A rapid versatile microassay for cellular retinol-binding protein using Lipidex-1000 microcolumns

A new, rapid and versatile microassay for cellular retinol-binding protein has been developed based on separation of bound and free ligand by means of Lipidex-1000, a hydrophobic Sephadex derivative. This requires quantitative manipulation of retinol in aqueous solution. The tendency of retinol to adhere to glass and plastic surfaces was overcome by addition of the detergent Ammonyx LO, which yields a micellar dispersion. Detergent concentrations up to 10 mM did not interfere with binding of retinol to Lipidex-1000 or binding protein. The binding capacity of Lipidex-1000 was found to exceed 400 nmol of retinol per ml of gel. Retinal pigment epithelium (RPE) cells were used as a source for cRBP (cellular retinol-binding protein). The binding protein is saturated with ligand by incubation for 60 min at room temperature at concentrations of free retinol over 180 nM. Separation of protein-bound retinol from free retinol is achieved via Lipidex-1000: protein-bound (specific and nonspecific) retinol is not retained and is eluted by buffer with the protein fraction. Free retinol is retained by Lipidex and is subsequently recovered by elution with methanol. Total recovery of ligand approaches 100%. Analysis time is about 4 hr for a maximum of ca. 50 samples. Nonspecific protein binding can be determined equally effectively either by incubation with 3 mM PCMBS or by addition of a 100-fold molar excess of nonlabeled retinol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Identification and partial characterization of a retinal pigment epithelial membrane receptor for plasma retinol-binding protein.

We have developed a membrane binding assay by which we have been able to characterize the interaction between 125I-labeled retinol-binding protein and its receptor in microsome fractions derived from retinal pigment epithelial cells. The binding of retinol-binding protein to the membranes was fast, with a dissociation constant in the range of 31-72 nM, and maximum binding occurred at neutral pH. Receptor binding sites were also found in microsome fractions of liver and kidney, whereas lung and muscle contained few, if any. Chemical cross-linking of retinol-binding protein to the microsomal membranes yielded a major molecular complex of Mr 86,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein responsible for binding of retinol-binding protein was identified as a Mr 63,000 protein using a label transfer cross-linking technique. Further characterization demonstrated that the receptor for retinol-binding protein is a terminally glycosylated membrane protein noncovalently associated with a high molecular weight complex.     

Purification and characterization of a murine cellular retinoic acid-binding protein

A cellular retinoic acid-binding protein from 1-day-old mouse pups has been purified to homogeneity. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE cellulose, and chromatofocusing on PBE9-4 ion exchange resin. The chromatofocusing step was most useful in removing the major contaminants, which were otherwise difficult to remove. The binding protein was finally subjected to two cycles of high performance liquid chromatography on a DEAE-5PW column to achieve homogeneity. The protein has an isoelectric point of 4.75 and consists of a single polypeptide, migrating with an apparent Mr of 14,600 in SDS--polyacrylamide gel electrophoresis. Amino-terminal sequence analysis showed that the mouse cellular retinoic acid-binding protein has a high percentage of amino acid identity with other retinoid-binding proteins. However, it is immunologically distinct from the cellular retinol-binding protein.     

Cellular retinoid-binding proteins in reginerating rat liver: demonstration of a novel cellular retinoid-binding protein

Changes in the levels of liver cellular aetinol- and retinoic acid-binding proteins were studied after partial (about 70%) hepatectomy for 14 days in the rat. It was found that a novel binding protein designated F-type appears transiently in liver cytosol 3 days after the operation. The appearance of this protein coincides with the peak level of the alpha 1-fetoproteain. In contrast, cellular retinoic acid-binding protein was detected only the first day after hepatectomy, whereas no significant change was observed in the level of the cellular retinol-binding protein during the entire observation period. [3H]Retinol or [3H]retinoic acid complexed with serum retinol-binding protein injected intravenously into vitamin A-deficient rats 1 day after the hepatectomy was recovered 5 min or 20 min later bound specifically to cellular retinol- or retinoic acid-binding protein, respectively. The results presented here strongly suggest that each of the three cellular retinoid-binding proteins plays a distinct role in cell proliferation and differentiation.     

