Specificity of cellular retinol-binding protein in the transfer of retinol to nuclei and chromatin

We have reported previously that cellular retinol-binding protein (CRBP) is able to transfer retinol to specific binding sites in nuclei and chromatin. In this report, we have examined the specificity of the interaction of the protein moiety of retinol-CRBP (R-CRBP) with chromatin and nuclei in the transfer process. We first determined the ability of apo-CRBP, apo-serum retinol-binding protein (RBP), and apo beta-lactoglobulin (BLG), all capable of retinol binding, to compete with R-CRBP in the transfer of retinol to chromatin and nuclei. Apo-CRBP was an effective competitor but apo-RBP and apo-BLG showed no competitive ability. On the other hand, cellular retinol-binding protein type II (CRBP(II], whose amino acid sequence shows a considerable similarity to CRBP, did compete for the transfer of retinol from the R-CRBP complex, but less effectively than CRBP. These results demonstrate that the interaction of the protein moiety of the R-CRBP complex with nuclei and chromatin is quite specific.     

Acyl-CoA-independent esterification of retinol bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine

Cellular retinol-binding protein (type II) (CRBP(II)), a newly described retinol-binding protein, is present in the small intestinal absorptive cell at high levels. Retinol (vitamin A alcohol) presented as a complex with CRBP(II) was found here to be esterified by microsomal preparations from rat small intestinal mucosa. The esterification observed utilized an endogenous acyl donor(s) and produced retinyl esters containing linoleate, oleate, palmitate, and stearate in a proportion quite similar to that previously reported for retinyl esters in lymph and isolated chylomicrons of rat. No dependence on endogenous or exogenous acyl-CoA could be demonstrated. The apparent Km for retinol-CRBP(II) in the reaction with endogenous acyl donor was 2.4 X 10(-7) M. Retinol presented as a complex with CRBP(II) was esterified more than retinol presented as a complex with cellular retinol-binding protein or retinol-binding protein, two other proteins known to bind retinol in vivo, but about the same as retinol presented bound to bovine serum albumin or beta-lactoglobulin. The ability of protein-bound retinol to be esterified was related to accessibility of the hydroxyl group, as judged by the ability of alcohol dehydrogenase to oxidize the bound retinol. However, whereas retinol bound to CRBP(II) was unavailable for esterification in any acyl-CoA-dependent reaction, retinol bound to bovine serum albumin was rapidly esterified in a reaction utilizing exogenous acyl-CoA. The results suggest that one of the functions of CRBP(II) is to accept retinol after it is absorbed or generated from carotenes in the small intestine and present it to the appropriate esterifying enzyme.     

Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein

We have investigated the esterification by liver membranes of retinol bound to cellular retinol-binding protein (CRBP). When CRBP carrying [3H]retinol as its ligand was purified from rat liver cytosol and incubated with rat liver microsomes, a significant fraction of the [3H]retinol was converted to [3H]retinyl ester. Esterification of the CRBP-bound [3H]retinol, which was maximal at pH 6-7, did not require the addition of an exogenous fatty acyl group. Indeed, when additional palmitoyl-CoA or coenzyme A was provided, the rate of esterification increased either very slightly or not at all. The esterification reaction had a Km for [3H]retinol-CRBP of 4 +/- 0.6 microM and a maximum velocity of 145 +/- 52 pmol/min/mg of microsomal protein (n = 4). The major products were retinyl palmitate/oleate and retinyl stearate in a ratio of approximately 2 to 1 over a range of [3H]retinol-CRBP concentrations from 1 to 8 microM. The addition of progesterone, a known inhibitor of the acyl-CoA:retinol acyltransferase reaction, consistently increased the rate of retinyl ester formation when [3H]retinol was delivered bound to CRBP. These experiments indicate that retinol presented to liver microsomal membranes by CRBP can be converted to retinyl ester and that this process, in contrast to the esterification of dispersed retinol, is independent of the addition of an activated fatty acid and produces a pattern of retinyl ester species similar to that observed in intact liver. A possible role of phospholipids as endogenous acyl donors in the esterification of retinol bound to CRBP is supported by our observations that depletion of microsomal phospholipid with phospholipase A2 prior to addition of retinol-CRBP decreased the retinol-esterifying activity almost 50%. Conversely, incubating microsomes with a lipid-generating system containing choline, CDP-choline, glycerol 3-phosphate, and an acyl-CoA-generating system prior to addition of retinol-CRBP increased retinol esterification significantly as compared to buffer-treated controls.     

