[Show abstract][Hide abstract] ABSTRACT: 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 (IC = 1 μM), 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) (IC = 20 μM) 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.
[Show abstract][Hide abstract] ABSTRACT: A microsomal retinol dehydrogenase (RoDH) that recognizes holo-cellular retinol binding protein (CRBP) as substrate is inhibited by phenylarsine oxide (IC50 = 3 microM) 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 (IC50 = 0.2 microM), 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 microM) of all-trans-retinoic acid, holo-cellular retinoic acid binding protein, apo-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 Km for retinal synthesis from holo-CRBP of 0.6 microM (Vmax = 115 pmol/min/mg protein) and a Km for reduction of retinal bound to CRBP of 0.6 microM (Vmax = 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.
No preview · Article · Sep 1995 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jul 1995 · The Journal of Steroid Biochemistry and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Mar 1995 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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 K of 0.9 μM, a value lower than the 5 μM 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.
Preview · Article · Feb 1995 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Apo-cellular retinol-binding protein (apoCRBP) activated the hydrolysis of endogenous retinyl esters in rat liver microsomes by a cholate independent retinyl ester hydrolase. A Michaelis-Menten relationship was observed between the apoCRBP concentration and the rate of retinol formation, with half-maximum stimulation at 2.6 +/- 0.6 microM (mean +/- S.D., n = 5). Two other retinol-binding proteins, bovine serum albumin and beta-lactoglobulin, acceptors for the rapid and spontaneous hydration of retinol from membranes, had no effect up to 90 microM. These data suggest activation of the hydrolase by apoCRBP directly, rather than by facilitating removal of retinol from membranes. The hydrolase responding was the cholate-independent/cholate-inhibited retinyl ester hydrolase as shown by: 60% inhibition of the apoCRBP effect by 3 mM cholate; apoCRBP enhancement of retinyl ester hydrolysis in liver microsomes that had no detectable cholate-enhanced activity; inhibition of cholate-dependent, but not apoCRBP-stimulated retinyl ester hydrolysis by rabbit anti-rat cholesteryl esterase. Compared to the rate (mean +/- S.D. of [n] different preparations) supported by 5 microM apoCRBP in liver microsomes of 6.7 +/- 3.7 pmol/min/mg protein , microsomes from rat lung, kidney, and testes had endogenous retinyl ester hydrolysis rates of 1.8 +/- 0.3 , 0.5 +/- 0.2 , and 0.3 +/- 0.2  pmol/min/mg protein, respectively. N-Ethylmaleimide and N-tosyl-L-phenylalanine chloromethyl ketone were potent inhibitors of apoCRBP-stimulated hydrolysis with IC50 values of 0.25 and 0.15 mM, respectively, but phenylmethylsulfonyl fluoride and diisopropyl-fluorophosphate were less effective with IC50 values of 1 mM, indicating the importance of imidazole and sulfhydryl groups to the activity. These data provide evidence of a physiological role for the cholate-independent hydrolase in retinoid metabolism and suggest that apoCRBP is a signal for retinyl ester mobilization.
Preview · Article · Dec 1991 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Holocellular retinol binding protein (holo-CRBP) was substrate for retinal synthesis at physiological pH with microsomes prepared from rat liver, kidney, lung, and testes. Four observations indicated that retinal synthesis was supported by holo-CRBP directly, rather than by the unbound retinol in equilibrium with CRBP. First, the rate of retinal synthesis with holo-CRBP exceeded the rate that was observed from the concentration of unbound retinol in equilibrium with CRBP. Second, NADP was the preferred cofactor only with holo-CRBP, supporting a rate about 3-fold greater than that of NAD. In contrast, with unbound retinol as substrate, similar rates of retinal formation were supported by either NAD or NADP. Third, the rate of retinal synthesis was not related to the decrease in the concentration of unbound retinol in equilibrium with holo-CRBP caused by increasing the concentration of apo-CRBP. Fourth, the rate of retinal synthesis increased with increases in the concentration of holo-CRBP as a fixed concentration of unbound retinol was maintained. This was achieved by increasing both apo-CRBP and holo-CRBP, but keeping constant the ratio apo-CRBP/holo-CRBP. Retinal formation from holo-CRBP displayed typical Michaelis-Menten kinetics with a Km about 1.6 microM, less than the physiological retinal concentration of 4-10 microM in the livers of rats fed diets with recommended vitamin A levels. The Vmax for retinal formation from holo-CRBP was 14-17 pmol min-1 (mg of protein)-1, a rate sufficiently high to generate adequate retinal to contribute significantly to retinoic acid synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
[Show abstract][Hide abstract] ABSTRACT: This article will address recent work on the physiological occurrence, biogenesis and metabolism of retinoic acid and summarize the data that retinoic acid is synthesized in situ in multiple tissues and cell types via enzymes or enzyme complexes that are distinct from the alcohol dehydrogenases. There is now considerable evidence that retinoic acid is an activated metabolite of retinol that supports the systemic functions of vitamin A in vivo. Many studies in vitro, for example, have shown that retinoic acid is the most potent naturally-occurring retinoid with an ED-50 in the range of 1 pM to 10 nM, depending on the assay system. This is below the tissue concentrations of retinoic acid which range from approximately 20-600 nM. Retinoic acid synthesis from retinol in the dog kidney cell line MDCK maintained in serum-free medium is inhibited by the prostanoid, PGE, and the phorbol ester, TPA. In tissues, one pathway of retinoic acid synthesis begins with apo-CRBP stimulating retinyl ester hydrolysis by a microsomal, cholate-independent retinyl ester hydrolase to form holo-CRBP. The holo-CRBP itself is used as substrate by an NADP-dependent, microsomal retinol dehydrogenase to generate retinal, which is converted into retinoic acid by a cytosolic NAD-dependent retinal dehydrogenase. Therefore, cellular retinol-binding protein (CRBP) apparently has at least 2 functions in retinoic acid synthesis: the apo form stimulates retinol mobilization from retinyl ester stores; the holo form delivers the retinol via direct transfer to dehydrogenase(s). Retinoic acid is converted into a mixture of at least 4 metabolites by testes microsomes which migrate closely on reverse-phase HPLC with 4-hydroxyretinoic acid, and may be mistaken for either 4-hydroxy or 4-oxo-retinoic acid. More rigorous analysis, however, shows that only one of them is 4-hydroxyretinoic acid, and another is 18-hydroxyretinoic acid. Two others remain unidentified. These metabolites are also formed in the presence of excess cellular retinoic acid-binding protein (CRABP), which increases the elimination half-life of retinoic acid, but does not prevent retinoic acid catabolism, suggesting that holo-CRABP may be a substrate for retinoic acid catabolism that modulates the steady-state concentrations of retinoic acid. Thus, both retinoid binding proteins, CRBP and CRABP, may each have direct roles as substrate in the biosynthesis and metabolism of retinoic acid, respectively.
Full-text · Article · Feb 1991 · Biomedecine [?] Pharmacotherapy