Microsomal Retinol Dehydrogenase Research Articles

Impact of Sample Matrix on Accuracy of Peptide Quantification: Assessment of Calibrator and Internal Standard Selection and Method Validation.

Protein quantification based on peptides using LC-MS/MS has emerged as a promising method to measure biomarkers, protein drugs, and endogenous proteins. However, the best practices for selection, optimization, and validation of the quantification peptides are not well established, and the influence of different matrices on protein digestion, peptide stability, and MS detection has not been systematically addressed. The aim of this study was to determine how biological matrices affect digestion, detection, and stability of peptides. The microsomal retinol dehydrogenase (RDH11) and cytosolic soluble aldehyde dehydrogenases (ALDH1As) involved in the synthesis of retinoic acid (RA) were chosen as model proteins. Considerable differences in the digestion efficiency, sensitivity, and matrix effects between peptides were observed regardless of the target protein's subcellular localization. The precision and accuracy of the quantification of RDH11 and ALDH1A were affected by the choice of calibration and internal standards. The final method using recombinant protein calibrators and stable isotope labeled (SIL) peptide internal standards was validated for human liver. The results demonstrate that different sample matrices have peptide, time, and matrix specific effects on protein digestion and absolute quantification.

Disease-associated variants of microsomal retinol dehydrogenase 12 (RDH12) are degraded at mutant-specific rates

Mutations in retinol dehydrogenase 12 (RDH12) cause severe retinal degeneration. However, some of the disease-associated RDH12 mutants retain significant catalytic activity, indicating the existence of additional pathophysiological mechanisms. This study demonstrates that the catalytically active T49M and I51N mutants undergo accelerated degradation, which results in their reduced cellular levels. Inhibition of proteasome leads to significant accumulation of ubiquitylated T49M and I51N. Furthermore, the degree of ubiquitylation strongly correlates with the half-lives of the proteins. These results suggest that the accelerated degradation of RDH12 mutants by the ubiquitin-proteasome system contributes to the pathophysiology and phenotypic variability associated with mutations in the RDH12 gene. Structured summaryMINT-7383581, MINT-7383598: RDH12 (uniprotkb:Q96NR8) physically interacts (MI:0915) with ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)

The regulation of retinoic acid formation.

Rat liver microsomal as well as cytosolic retinol dehydrogenases from cellular retinal-binding protein (CRBP)-retinal from CRBP-retinol. A cytosolic dehydrogenase transforms retinal into retinoic acid. Apo-CRBP and ethanol inhibit the cytosolic but not the microsomal retinol dehydrogenase, the latter being the primary enzyme in the rate-limiting step in retinoic acid synthesis.

Medium- and short-chain dehydrogenase/reductase gene and protein families : Medium-chain and short-chain dehydrogenases/reductases in retinoid metabolism.

Retinoic acid (RA), the most active retinoid, is synthesized in two steps from retinol. The first step, oxidation of retinol to retinaldehyde, is catalyzed by cytosolic alcohol dehydrogenases (ADHs) of the medium-chain dehydrogenase/reductase (MDR) superfamily and microsomal retinol dehydrogenases (RDHs) of the short-chain dehydrogenase/reductase (SDR) superfamily. The second step, oxidation of retinaldehyde to RA, is catalyzed by several aldehyde dehydrogenases. ADH1 and ADH2 are the major MDR enzymes in liver retinol detoxification, while ADH3 (less active) and ADH4 (most active) participate in RA generation in tissues. Several NAD(+)- and NADP(+)-dependent SDRs are retinoid active. Their in vivo contribution has been demonstrated in the visual cycle (RDH5, RDH12), adult retinoid homeostasis (RDH1) and embryogenesis (RDH10). K(m) values for most retinoid-active ADHs and RDHs are close to 1 microM or lower, suggesting that they participate physiologically in retinol/retinaldehyde interconversion. Probably none of these enzymes uses retinoids bound to cellular retinol-binding protein, but only free retinoids. The large number of enzymes involved in the two directions of this step, also including aldo-keto reductases, suggests that retinaldehyde levels are strictly regulated.