Esterification of retinol in rat liver. Possible participation by cellular retinol-binding protein and cellular retinol-binding protein II

Retinol bound to cellular retinol-binding protein (CRBP) was available for esterification by liver microsomes in the absence of exogenous acyl donors. Moreover, exogenous acyl-CoA gave little or no stimulation of ester production over what was observed with the endogenous acyl donor. In contrast, unbound retinol was esterified in an acyl-CoA-dependent reaction. The presence of two different enzyme activities, acyl-CoA-dependent and -independent, was demonstrated by differential sensitivities to several enzyme inhibitors. The enzyme reaction with retinol-CRBP and endogenous acyl donor produced retinyl esters normally found in vivo in liver. In addition, rates of esterification with this system were sufficient to maintain liver stores. Liver also contains cellular retinol-binding protein, type II (CRBP(II] during the perinatal period. Radioimmunoassay revealed highest levels of CRBP(II) in liver 3-4 days after birth. Examination of retinol esterification by microsomes from the liver of 3-day-old rats revealed a retinyl ester synthase activity with lower Km and higher Vmax than that found in the adult. The activity could use either retinol-CRBP or retinol-CRBP(II) and an endogenous acyl donor. The microsomes from 3-day-old liver had greater esterifying ability than microsomes from adult liver, perhaps due to the presence of two retinyl ester synthase enzymes.     

Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins.

Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in Km values (0.63 and 0.44 microM, respectively) similar to those obtained for esterification of retinol-CRBP (0.20 and 0.78 microM, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32 microM, respectively). Because Kd values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10-(-10) M, these similar Km values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a KI (0.21 microM) lower than the Km for CRBP-retinol (0.78 microM). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP(II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoli cells accumulated [3H]retinol in a time- and temperature-dependent manner. At 32 degrees C, the rate of retinol accumulation was biphasic. Accumulation was linear for approximately 1 h, but then accumulation continued at a linear but decreased rate for 23 h. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/micrograms of cellular DNA. This amount of retinol was approximately equal to the cellular content of cellular retinol-binding protein (CRBP). Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [3H]retinol-RBP for [3H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [3H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 microM, suggesting that any change in the normal circulating retinol-RBP level (approximately 2 microM) would directly affect the rate of retinol accumulation. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. In addition, energy inhibitors and lysosomal poisons had no effect on [3H]retinol accumulation, indicating that RBP delivery of retinol to Sertoli cells did not occur by endocytosis of the retinol-RBP complex. Competition studies indicated, however, that protein recognition is important in the retinol uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two homologous rat cellular retinol-binding proteins differ in local conformational flexibility