Vitamin A and thyroxine carrier proteins in chicken plasma: steady-state control of the plasma level of free retinol-binding protein and free thyroxine.

1. The binding parameters of prealbumin-2 with retinol-binding protein and thyroxine (T4) revealed the existence of distinct and multiple sites for both retinol-binding protein and T4. 2. From the analysis of binding parameters of retinol-binding protein with prealbumin-2 it is clear that under steady-state conditions about 99% of the holo-retinol-binding protein remains bound to prealbumin-2. 3. Equilibrium dialysis studies on binding properties of thyroid hormones with prealbumin-2 revealed that it has a single high affinity site and three low affinity sites. 4. The occurrence of three carrier proteins for thyroid hormones, thyroxine-binding globulin, prealbumin-2 and albumin has been demonstrated. However, the chicken thyroxine-binding globulin differs from human thyroxine-binding globulin by being relatively less acidic and occurring at a two-fold lower concentration. But the thyroid hormone binding parameters are comparable. 5. Highly sensitive methods were developed for determination of T4 binding capacities of the various proteins and plasma level of total T4 by fractionation of carrier proteins and further quantitatively employing in electrophoresis and equilibrium dialysis. 6. The thyroxine-binding proteins were found to be of two types, one (viz., thyroxine-binding globulin) of great affinity but of low binding capacity, which mainly acts as reservoir of T4, and another (viz., prealbumin-2) of low affinity but of high binding capacity, which can participate predominantly in the control of the free T4 pool.     

Synthesis and secretion of a novel binding protein for retinol by a cell line derived from Sertoli cells

An established cell line (TM-4) derived from murine Sertoli cells, the major supportive cell type of the testes, secretes a protein that binds retinol when grown in serum-free chemically defined medium. The protein that binds retinol is trypsin-sensitive and has an apparent Kd for retinol of 54 nM. Cholesterol, retinyl acetate, or UV-irradiated retinol at levels 100-fold in excess of retinol are poor competitors of [3H]retinol binding. Retinoic acid at a 100-fold molar excess inhibited [3H]retinol binding by 71%. In contrast, excess unlabeled retinol completely inhibits [3H]retinol binding. More than 80% of the total retinol-binding activity in confluent cultures is found in the culture medium. Prior to incubation with retinol, the protein that binds retinol has an apparent Mr of less than 150,000 by column chromatography; however, after incubation with retinol the protein that binds retinol exhibits an apparent Mr of 2 X 10(6) or greater and a sedimentation coefficient greater than 4 S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the major iodinatable component of the aggregated protein that binds retinol has an apparent Mr of 70,000. The secreted protein that binds retinol is not immunologically cross-reactive with either serum or cellular retinol-binding protein or transferrin. These findings suggest that Sertoli cells may secrete a protein that binds retinol. Such a protein could be involved in the transport of retinol either to the lumen of the seminiferous tubules or to the developing germ cells themselves.     

Distributions of retinoids, retinoid-binding proteins and related parameters in different types of liver cells isolated from young and old rats