Analysis of the NADH‐dependent retinaldehyde reductase activity of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution of the retinol dehydrogenase family

In vertebrates, multiple microsomal retinol dehydrogenases are involved in reversible retinol/retinal interconversion, thereby controlling retinoid metabolism and retinoic acid availability. The physiologic functions of these enzymes are not, however, fully understood, as each vertebrate form has several, usually overlapping, biochemical roles. Within this context, amphioxus, a group of chordates that are simpler, at both the functional and genomic levels, than vertebrates, provides a suitable evolutionary model for comparative studies of retinol dehydrogenase enzymes. In a previous study, we identified two amphioxus enzymes, Branchiostoma floridae retinol dehydrogenase 1 and retinol dehydrogenase 2, both candidates to be the cephalochordate orthologs of the vertebrate retinol dehydrogenase enzymes. We have now proceeded to characterize these amphioxus enzymes. Kinetic studies have revealed that retinol dehydrogenase 1 and retinol dehydrogenase 2 are microsomal proteins that catalyze the reduction of all-trans-retinaldehyde using NADH as cofactor, a remarkable combination of substrate and cofactor preferences. Moreover, evolutionary analysis, including the amphioxus sequences, indicates that Rdh genes were extensively duplicated after cephalochordate divergence, leading to the gene cluster organization found in several mammalian species. Overall, our data provide an evolutionary reference with which to better understand the origin, activity and evolution of retinol dehydrogenase enzymes.

The C-terminal region of cis-retinol/androgen dehydrogenase 1 (CRAD1) confers ER localization and in vivo enzymatic function

Retinoic acid is generated from retinol (vitamin A) by the sequential actions of two different classes of enzymes, retinol dehydrogenases and retinal dehydrogenases. Several enzymes implicated in this process have been identified and characterized in vitro. However, our understanding of the cell biological function and regulation of this process is limited. To get further knowledge regarding the regulation of RA biosynthesis, we have determined possible regulatory mechanisms at the transcriptional and post-transcriptional levels for the prototypic microsomal retinol dehydrogenase cis-retinol/androgen dehydrogenase 1 (CRAD1). We note that the expression and stability of the enzyme are only moderately controlled by the retinoid status. Instead, we find that the cytosolic tail dramatically affects the activity of the enzyme, and we have mapped the structural elements required for ER retention and in vivo functional activity, respectively. Although inactive tail-deletion mutants display an abnormal subcellular localization, restoration of ER localization per se is not sufficient for enzymatic activity suggesting that additional trans-acting components interacting with, or modifying, the cytosolic tail are required for controlling the activity of the enzyme in vivo.

Oxidative 3α-hydroxysteroid dehydrogenase activity of human type 10 17β-hydroxysteroid dehydrogenase

In vitro enzyme assays have demonstrated that human type 10 17β-hydroxysteroid dehydrogenase (17β-HSD10) catalyzes the oxidation of 5α-androstane-3α,17β-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17β-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17β-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17β-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17β-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3α-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17β-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.

Expression pattern and biochemical characteristics of a major epidermal retinol dehydrogenase

The biological functions of vitamin A in the epidermis are mediated by all- trans retinoic acid, which is biosynthesized from retinol in two oxidative reactions. The first step involves enzymatic conversion of retinol to retinaldehyde. The physiological significance and relative contributions of the various retinol dehydrogenases to the oxidation of retinol in epidermal cells remain unclear. We report the characterization of a retinol dehydrogenase/reductase of the SDR superfamily, hRoDH-E2, which is abundantly expressed in the epidermis, epidermal appendages and in cultured epidermal keratinocytes. Both in live keratinocytes and in isolated keratinocyte microsomes, where the enzyme normally localizes, hRoDH-E2 functions as a bona fide retinol dehydrogenase. In the prevailing oxidative reaction it recognizes both free- and CRBP-bound retinol, and shows preference toward NADP as a co-substrate. In comparison, hRoDH-E2 retinol dehydrogenase activity in the simple epithelial HEK 293 cells is much lower and in CHO cells is non-existent. hRoDH-E2 transcripts are distributed throughout the epidermal layers but are more abundant in the basal cells. In contrast, the protein is detected predominantly in the basal and the most differentiated living layers. Its synthesis is negatively regulated by retinoic acid. The biochemical properties and the differential expression of hRoDH-E2 in the strata where retinoic acid signaling is critical for epidermal homeostasis support a conclusion that hRoDH-E2 bears the characteristics of the major microsomal retinol dehydrogenase activity in the epidermal keratinocytes in physiological circumstances.

