retinaldehyde had been added did not result in a reduction of the specific activity of recovered 11-cis-retinaldehyde, ruling out a bleaching regeneration cycle. The quantum efficiency of photoisomerization for CRALBP X 11-cis-retinaldehyde was determined by comparing the rate of photoisomerization of 11-cis-retinaldehyde bound to purified CRALBP and opsin. The low value obtained (0.07), coupled with a low molar extinction coefficient (15,400 M-1 cm-1), results in a photosensitivity only about 4% that of rhodopsin. CRALBP binds 9-cis- and 11-cis-retinaldehyde, producing complexes with absorption maxima at 405 and 425 nm, respectively. No complexes were detected with 13-cis- and all-trans-retinaldehyde. Following incubation of CRALBP X 11-cis-retinol with an equimolar mixture of 9-, 11-, 13-cis-, and all-trans-retinaldehydes, only 11-cis-retinaldehyde and residual 11-cis-retinol are present on the protein following separation from excess retinoids. A similar result is obtained following incubation of CRALBP X 11-cis-retinol with mixtures of 9- and 11-cis-retinaldehyde ranging in composition from 9:1 to 1:9 (9-cis-:11-cis-,mol/mol). The results indicate that CRALBP X 11-cis-retinol is sufficiently stereoselective in its binding properties to warrant consideration as a component of the mechanism for the generation of 11-cis-retinaldehyde in the dark. 相似文献
13.
9-cis-Retinoic acid activates retinoid X receptors, which serve as heterodimeric partners with other nuclear hormone receptors, yet the enzymology of its physiological generation remains unclear. Here, we report the identification and molecular/enzymatic characterization of a previously unknown member of the short-chain dehydrogenase/reductase family, CRAD3 (cis-retinoid/androgen dehydrogenase, type 3), which catalyzes the first step in 9-cis-retinoic acid biosynthesis, the conversion of 9-cis-retinol into 9-cis-retinal. CRAD3 shares amino acid similarity with other retinoid/steroid short-chain dehydrogenases/reductases: CRAD1, CRAD2, and RDH4. Relative to CRAD1, CRAD3 has greater 9-cis-retinol/all-trans-retinol discrimination and lower efficiency as an androgen dehydrogenase. CRAD3 has apparent efficiency (V/K(m)) for 9-cis-retinol about equivalent to that for CRAD1 and 3 orders of magnitude greater than that for RDH4. (CRAD2 does not recognize 9-cis-retinol as a substrate). CRAD3 contributes to 9-cis-retinoic acid production in intact cells, in conjunction with each of three retinal dehydrogenases that recognize 9-cis-retinal (RALDH1/AHD2, RALDH2, and ALDH12). Liver and kidney, two tissues reportedly with the highest concentrations of 9-cis-retinoids, show the most intense mRNA expression of CRAD3, but expression also occurs in testis, lung, small intestine, heart, and brain. These data are consistent with the participation of CRAD3 in the biogeneration of 9-cis-retinoic acid. 相似文献
14.
Because only retinol and not all-trans-retinoic acid (atRA) can satisfy all of the functions of vitamin A, we have investigated the retinol metabolites in tissues of vitamin A-deficient (VAD) rats responding to a radioactive dose of [20-(3)H]all-trans-retinol. As expected, atRA is the major vitamin A metabolite present in the target tissues of VAD rats given a physiological dose (1 microg) of [20-(3)H]all-trans-retinol (atROL). Both atROL and atRA were detected by high-performance liquid chromatographic (HPLC) analysis of the radioactivity extracted from the liver, kidney, small intestine, lung, spleen, bone, skin, or testis of these animals. Novel retinol metabolites were observed in the aqueous extracts from the testis, lung, and skin. However, these metabolites were detected in very small amounts and were not characterized further. Importantly, neither 9-cis-retinoic acid (9cRA), 9-cis-retinol (9cROL), nor 13-cis-retinoic acid (13cRA) was present in detectable amounts. The amounts of atRA varied in each tissue, ranging from 0.29 +/- 0.05 fmol of RA/g of tissue in the femurs to 12.9 +/- 4.3 fmol of RA/g of tissue in the kidneys. The absence of 9cRA in vivo was not due to degradation of this retinoid during the extraction procedure or HPLC analysis of the extracted radioactivity. As atROL completely fulfills all of the physiological roles of vitamin A, and 9cRA is not detected in any of the tissues analyzed, these results suggest that 9cRA may have no physiological relevance in the rat. 相似文献
15.