Cellular retinol-binding protein I (CRBP I) and cellular retinol-binding protein II (CRBP II) are closely homologous proteins that play distinct roles in the maintenance of vitamin A homeostasis. The solution structure and dynamics of CRBP I and CRBP II were compared by multidimensional NMR techniques. These studies indicated that differences in the mean backbone structures of CRBP I and CRBP II were localized primarily to the alphaII helix. Intraligand NOE cross-peaks were detected for the hydroxyl proton in the NOESY spectrum of CRBP I-bound retinol, but not for CRBP II-bound retinol, indicating that the conformational dynamics of retinol binding are different for these two proteins. As determined by Lipari-Szabo formalism, both the apo and holo forms of CRBP I and CRBP II are conformationally rigid on the pico- to nanosecond timescale. transverse relaxation optimized spectroscopy-Carr-Purcell-Meiboom-Gill -based 15N relaxation dispersion experiments at both 500 MHz and 600 MHz magnetic fields revealed that 84 and 62 residues for apo-CRBP I and II, respectively, showed detectable conformational exchange on a micro- to millisecond timescale, in contrast to three and seven residues for holo-CRBP I and II, respectively. Thus binding of retinol markedly reduced conformational flexibility in both CRBP I and CRBP II on the micro- to millisecond timescale. The 15N relaxation dispersion curves of apo-CRBP I and II were fit to a two-state conformational exchange model by a global iterative fitting process and by an individual (residue) fitting process. In the process of carrying out the global fit, more than half of the residue sites were eliminated. The individual chemical exchange rates k(ex), and chemical shift differences, Deltadelta, were increased in the putative portal region (alphaII helix and betaC-betaD turn) of apo-CRBP II compared to apo-CRBP I. These differences in conformational flexibility likely contribute to differences in how CRBP I and CRBP II interact with ligands, membranes and retinoid metabolizing enzymes.     

Increased neonatal mortality in mice lacking cellular retinol-binding protein II

Cellular retinol-binding protein II (CRBP II) is a member of the cellular retinol-binding protein family, which is expressed primarily in the small intestine. To investigate the physiological role of CRBP II, the gene encoding CRBP II was inactivated. The saturable component of intestinal retinol uptake is impaired in CRBP II(-/-) mice. The knockout mice, while maintained on a vitamin A-enriched diet, have reduced (40%) hepatic vitamin A stores but grow and reproduce normally. However, reducing maternal dietary vitamin A to marginal levels during the latter half of gestation results in 100% mortality/litter within 24 h after birth in the CRBP II(-/-) line but no mortality in the wild type line. The neonatal mortality in heterozygote offspring of CRBP II(-/-) dams (79 +/- 21% deaths/litter) was increased as compared with the neonatal mortality in heterozygote offspring of wild type dams (29 +/- 25% deaths per litter, p < 0.05). Maternal CRBP II was localized by immunostaining in the placenta at 18 days postcoitum as well as in the small intestine. These studies suggest that both fetal as well as maternal CRBP II are required to ensure adequate delivery of vitamin A to the developing fetus when dietary vitamin A is limiting.     

Cellular retinol-binding protein (type two) is abundant in human small intestine

Human small intestine was found to contain a retinol-binding protein similar to the gut-specific cellular retinol-binding protein, type two [CRBP (II)], described in the rat. This newly detected human protein was immunochemically distinct from human cellular retinol binding protein previously described but immunochemically similar to rat CRBP (II). The partially purified protein bound retinol and exhibited fluorescence excitation and emission spectra distinct from those spectra for retinol bound to pure human CRBP but similar to the spectra for retinol bound to rat CRBP (II). Human CRBP (II) could be localized to the villus-associated enterocytes by immunohistochemistry, using antiserum against rat CRBP (II). The protein was abundant representing 0.4% of the total soluble protein in a jejunum mucosal extract. This protein may play an important role in the absorption and necessary intestinal metabolism of vitamin A.     

Ligand binding and structural analysis of a human putative cellular retinol-binding protein