Biosynthesis of 9-cis-Retinoic Acidin Vivo: THE ROLES OF DIFFERENT RETINOL DEHYDROGENASES AND A STRUCTURE-ACTIVITY ANALYSIS OF MICROSOMAL RETINOL DEHYDROGENASES

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.

Characterization of a microsomal retinol dehydrogenase gene from amphioxus: retinoid metabolism before vertebrates

Amphioxus, a member of the subphylum Cephalochordata, is thought to be the closest living relative to vertebrates. Although these animals have a vertebrate-like response to retinoic acid, the pathway of retinoid metabolism remains unknown. Two different enzyme systems — the short chain dehydrogenase/reductases and the cytosolic medium-chain alcohol dehydrogenases (ADHs) — have been postulated in vertebrates. Nevertheless, recent data show that the vertebrate-ADH1 and ADH4 retinol-active forms originated after the divergence of cephalochordates and vertebrates. Moreover, no data has been gathered in support of medium-chain retinol active forms in amphioxus. Then, if the cytosolic ADH system is absent and these animals use retinol, the microsomal retinol dehydrogenases could be involved in retinol oxidation. We have identified the genomic region and cDNA of an amphioxus Rdh gene as a preliminary step for functional characterization. Besides, phylogenetic analysis supports the ancestral position of amphioxus Rdh in relation to the vertebrate forms.

Alcohol and retinoid metabolism

See article on page 825 The complex interactions between the metabolism of retinoids and ethanol have been reported for a long time. Clinically, chronic ethanol consumption leads to vitamin A...

Cloning and Characterization of the CYP2D1-Binding Protein, Retinol Dehydrogenase

A CYP2D1-binding protein, 29 k-protein (p29), has been isolated and its N-terminal amino acid sequence has been reported (Ohishiet al. (1993)Biochim. Biophys. Acta1158, 227–236). In this study, p29 cDNA was isolated by PCR with oligonucleotide probes designed from the N-terminal amino acid sequence and p29 was found to be a microsomal retinol dehydrogenase, a member of the short-chain alcohol dehydrogenase family which metabolize hydroxysteroids and prostaglandins. CYP2D1 and p29 were expressed inSaccharomyces cerevisiaeto characterize these proteins. CYP2D1 had an absorption maximum at 448 nm in a CO-reduced form. Expressed p29 in yeast cells was detected with anti-p29 antibody. Solubilized CYP2D1 and p29 from yeast microsomes were mixed and applied to an anti-CYP2D1 antibody-binding column. Both proteins were retained in the column and eluted with glycine buffer (pH 2.8). However, when applied alone, p29 was not retained in the column. The findings indicated that CYP2D1 bound tightly with p29. Catalytic activities of p29 expressed in yeast were investigated. p29 had retinal reductase activity in the presence of NADPH. Addition of CYP2D1 and NADPH-P450 reductase increased the retinal reductase activity of p29. These findings suggest that the complex of CYP2D1, p29, and NADPH-P450 reductase has an important role in the metabolism of retinoids.

Enzymatic characteristics of retinal dehydrogenase type I expressed in Escherichia coli

We expressed RalDH(I) in Escherichia coli and have shown that it functions in vitro with the complex CRBP-retinal (cellular retinol-binding protein) as substrate, either generated in situ from the complex CRBP-retinol and microsomal retinol dehydrogenases or provided directly as CRBP-retinal. Recombinant RalDH(I) had kinetic constants with CRBP-retinal of: Hill coefficient 1.8; K 0.5 0.8 μM; and V m 1.5 nmol/min/mg of protein at 25°C. Apo-CRBP inhibited the reaction with CRBP-retinal with an IC 50 of 1.4 μM. Citral inhibited RalDH(I) with an IC 50 of ∼1 μM compared to an IC 50 of ∼12 μM for RalDH(II), but did not serve as substrate for RalDH(I). RalDH(I) did not catalyze efficiently the dehydrogenation of acetaldehyde, but showed higher V max/ K m values for hexanal, octanal, decanal and benzaldehyde than for either propanal or retinal. These data extend the characterization of RalDH(I), show that apo-CRBP competes with holo-CRBP as substrate for RalDH(I), and expand insight into the pathways of retinoic acid biogenesis from the most abundant substrates in vivo, retinoid-liganded CRBP.