Disruption of the 11-cis-retinol dehydrogenase gene leads to accumulation of cis-retinols and cis-retinyl esters 总被引:1,自引:0,他引:1
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Driessen CA Winkens HJ Hoffmann K Kuhlmann LD Janssen BP Van Vugt AH Van Hooser JP Wieringa BE Deutman AF Palczewski K Ruether K Janssen JJ 《Molecular and cellular biology》2000,20(12):4275-4287
To elucidate the possible role of 11-cis-retinol dehydrogenase in the visual cycle and/or 9-cis-retinoic acid biosynthesis, we generated mice carrying a targeted disruption of the 11-cis-retinol dehydrogenase gene. Homozygous 11-cis-retinol dehydrogenase mutants developed normally, including their retinas. There was no appreciable loss of photoreceptors. Recently, mutations in the 11-cis-retinol dehydrogenase gene in humans have been associated with fundus albipunctatus. In 11-cis-retinol dehydrogenase knockout mice, the appearance of the fundus was normal and punctata typical of this human hereditary ocular disease were not present. A second typical symptom associated with this disease is delayed dark adaptation. Homozygous 11-cis-retinol dehydrogenase mutants showed normal rod and cone responses. 11-cis-Retinol dehydrogenase knockout mice were capable of dark adaptation. At bleaching levels under which patients suffering from fundus albipunctatus could be detected unequivocally, 11-cis-retinol dehydrogenase knockout animals displayed normal dark adaptation kinetics. However, at high bleaching levels, delayed dark adaptation in 11-cis-retinol dehydrogenase knockout mice was noticed. Reduced 11-cis-retinol oxidation capacity resulted in 11-cis-retinol/13-cis-retinol and 11-cis-retinyl/13-cis-retinyl ester accumulation. Compared with wild-type mice, a large increase in the 11-cis-retinyl ester concentration was noticed in 11-cis-retinol dehydrogenase knockout mice. In the murine retinal pigment epithelium, there has to be an additional mechanism for the biosynthesis of 11-cis-retinal which partially compensates for the loss of the 11-cis-retinol dehydrogenase activity. 11-cis-Retinyl ester formation is an important part of this adaptation process. Functional consequences of the loss of 11-cis-retinol dehydrogenase activity illustrate important differences in the compensation mechanisms between mice and humans. We furthermore demonstrate that upon 11-cis-retinol accumulation, the 13-cis-retinol concentration also increases. This retinoid is inapplicable to the visual processes, and we therefore speculate that it could be an important catabolic metabolite and its biosynthesis could be part of a process involved in regulating 11-cis-retinol concentrations within the retinal pigment epithelium of 11-cis-retinol dehydrogenase knockout mice. 相似文献
16.
Some members of the human alcohol dehydrogenase (ADH) family possess retinol dehydrogenase activity and may thus function in production of the active nuclear receptor ligand retinoic acid. Many diverse natural forms of retinol exist including all-trans-retinol (vitamin A(1)), 9-cis-retinol, 3,4-didehydroretinol (vitamin A(2)), 4-oxo-retinol, and 4-hydroxy-retinol as well as their respective carboxylic acid derivatives which are active ligands for retinoid receptors. This raises the question of whether ADHs can accommodate all these different retinols and thus participate in the activation of several retinoid ligands. The crystal structures of human ADH1B and ADH4 provide the opportunity to examine their active sites for potential binding to many diverse retinol structures using molecular docking algorithms. The criteria used to score successful docking included achievement of distances of 1.9-2.4 A between the catalytic zinc and the hydroxyl oxygen of retinol and 3.2-3.6 A between C-4 of the coenzyme NAD and C-15 of retinol. These distances are sufficient to enable hydride transfer during the oxidation of an alcohol to an aldehyde. By these criteria, all-trans-retinol, 4-oxo-retinol, and 4-hydroxy-retinol were successfully docked to both ADH1B and ADH4. However, 9-cis-retinol and 3,4-didehydroretinol, which have more restrictive conformations, were successfully docked to only ADH4 which possesses a wider active site than ADH1B and more easily accommodates the C-19 methyl group. Furthermore, docking of all retinols was more favorable in the active site of ADH4 rather than ADH1B as measured by force field and contact scores. These findings suggest that ADH1B has a limited capacity to metabolize retinols, but that ADH4 is well suited to function in the metabolism of many diverse retinols and is predicted to participate in the synthesis of the active ligands all-trans-retinoic acid, 9-cis-retinoic acid, 3, 4-didehydroretinoic acid, 4-oxo-retinoic acid, and 4-hydroxy-retinoic acid. 相似文献
17.