The levels of retinoids, retinol-binding protein, cellular retinol-binding protein, cellular retinoic-acid-binding protein, transthyretin and the activities of retinyl palmitate hydrolase and cholesteryl oleate hydrolase were determined in purified parenchymal, fat-storing, endothelial and Kupffer cell preparations, and in liver homogenates from young adult (6-month-old) and old (36-month-old) rats. Retinoid levels were also determined in the plasma from young and old rats. Retinoid contents were determined by HPLC. The binding proteins and transthyretin were measured by specific radioimmunoassays; retinyl palmitate and cholesterol oleate hydrolases were measured by sensitive microassays. The retinoid content of both the liver homogenates and of the fat-storing, and parenchymal cell preparations increased between 6 months and 36 months of age. The cellular distribution of retinoids was similar for the two age groups analyzed with the fat-storing cells being the main retinoid storage sites in the rat liver. Concentrations of retinol-binding protein and transthyretin were high in parenchymal cell preparations. Cellular retinol-binding protein was enriched both in parenchymal and in fat-storing cell preparations; the highest concentrations of cellular retinoic-acid-binding protein were present in fat-storing cell preparations. No major differences were observed between the two age groups in the cellular concentrations and distributions of any of these binding proteins. High activity of cholesterol oleate hydrolase was measured in parenchymal and in Kupffer cell preparations; endothelial cell preparations also contained considerable activities. The distribution of this activity over the various cell types reflects their role in lipoprotein metabolism. Retinyl palmitate hydrolase activity was specifically enriched in parenchymal and in fat-storing cell preparations, consistent with the roles of these cells in retinoid metabolism. No major differences were observed between the two age groups in the cellular distributions of the two hydrolase activities. This study indicates that no major changes occur in the retinoid-related parameters analyzed with age, suggesting that rat liver retinoid metabolism does not change dramatically with age and that retinoid homeostasis is maintained.     

Novel retinoid-binding proteins from filarial parasites.

The present study deals with the discovery and partial characterization of specific binding proteins for retinol and retinoic acid from filarial parasites (worms of the superfamily Filarioidea), including those from two species of Onchocerca. These binding proteins, which are distinct in their physicochemical properties and in the mode of ligand interactions from the host-tissue retinoid-binding proteins, may be involved in the mediation of the putative biological roles of retinoids in the control of parasitic growth, differentiation and reproduction. Parasite retinol-binding protein and retinoic acid-binding protein exhibited specificity for binding retinol and retinoic acid respectively. Both the binding proteins showed an s20,w value of 2.0 S. On gel filtration, both proteins were retarded to a position corresponding to the same molecular size (19.0 kDa). On preparative columns, the parasite binding proteins exhibited isoelectric points at pH 5.7 and 5.75. Unlike the retinoid-binding proteins of mammalian and avian origin, the parasite retinoid-binding proteins showed a lack of mercurial sensitivity in ligand binding. The comparative amounts of retinoic acid-binding protein in five parasites, Onchocerca volvulus, Onchocerca gibsoni, Dipetalonema viteae, Brugia pahangi and Dirofilaria immitis, were between 2.7 and 3.1 pmol of retinoic acid bound/mg of extractable protein. However, the levels of parasite retinol-binding protein were between 4.8 and 5.8 pmol/mg, which is considerably higher than the corresponding levels of cellular retinol-binding protein of mammalian and avian origin. Both retinol- and retinoic acid-binding-protein levels in O. volvulus-infected human nodules and O. gibsoni-infected bovine nodules were similar to their levels in mammalian tissues. Also, these nodular binding proteins, like the host-binding proteins, exhibited mercurial sensitivity to ligand interactions.     

视黄醇结合蛋白及其基因的分子生物学

视黄醇结合蛋白（RBP）是一类维生素A（VitA）的运载蛋白,参与血清和细胞内视黄醇/视黄酸的转运,是疏水小分子结合蛋白家族的成员。这类RBP主要在肝脏中合成并释放入血液进而进入各种组织。血清RBP通过与视黄醇、前白蛋白及细胞表面受体相互作用,在VitA 的储存、代谢、转运到周围靶器官中具有重要功能;细胞RBP则主要在细胞内发挥类似作用。本文介绍了视黄醇结合蛋白的作用机理、组织定位和发育性表达,还介绍了视黄醇结合蛋白基因的结构、染色体定位以及与动物繁殖性能的关系。Abstract: Retinol-binding proteins (RBPs) are a kind of circulating carrier proteins for serum and cellular retinol and retinol acid, which are lipid-soluble vitamins, and are members of hydrophobic binding protein family. Serum RBPs were synthesized primarily in liver, then was released into blood streams, and then to various tissues. Under the interaction with substances such as retinol, pre-albumin and the receptors of cellular surface, they play important roles in storage, metabolism of VitA and transport of VitA to the target cells. Cellular RBPs play the similar function as serum RBPs in intracell. This review introduces action mechanism, tissue localization and developmental expression of retinol-binding proteins. This review also introduces the structure, chromosome mapping and their relationships with reproductive performance of retinol-binding protein genes.     