Cloning of a cDNA Encoding an Aldehyde Dehydrogenase and Its Expression in Escherichia coli: RECOGNITION OF RETINAL AS SUBSTRATE

The biosynthesis of the hormone retinoic acid from retinol (vitamin A) involves two sequential steps, catalyzed by retinol dehydrogenases and retinal dehydrogenases, respectively. This report describes the cloning of a cDNA encoding a heretofore unknown aldehyde dehydrogenase from a rat testis library and its expression in Escherichia coli. This enzyme has been designated retinal dehydrogenase, type II, RalDH(II). The deduced amino acid sequence of RalDH(II) had the highest identity with mammalian aldehyde dehydrogenases that feature low Km values (microM) for retinal: human ALDH1 (72.2%), rat retinal dehydrogenase, type I (71.5%), bovine retina (72.7%), and mouse AHD-2 (71.5%). RalDH(II) expressed in E. coli recognizes as substrates free retinal, with a Km of approximately 0.7 microM, and cellular retinol-binding protein-bound retinal, with a Km of approximately 0.2 microM. RalDH(II) also can utilize as substrate retinal generated in situ by microsomal retinol dehydrogenases, from the physiologically most abundant substrate: retinol bound to cellular retinol-binding protein. Rat testis expresses RalDH(II) mRNA most abundantly, followed by (relative to testis): lung (6.7%), brain (6.3%), heart (5.2%), liver (4.4%), and kidney (2.7%). RalDH(II) does not recognize citral, benzaldehyde, acetaldehyde, and propanal efficiently as substrates, but does metabolize octanal and decanal efficiently. These data support a function for RalDH(II) in the pathway of retinoic acid biogenesis.

Cellular Retinol-binding Protein-supported Retinoic Acid Synthesis: RELATIVE ROLES OF MICROSOMES AND CYTOSOL

This study shows that microsomal retinol dehydrogenases, versus cytosolic retinol dehydrogenases, provide the quantitatively major share of retinal for retinoic acid (RA) biogenesis in rat tissues from the predominant substrate available physiologically, holo-cellular retinol-binding protein, type I (CRBP). With holo-CRBP as substrate in the absence of apo-CRBP microsomal retinol dehydrogenases have the higher specific activity and capacity to generate retinal used for RA synthesis by cytosolic retinal dehydrogenases. In the presence of apo-CRBP, a potent inhibitor of cytosolic retinol dehydrogenases (IC50 = approximately 1 microM), liver microsomes provide 93% of the total retinal synthesized in a combination of microsomes and cytosol. Cytosolic retinol dehydrogenase(s) and the isozymes of alcohol dehydrogenase expressed in rat liver had distinct enzymatic properties; yet ethanol inhibited cytosolic retinol dehydrogenase(s) (IC50 = 20 microM) while stimulating RA synthesis in a combination of microsomes and cytosol. At least two discrete forms of cytosolic retinol dehydrogenase were observed: NAD- and NADP-dependent forms. Multiple retinal dehydrogenases also were observed and were inhibited partially by apo-CRBP. These results provide new insights into pathways of RA biogenesis and provide further evidence that they consist of multiple enzymes that recognize both liganded and nonliganded states of CRBP.

Involvement of alcohol dehydrogenase, short-chain dehydrogenase/reductase, aldehyde dehydrogenase, and cytochrome P450 in the control of retinoid signaling by activation of retinoic acid synthesis.