Binding specificities of cellular retinol-binding protein and cellular retinol-binding protein, type II 总被引:1,自引:0,他引:1
Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type ii (CRBP(II] are cytoplasmic proteins that bind trans-retinol as an endogenous ligand. These proteins are structurally similar having greater than 50% sequence homology. Employing fluorescence, absorbance, and competition studies, the ability of pure preparations of CRBP(II) and CRBP to bind various members of the vitamin A family has been examined. In addition to trans-retinol, CRBP(II) was able to form high affinity complexes (K'd less than 5 X 10(-8) M) with 13-cis-retinol, 3-dehydroretinol, and all-trans-retinaldehyde. CRBP bound those retinol isomers with similar affinities, but did not bind trans-retinaldehyde. Neither protein bound retinoic acid nor 9-cis- and 11-cis-retinol. The spectra of 13-cis-retinol and 3-dehydroretinol, when bound, were shifted and displayed fine structure compared to their spectra in organic solution. However, the lambda max and fluorescent yield of a particular ligand were different when bound to CRBP(II) versus CRBP. It appears that CRBP(II) and CRBP bind trans-retinol, 13-cis-retinol, and 3-dehydroretinol in a planar configuration. However, the binding sites of CRBP(II) and CRBP are clearly distinct based on the observed spectral differences of the bound ligands and the observations that only CRBP(II) could bind trans-retinaldehyde. The ability of CRBP(II) to bind trans-retinaldehyde suggests a physiological role for the protein in accepting retinaldehyde generated from the cleavage of beta-carotene in the absorptive cell. 相似文献
18.
19.
Romert A Tuvendal P Tryggvason K Dencker L Eriksson U 《Experimental cell research》2000,256(1):338-345
The murine retinol dehydrogenase RDH4 oxidizes several cis-isomers of retinol into their corresponding aldehydes. We have determined the structure of the murine gene, investigated the temporal and spatial expression of the enzyme, and analyzed the membrane topology of the enzyme. The gene has four translated exons, and several alternatively spliced exons in the 5'-untranslated region were identified. Immunohistochemical analysis showed expression of RDH4 in developing and adult mouse eye, particularly in the retinal pigment epithelium. In nonocular adult tissues, including liver, kidney, lung, and skin, RDH4 expression was widespread. The results suggest that RDH4 may have a dual and tissue-specific role in oxidation of 9-cis- and 11-cis-isomers of retinol into 9-cis-retinal and 11-cis-retinal, respectively. Furthermore, the lumenal orientation of the enzyme domain in the ER suggests that oxidation of both cis-isomers of retinol occurs in the ER. 相似文献
20.
Interaction of 11-cis-retinol dehydrogenase with the chromophore of retinal g protein-coupled receptor opsin 总被引:1,自引:0,他引:1
Vertebrate opsins in both photoreceptors and the retinal pigment epithelium (RPE) have fundamental roles in the visual process. The visual pigments in photoreceptors are bound to 11-cis-retinal and are responsible for the initiation of visual excitation. Retinochrome-like opsins in the RPE are bound to all-trans-retinal and play an important role in chromophore metabolism. The retinal G protein-coupled receptor (RGR) of the RPE and Müller cells is an abundant opsin that generates 11-cis-retinal by stereospecific photoisomerization of its bound all-trans-retinal chromophore. We have analyzed a 32-kDa protein (p32) that co-purifies with bovine RGR from RPE microsomes. The co-purified p32 was identified by mass spectrometric analysis as 11-cis-retinol dehydrogenase (cRDH), and enzymatic assays have confirmed the isolation of an active cRDH. The co-purified cRDH showed marked substrate preference to 11-cis-retinal and preferred NADH rather than NADPH as the cofactor in reduction reactions. cRDH did not react with endogenous all-trans-retinal bound to RGR but reacted specifically with 11-cis-retinal that was generated by photoisomerization after irradiation of RGR. The reduction of 11-cis-retinal to 11-cis-retinol by cRDH enhanced the net photoisomerization of all-trans-retinal bound to RGR. These results indicate that cRDH is involved in the processing of 11-cis-retinal after irradiation of RGR opsin and suggest that cRDH has a novel role in the visual cycle. 相似文献