N-(4-hydroxyphenyl)retinamide (Fenretinide) and nephrectomy alter normal plasma retinol-binding protein metabolism

We have reported previously that injecting vitamin A-deficient rats with N-(4-hydroxyphenyl)retinamide causes a significant reduction in the liver retinol-binding protein concentration and a 2 fold rise in the kidney retinol-binding protein concentration. This presumably reflects a rapid translocation of retinol-binding protein from the liver to the kidney through the plasma, although no rise in plasma retinol-binding protein is detected. In the present studies, nephrectomized rats were used to determine if retinol-binding protein accumulating in kidneys passes through the plasma. Bilateral nephrectomy in control rats caused the plasma retinol-binding protein concentration to approximately double by 5 hr postsurgery. However, nephrectomy plus N-(4-hydroxyphenyl)retinamide treatment did not result in an increase in the plasma retinol-binding protein concentration. Therefore, the lowering of liver retinol-binding protein concentration in response to N-(4-hydroxyphenyl)retinamide treatment was not accounted for by an accumulation of retinol-binding protein in the plasma compartment. Interestingly, the muscle retinol-binding protein concentration increased with nephrectomy plus N-(4-hydroxyphenyl)retinamide treatment. The ratio of muscle retinol-binding protein:plasma retinol-binding protein in vitamin A-deficient nephrectomized rats treated with N-(4-hydroxyphenyl)retinamide was significantly higher than in comparable rats treated with either carrier or retinol. We conclude that in vivo N-(4-hydroxyphenyl)retinamide induces the secretion of retinol-binding protein from the liver. Since the N-(4-hydroxyphenyl)retinamide-retinol-binding protein complex does not bind with transthyretin it rapidly leaves the plasma. In non-nephrectomized rats this complex is rapidly filtered by the kidney. Nephrectomizing rats causes the retinol-binding protein secreted in response to N-(4-hydroxyphenyl)retinamide to diffuse into interstitial fluid.     

ATP stimulates binding of retinol-cellular retinol binding protein complex to DNA-cellulose

In calf uterus cytosol a cellular retinol-binding protein (cRBP) was detected which was found to bind to DNA-cellulose. The binding to DNA-cellulose could be enhanced by ATP in a dose-dependent manner. ATP treatment did not change the physico-chemical properties of the retinol-cRBP complex. Our findings suggest a role for ATP in the binding of retinol-cRBP complex to DNA.     

Complete sequence and model for the A2 subunit of the carotenoid pigment complex, crustacyanin

The complete sequence has been determined for the A2 subunit of crustacyanin, an astaxanthin-binding protein from the carapace of the lobster Homarus gammarus. The polypeptide chain is 174 residues long and is similar to proteins of the retinol-binding protein superfamily. Some regions of the sequence are most similar to the retinol-binding protein, beta-lactoglobulin subgroup, while the disulphide bonding pattern is more akin to that seen in the porphyrin binding proteins insecticyanin and bilin-binding protein. It is beginning to appear as though this superfamily of proteins, characterized by a similar gross structural framework, may be further subdivided into interrelated subclasses. Model building based on the coordinates of the known structure of human plasma retinol-binding protein and on empirical prediction algorithms has allowed the putative identification of side-chains which line the binding cavity. This pocket is larger than in retinol binding protein and beta-lactoglobulin but does not allow the carotenoid to adopt a folded conformation. The amino acid composition of the pocket does not support a 'charge-shift'-type hypothesis to support the bathochromic shift phenomenon which takes place on interaction of the chromophore with the protein. Instead aromatic side-chains may play a prominent role.     