The effects of vitamin A (retinol) on growth and development are mediated by the active metabolite retinoic acid which controls a nuclear receptor signaling pathway. While elegant work on the retinoic acid receptor family has focused attention upon how the receptor controls this pathway, there now exists a relatively large gap in our understanding of how retinol is activated to form the ligand. During vertebrate embryogenesis and in adult organs retinoic acid is detected in a distinct spatiotemporal pattern, suggesting that it is produced from retinol in a regulated fashion. Enzymes involved in retinol and retinal metabolism are likely candidates for regulators of tissue retinoic acid levels. Members of the alcohol dehydrogenase and short-chain dehydrogenase/reductase enzyme families catalyze the reversible interconversion of retinol and retinal, the rate-limiting step, whereas members of the aldehyde dehydrogenase and cytochrome P450 enzyme families catalyze the irreversible oxidation of retinal to retinoic acid. The identification of enzymes likely to catalyze retinol oxidation in vivo has been particularly controversial, and this is made even more difficult by the reversible nature of this reaction. Taking into account enzymatic properties and coenzyme preferences, a case can be made that class IV alcohol dehydrogenase catalyzes retinol oxidation to provide retinal for retinoic acid synthesis, whereas microsomal retinol dehydrogenase (a short-chain dehydrogenase/reductase) catalyzes the reduction of retinal to retinol to promote retinoid storage. Further studies on these enzyme families will allow this layer of control in the retinoid signaling pathway to be understood.

Effects of Sulfhydryl Reagents, Retinoids, and Solubilization on the Activity of Microsomal Retinol Dehydrogenase

A microsomal retinol dehydrogenase (RoDH) that recognizes hole-cellular retinol binding protein (CRBP) as substrate is inhibited by phenylarsine oxide (IC 50 = 3 μM) in the presence of 10 mM cysteine. Inhibition was reversible with dithiothreitol, indicating that two cysteine residues in close proximity are essential for RoDH activity. Bromophenylarsine oxide was an irreversible inhibitor (IC 50 = 0.2 μM)) suggesting that a nucleophile lies close to the two cysteine residues. N-Ethylmaleimide inhibited reactions supported by holo-CRBP, but not from free retinol, suggesting that it obstructed holo-CRBP access to RoDH without affecting the catalytic site. RoDH activity was similar in microsomes from vitamin A-sufficient or vitamin A-deficient rats and was not inhibited by relatively high concentrations (5 μM) of all- trans-retinoic acid, holo-cellular retinoic acid binding protein, ape-cellular retinoic acid binding protein, or 9- cis-retinoic acid. Triton X-100 stimulated RoDH activity eightfold at a detergent to protein ratio of 0.25 to 1 (w/w). A combination of Tween 80, Brij 92, and Triton X-100 (2:1:2) stimulated RoDH activity eightfold at a detergent to protein ratio of 2.5 to 1 (w/w). Detergent-solubilized RoDH, partially purified through a PAO-Sepharose resin, preferred NADP(H) as cofactor, had a K m for retinal synthesis from holo-CRBP of 0.6 μM ( V max = 115 pmol/min/mg protein) and a K m for reduction of retinal bound to CRBP of 0.6 μM ( V max = 613 pmol/min/mg protein). This work provides further insight into microsomal RoDH and strengthens the evidence of an interaction between RoDH and holo-CRBP.

Enzymes and binding proteins affecting retinoic acid concentrations.

Free retinoids suffer promiscuous metabolism in vitro. Diverse enzymes are expressed in several subcellular fractions that are capable of converting free retinol (retinol not sequestered with specific binding proteins) into retinal or retinoic acid. If this were to occur in vivo, regulating the temporal-spatial concentrations of functionally-active retinoids, such as RA (retinoic acid), would be enigmatic. In vivo, however, retinoids occur bound to high-affinity, high-specificity binding proteins, including cellular retinol-binding protein, type I (CRBP) and cellular retinoic acid-binding protein, type I (CRABP). These binding proteins, members of the superfamily of lipid binding proteins, are expressed in concentrations that exceed those of their ligands. Considerable data favor a model pathway of RA biosynthesis and metabolism consisting of enzymes that recognize CRBP (apo and holo) and holo-CRABP as substrates and/or affecters of activity. This would restrict retinoid access to enzymes that recognize the appropriate binding protein, imparting specificity to RA homeostasis; preventing, e.g. opportunistic RA synthesis by alcohol dehydrogenases with broad substrate tolerances. An NADP-dependent microsomal retinol dehydrogenase (RDH) catalyzes the first reaction in this pathway. RDH recognizes CRBP as substrate by the dual criteria of enzyme kinetics and chemical crosslinking. A cDNA of RDH has been cloned, expressed and characterized as a short-chain alcohol dehydrogenase. Retinal generated in microsomes from holo-CRBP by RDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase (RalDH). RalDH has been purified, characterized with respect to substrate specificity, and its cDNA has been cloned. CRABP is also important to modulating the steady-state concentrations of RA, through sequestering RA and facilitating its metabolism, because the complex CRABP/RA acts as a low Km substrate.