Three cellular retinol-binding protein (CRBP) types (CRBP I, II, and III) with distinct tissue distributions and retinoid binding properties have been structurally characterized thus far. A human binding protein, whose mRNA is expressed primarily in kidney, heart, and transverse colon, is shown here to be a CRBP family member (human CRBP IV), according to amino acid sequence, phylogenetic analysis, gene structure organization, and x-ray structural analysis. Retinol binding to CRBP IV leads to an absorption spectrum distinct from a typical holo-CRBP spectrum and is characterized by an affinity (K(d) = approximately 200 nm) lower than those for CRBP I, II, and III, as established in direct and competitive binding assays. As revealed by mutagenic analysis, the presence in CRBP IV of His(108) in place of Gln(108) is not responsible for the unusual holo-CRBP IV spectrum. The 2-A resolution crystal structure of human apo-CRBP IV is very similar to those of other structurally characterized CRBPs. The side chain of Tyr(60) is present within the binding cavity of the apoprotein and might affect the interaction with the retinol molecule. These results indicate that human CRBP IV belongs to a clearly distinct CRBP subfamily and suggest a relatively different mode of retinol binding for this binding protein.     

Binding specificities of cellular retinol-binding protein and cellular retinol-binding protein, type II

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.     

Identification and structural analysis of a zebrafish apo and holo cellular retinol-binding protein

Cellular retinol-binding proteins (CRBPs) are cytoplasmic retinol-specific binding proteins. Mammalian CRBPs have been thoroughly characterised previously. Here we report on the identification and X-ray structural analysis of the apo (1.7A resolution) and holo (1.4A resolution) forms of a zebrafish CRBP. According to amino acid sequence and structure analyses, the zebrafish CRBP that we have identified resembles closely mammalian CRBP II, suggesting that it is the zebrafish orthologue of this mammalian CRBP type. Zebrafish CRBP forms a tight complex with all-trans retinol, producing an absorption spectrum similar to those of mammalian holo-CRBPs, albeit slightly blue-shifted. The superposition of the alpha-carbon atoms of the liganded (complexed with retinol) and unliganded forms of zebrafish CRBP shows significant differences in correspondence of the betaC-betaD (residues 55-58) and betaE-betaF (residues 74-77) turns, providing evidence for the occurrence of conformational changes accompanying retinol binding/release. Remarkable and well-defined ligand-dependent conformational changes in the protein region comprising the two beta-turns affect both the main chain and the side-chains of several residues. The two beta-turns project towards the interior of the cavity devoid of ligand of the apoprotein. The side-chains of F57, Y60 and L77 change substantially their orientation and position in the apoprotein relative to the holoprotein. In the beta-barrel internal cavity of apo-CRBP they occupy some of the space that is otherwise occupied by bound retinol in holo-CRBP, and are displaced from these positions on ligand binding. These results indicate that a flexible area encompassing the betaC-betaD and betaE-betaF turns may serve as the ligand portal and that these turns undergo conformational changes associated with the not yet clarified mechanism of retinol binding and release in CRBPs.     

Characterization of a cellular retinol-binding protein from lamprey, Lethenteron japonicum

Lampreys are ancestral representatives of vertebrates known as jawless fish. The Japanese lamprey, Lethenteron japonicum, is a parasitic member of the lampreys known to store large amounts of vitamin A within its body. How this storage is achieved, however, is wholly unknown. Within the body, the absorption, transfer and metabolism of vitamin A are regulated by a family of proteins called retinoid-binding proteins. Here we have cloned a cDNA for cellular retinol-binding protein (CRBP) from the Japanese lamprey, and phylogenetic analysis suggests that lamprey CRBP is an ancestor of both CRBP I and II. The lamprey CRBP protein was expressed in bacteria and purified. Binding of the lamprey CRBP to retinol (Kd of 13.2 nM) was identified by fluorimetric titration. However, results obtained with the protein fluorescence quenching technique indicated that lamprey CRBP does not bind to retinal. Northern blot analysis showed that lamprey CRBP mRNA was ubiquitously expressed, although expression was most abundant in the intestine. Together, these results suggest that lamprey CRBP has an important role in absorbing vitamin A from the blood of host animals.     