Vitamin A receptors. I. Comparison of retinol binding to serum retinol-binding protein and to tissue receptors in chick retina and pigment epithelium.

1. A simple, efficient three-step method for purification of serum retinol-binding-protein is described with homogeneity obtained after chromatography on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. 2. Evidence is presented indicating that retinol receptors present in the cytosol fraction of chick retina and pigment epithelium are separate and distinct from purified retinol-binding protein. Fluorescence characteristics are different in tissue cytosol and serum as assessed by sucrose density gradient analysis. Tissue retinol receptors do not interact with human serum prealbumin although the prealbumin readily complexes with purified chicken retinol-binding protein. Likewise, no binding to serum retinol-binding protein antibody could be detected by sucrose density gradient analysis, in immunoprecipitation experiments or by double immunodiffusion. It thus appears that specific retinol receptors are present in neural retina and pigment epithelium that are different from serum retinol-binding protein.     

Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol

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.     

Vitamin A transport in plasma of the non-mammalian vertebrates: isolation and partial characterization of piscine retinol-binding protein

Studies were conducted to explore vitamin A transport in the non-mammalian vertebrates, especially Pisces, Amphibia, and Reptilia, and to isolate and partially characterize piscine retinol-binding protein. Retinol-containing proteins in fresh plasma obtained from bullfrogs and a turtle exhibited similar properties to those found in mammalian and chicken plasma: i.e., molecular weight of about 60,000-80,000 as estimated by gel filtration and binding affinity to prealbumin on human prealbumin-Sepharose affinity chromatography. In sharp contrast, vitamin A-containing proteins in plasma from larvae of bullfrogs as well as three fishes (carp, blue sharks, and young yellowtails) appeared to be present in plasma as monomeric retinol-binding proteins without any affinity to human prealbumin. On the other hand, plasma vitamin A in the lamprey (Cyclostomes) was found to exist exclusively as an ester form in association with the lipoproteins of hydrated density less than 1.21 g/ml. Piscine retinol-binding protein was isolated from pooled plasma of young yellowtails and was converted (1000-fold purification) to a homogeneous component by a procedural sequence that included gel filtration on Sephadex G-100, chromatography on SP-Sephadex, gel isoelectric focusing, and, finally, polyacrylamide gel electrophoresis. Purified piscine retinol-binding protein showed physico-chemical properties distinctly different from the mammalian and chicken retinol-binding proteins examined, i.e., a smaller molecular weight of approximately 16,000, a lower isoelectric point of 4.3, a prealbumin mobility on analytical polyacrylamide gel electrophoresis, and a lack of binding affinity for human prealbumin; however, it displayed similar characteristics in two ways: a 1:1 molar complex with retinol, and a high content of tryptophan (four residues). These results strongly suggest that the piscine retinol-binding protein is a prototype of the specific vitamin A-transporting protein in plasma of the vertebrates, being modified later in evolution, during phylogenetic development of the vertebrates, to acquire a binding site for prealbumin on the molecule.     

Homeostatic regulation of free retinol-binding protein and free thyroxine pools of plasma by their plasma carrier proteins in chicken