Characterization of a microsomal retinol dehydrogenase: a short-chain alcohol dehydrogenase with integral and peripheral membrane forms that interacts with holo-CRBP (type I).

Integral and peripheral forms of a microsomal retinol dehydrogenase (RoDH) have been distinguished in rat liver through differences in solubility, behavior toward affinity resins, and phase partitioning with Triton X-114. Despite physical differences, polyclonal antibodies raised against integral RoDH recognized peripheral RoDH. No obvious differences were observed in substrate specificity between the two forms. Integral and peripheral RoDH catalyzed retinal synthesis from all-trans-retinol bound to cellular retinol-binding protein, type I (CRBP), with similar Km values of 0.6 and 0.4 microM, respectively. Both also discriminated against CRBP-bound all-trans-3,4-didehydroretinol and against 9-cis-retinol. Phenylarsine oxide inhibited both forms with IC50 values of 5 microM (integral) and 15 microM (peripheral). The more stable peripheral form has been reduced to two major polypeptides that migrate as 34 and 54 kDa bands on SDS-PAGE. The active site of this form has been associated with the 34 kDa polypeptide by covalent binding and inactivation with phenylarsine oxide and by cross-linking to holo-CRBP. Cross-linking required cofactor and was maximum with NADP, consistent with the ordered bisubstrate reaction mechanism of an NADP-supported dehydrogenase. The 34 kDa polypeptide has a subunit molecular weight and other attributes typical of short-chain alcohol dehydrogenases (SCAD) including the highly-conserved SCAD sequence WXLVNNAG, Zn2+ independence; inhibition by carbenoxolone (IC50 = 55 microM), and insensitivity to inhibition by ethanol and 4-methylpyrazole. Tight association between the 34 and 54 kDa polypeptides was demonstrated by their coelution through several columns and the precipitation of RoDH activity with either anti-34 kDa or anti-54 kDa antisera. Because SCAD normally occur as homomultimers, however, the 54 kDa polypeptide is not likely to be a subunit of the peripheral form. This work provides new evidence that the retinol-CRBP "cassette" serves as a substrate for a microsomal RoDH and further characterizes the RoDH.

Cloning of a cDNA for Liver Microsomal Retinol Dehydrogenase: A TISSUE-SPECIFIC, SHORT-CHAIN ALCOHOL DEHYDROGENASE

Retinoic acid, a hormone biosynthesized from retinol, controls numerous biological systems by regulating eukaryotic gene expression from conception through death. This work reports the cloning and expression of a liver cDNA encoding a microsomal retinol dehydrogenase (RoDH), which catalyzes the primary and rate-limiting step in retinoic acid synthesis. The predicted amino acid sequence and biochemical data obtained from the recombinant enzyme verify it as a short-chain alcohol dehydrogenase. Like microsomal RoDH, the recombinant enzyme recognized as substrate retinol bound to cellular retinol-binding protein, had higher activity with NADP rather than NAD, was stimulated by ethanol or phosphatidylcholine, was not inhibited by 4-methylpyrazole, was inhibited by phenylarsine oxide and carbenoxolone and localized to microsomes. RoDH recognized the physiological form of retinol, holocellular retinol-binding protein, with a Km of 0.9 microM, a value lower than the approximately 5 microM concentration of holocellular retinol binding protein in liver. Northern and Western blot analyses revealed RoDH expression only in rat liver, despite enzymatic activity in liver, brain, kidney, lung, and testes. These data suggest that tissue-specific isozyme(s) of short chain alcohol dehydrogenases catalyze the first step in retinoic acid biogenesis and further strengthen the evidence that the "cassette" of retinol bound to cellular retinol-binding protein serves as a physiological substrate.