Interaction of the retinol/cellular retinol-binding protein complex with isolated nuclei and nuclear components

Retinol (vitamin A alcohol) is involved in the proper differentiation of epithelia. The mechanism of this involvement is unknown. We have previously reported that purified cellular retinol-binding (CRBP) will mediate specific binding of retinol to nuclei isolated from rat liver. We now report that pure CRBP delivers retinol to the specific nuclear binding sites without itself remaining bound. Triton X-100-treated nuclei retain the majority of these binding sites. CRBP is also capable of delivering retinol specifically to isolated chromatin with no apparent loss of binding sites, as compared to whole nuclei. CRBP again does not remain bound after transferring retinol to the chromatin binding sites. When isolated nuclei are incubated with [3H]retinol- CRBP, sectioned, and autoradiographed, specifically bound retinol is found distributed throughout the nuclei. Thus, CRBP delivers retinol to the interior of the nucleus, to specific binding sites which are primarily, if not solely, on the chromatin. The binding of retinol to these sites may affect gene expression.     

Retinoic acid rapidly induces lung cellular retinol-binding protein mRNA levels in retinol deficient rats

Retinol deficiency resulted in decreased mRNA levels for cellular retinol-binding protein (CRBP) in the lungs and the testes. The level of lung CRBP mRNA increased 2.3-fold one hour after oral administration of retinoic acid to retinol deficient rats. In contrast, testicular CRBP mRNA level was not influenced. Our data indicate that retinoic acid regulates CRBP mRNA level in the whole animal and this rapid effect suggests a role for CRBP in the mechanism of vitamin A action at genomic level.     

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.     

Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are 132-residue cytosolic proteins which have 56% amino acid sequence identity and bind all-trans-retinol as their endogenous ligand. They belong to a family of cytoplasmic proteins which have evolved to bind distinct hydrophobic ligands. Their patterns of tissue-specific and developmental regulation are distinct. We have compared the ligand binding properties of rat apo-CRBP and apo-CRBP II that have been expressed in Escherichia coli. Several observations indicate that the E. coli-derived apoproteins are structurally similar to the native rat proteins: they co-migrate on isoelectric focusing gels; and when complexed with all-trans-retinol, their absorption and excitation/emission spectra are nearly identical to those of the authentic rat holoproteins. Comparative lifetime and acrylamide quenching studies suggest that there are differences in the conformations of apo-CRBP and apo-CRBP II. The interaction of E. coli-derived apo-CRBP and apo-CRBP II with a variety of retinoids was analyzed using spectroscopic techniques. Both apoproteins formed high affinity complexes with all-trans-retinol (K'd approximately 10 nM). In direct binding assays, all-trans-retinal bound to both apoproteins (K'd approximately 50 nM for CRBP; K'd approximately 90 nM for CRBP II). However, all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP. These observations suggests that there is a specific yet distinct interaction between these two proteins and all-trans-retinal. Apo-CRBP and apo-CRBP II did not demonstrate significant binding to either retinoic acid or methyl retinoate, an uncharged derivative of all-trans-retinoic acid. This indicates that the carboxymethyl group of methyl retinoate cannot be sterically accommodated in their binding pockets and that failure to bind retinoic acid probably is not simply due to the negative charge of its C-15 carboxylate group. Finally, neither all-trans-retinol nor retinoic acid bound to E. coli-derived rat intestinal fatty acid-binding protein, a homologous protein whose tertiary structure is known. Together, the data suggest that these three family members have acquired unique functional capabilities.     

Evidence for a lecithin-retinol acyltransferase activity in the rat small intestine