The mechanism underlying homeostatic regulation of the plasma levels of free retinol-binding protein and free thyroxine, the systemic distribution of which is of great importance, has been investigated. A simple method has been developed to determine the rate of dissociation of a ligand from the binding protein. Analysis of the dissociation process of retinol-binding protein from prealbumin-2 reveals that the free retinol-binding protein pool undergoes massive flux, and the prealbumin-2 participates in homeostatic regulation of the free retinol-binding protein pool.Studies on the dissociation process of thyroxine from its plasma carrier proteins show that the various plasma carrier proteins share two roles. Of the two types of protein, the thyroxine-binding globulin (the high affinity binding protein) contributes only 27% of the free thyroxine in a rapid transition process, despite its being the major binding protein. But prealbumin-2, which has lower affinity towards thyroxine, participates mainly in a rapid flux of the free thyroxine pool. Thus thyroxine-binding globulin acts predominantly as a plasma reservoir of thyroxine, and also probably in the ‘buffering’ action on plasma free thyroxine level, in the long term, while prealbumin-2 participates mainly in the maintainance of constancy of free thyroxine levels even in the short term. The existence of these two types of binding protein facilitates compensation for the metabolic flux of the free ligand and maintenance of the thyroxine pool within a very narrow range.     

Homeostatic regulation of free retinol-binding protein and free thyroxine pools of plasma by their plasma carrier proteins in chicken.

The mechanism underlying homeostatic regulation of the plasma levels of free retinol-binding protein and free thyroxine, the systemic distribution of which is of great importance, has been investigated. A simple method has been developed to determine the rate of dissociation of a ligand from the binding protein. Analysis of the dissociation process of retinol-binding protein from prealbumin-2 reveals that the free retinol-binding protein pool undergoes massive flux, and that prealbumin-2 participates in homeostatic regulation of the free retinol-binding protein pool. Studies on the dissociation process of thyroxine from its plasma carrier proteins show that the various plasma carrier proteins share two roles. Of the two types of protein, the thyroxine-binding globulin (the high affinity binding protein) contributes only 27% of the free thyroxine in a rapid transition process, despite its being the major binding protein. But prealbumin-2, which has lower affinity towards thyroxine, participates mainly in a rapid flux of the free thyroxine pool. Thus thyroxine-binding globulin acts predominantly as a plasma reservoir of thyroxine, and also probably in the 'buffering' action on plasma free thyroxine level, in the long term, while prealbumin-2 participates mainly in the maintenance of constancy of free thyroxine levels even in the short term. The existence of these two types of binding protein facilitates compensation for the metabolic flux of the free ligand and maintenance of the thyroxine pool within a very narrow range.     

Retinoic acid-binding protein in rat tissue. Partial purification and comparison to rat tissue retinol-binding protein.

When the 100,000 X g supernatant fractions of several rat organs are incubated with all-trans-[3H]retinoic acid, a binding component for retinoic acid with a sedimentation coefficient of 2 S can be detected by sucrose gradient centrifugation. This tissue binding protein for retinoic acid is distinct from the tissue binding protein for retinol which has been previously described. The tissue retinoic acid-binding protein has been partially purified from rat testis and this partially purified protein would appear to have a molecular weight of 14,500 as determined by gel filtration and high binding specificity for all-trans-retinoic acid. Binding of [3H]retinoic acid is not diminished by a 200-fold molar excess of retinal, retinol, or oleic acid but is reduced by a 200-fold excess of unlabeled retinoic acid. Tissue retinoic acid-binding protein can be detected in extracts of brain, eye, ovary, testis, and uterus but is apparently absent in heart muscle, small intestine, kidney, liver, lung, gastrocnemious muscle, serum, and spleen. This distribution is different than that observed for the tissue retinol-binding protein. Tissue retinol-binding protein was also purified extensively from rat testis. The partially purified protein has an apparent molecular weight of 14,000 and high binding specificity for all-trans-[3H]retinol as only unlabeled all-trans-retinol but not retinal, retinoic acid, retinyl acetate, retinyl palmitate, or oleic acid could diminish binding of the 3H ligand under the conditions employed. The partially purified protein has a fluorescence excitation spectrum with lambda max at 350 nm. In contrast, the retinol-binding protein isolated from rat serum and described by others has a fluorescence excitation spectrum with lambda max at 334 nm and an apparent molecular weight of 19,000. When partially purified tissue retinol-binding protein is extracted with heptane, the heptane extract has a fluorescence excitation spectrum similar to that of all-trans-retinol.