Cellular retinol-binding protein, type II (CRBP (II] is an abundant protein of the mature enterocytes of the small intestine. It has been shown to direct retinol to an acyl-CoA-independent esterifying activity that utilizes an endogenous acyl donor (Ong, D.E., Kakkad, B., and MacDonald, P.N. (1987) J. Biol. Chem. 262, 2729-2736). Here we report that this activity in intestinal microsomes will catalyze the transfer of acyl moieties from exogenous phosphatidylcholine (PC) to retinol-CRBP(II) to produce retinyl esters. The microsomal activity displayed positional selectivity as only the sn-1-acyl moiety of PC was transferred to retinol-CRBP(II). The retinyl ester synthase was selective for PC substrates as acyl transfer from phosphatidylethanolamine, phosphatidic acid, or free fatty acid to retinol-CRBP(II) was not observed. Some formation of retinyl esters was observed with exogenous acyl-CoA, but the amount produced was considerably lower than ester formation from exogenous PC and could be shown to be due to a different enzyme activity. Inhibitor studies clearly distinguished between the enzyme activities responsible for the acyl-CoA-dependent esterification and the phosphatidylcholine-dependent esterification of retinol. The results provide strong evidence that retinol-CRBP(II) esterification in the intestine proceeds via a phosphatidylcholine-dependent transacylase mechanism similar to that established for the esterification of cholesterol by lecithin-cholesterol acyltransferase.     

Interactions of retinol with binding proteins: studies with rat cellular retinol-binding protein and with rat retinol-binding protein

The interactions of retinol with rat cellular retinol-binding protein (CRBP) and with rat serum retinol-binding protein (RBP) were studied. The equilibrium dissociation constants of the two retinol-protein complexes (Kd) were found to be 13 x 10(-9) and 20 x 10(-9) M for CRBP and for RBP, respectively. The kinetic parameters governing the interactions of retinol with the two binding proteins were also studied. It was found that although the equilibrium dissociation constants of the two retinol-protein complexes were similar, retinol interacted with CRBP 3-5-fold faster than with RBP; the rate constants for dissociation of retinol from CRBP and from RBP (koff) were 0.57 and 0.18 min-1, respectively. The rate constants for association of retinol with the two proteins (kon) were calculated from the expression: Kd = koff/kon. The kon's for retinol associating with CRBP and with RBP were found to be 4.4 x 10(7) and 0.9 x 10(7) M-1 min-1, respectively. The data suggest that the initial events of uptake of retinol by cells are not rate-limiting for this process and that the rate of uptake is probably determined by the rate of metabolism of this ligand. The data indicate further that the distribution of retinol between RBP in blood and CRBP in cytosol is at equilibrium and that intracellular levels of retinol are regulated by the levels of CRBP.     

Reduction of retinaldehyde bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine

Cellular retinol-binding protein, type II (CRBP (II], an abundant protein of the rat small intestine, has recently been shown to be able to bind retinaldehyde in addition to retinol (MacDonald, P.N., and Ong, D. E. (1987) J. Biol. Chem. 262, 10550-10556). Retinaldehyde is produced in the intestine by oxidative cleavage of beta-carotene. The next step in the intestinal metabolism of vitamin A is the reduction of retinaldehyde to retinol which is then esterified for incorporation into chylomicrons. In the present study retinaldehyde bound to CRBP(II) was found to be available for reduction by microsomal preparations from rat small intestinal mucosa. The microsomal activity was about 8 times greater than the activity observed for an equal amount of cytosolic protein. Retinaldehyde reduction utilized either NADH or NADPH as cofactor, with NADH being slightly more effective. The apparent Km for retinaldehyde-CRBP(II) was 0.5 microM, and the Vmax was approximately 300 pmol/min/mg protein, a rate more than sufficient for the needs of the animal. The product retinol remained complexed to CRBP(II). The microsomal enzyme activity reduced free and bound retinaldehyde to approximately the same extent, although the aldehyde function of retinaldehyde bound to CRBP(II) was less accessible to chemical reducing agents than that of free retinaldehyde. Retinol bound to CRBP(II) could not be oxidized by the microsomal activity in the presence of NAD+, while free retinol or retinol bound to bovine serum albumin was oxidized to retinaldehyde. The more favorable reduction versus oxidation of retinoid bound to CRBP(II) consequently favored the reaction known to be required for the ultimate conversion of beta-carotene to retinyl esters for export from the gut.