Transport of retinol in the duck plasma

Retinol-binding protein and prealbumin were isolated from duck plasma by chromatography on DEAE-cellulose-and DEAE-Sephadex A-50, gel filtration on Sephadex G-100 and preparative Polyacrylamide gel electrophoresis. The molecular weights of the retinol-binding protein-prealbumin complex, prealbumin and retinol-binding protein were found to be 75,000, 55,0000 and 20,000, respectively. On sodium dodecyl sulphate Polyacrylamide gel electrophoresis, prealbumin dissociated into identical subunits exhibiting a molecular weight of 13,500. Retinol-binding protein exhibited microheterogeneity on electrophoresis, whereas prealbumin moved as a single band unlike the multiple bands observed in chicken and rat. The ultraviolet and fluorescence spectra of the two proteins were similar to those isolated from other species. No carbohydrate moiety was detected in either retinol-binding protein or prealbumin. Duck retinol-binding protein and prealbumin showed cross-reactivity with their counterparts in chicken but differed immunologically from those of goat and man. Retinol-binding protein and prealbumin could be dissociated at low ionic strength, in 2M urea, by CM-sephadex chromatography or on preparative electrophoresis. Although the transport of retinol in duck plasma is mediated by carrier proteins as in other species, it is distinguished by the absence of microheterogeneity in prealbumin and of an apo-retinol-binding protein form that could be transported in the plasma.     

Purification and partial characterization of a cellular retinol-binding protein from human liver

A protein with binding specificity for retinol was purified from human liver. [³H]Retinol was added to liver extracts and the [³H]retinol-binding protein isolated by conventional chromatographic techniques including ion-exchange chromatography on DEAE-Sepharose, gel filtration on Sephadex G-75 and G-50 and preparative isoelectric focusing. The yield was 10–15% in different preparations and the degree of purification was about 3000-fold. The purified protein had a molecular weight of about 15 000 as estimated from both gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulphate and was homogeneous in several electrophoretic systems. Isoelectric focusing of the purified protein gave a doublet band. Only one fluorescent band at pH 4.70 was seen if the protein solution was incubated with excess retinol prior to isoelectric focusing. The isolated protein did not react with antiserum to the retinol-binding protein of plasma. The amino acid composition and the amino terminal amino acid sequence for the first sixteen amino acids of the purified protein differed significantly from that of the plasma retinol-binding protein.     

Purification of a bovine counterpart to human prealbumin and its interaction with a bovine retinol-binding protein

A bovine counterpart to human prealbumin was purified from bovine serum by thiol-disulfide exchange chromatography on thiol-Sepharose 4B and affinity chromatography on human retinol-binding protein linked to Sepharose 4B. The bovine prealbumin had alpha1-mobility on agarose gel electrophoresis at pH 8.6. It has the same molecular weight as human prealbumin on gel filtration and consisted of subunits with a molecular weight of 12 500. This is compatible with a tetrameric structure for the bovine protein. Antiserum against human prealbumin cross-reacted with bovine prealbumin and vice versa. The bovine prealbumin formed at high ionic strength complexes with another bovine serum protein which were dissociated at low ionic strength. This property was used to isolate a protein from bovine serum, by chromatography on bovine prealbumin linked to Sepharose which cross-reacted with antiserum against human retinol-binding protein; had a molecular weight of 21 000 and alpha 2-mobility on agarose gel electrophoresis. It was concluded that the latter protein was a bovine retinol-binding protein.     

Interaction of diethylstilbestrol and ioxynil with transthyretin in chicken serum

The association of suspected endocrine-disrupting chemicals (EDCs), diethylstilbestrol (DES), ioxynil and pentachlorophenol (PCP), with chicken serum proteins was investigated in relation to thyroid system disruption. All of these chemicals strongly inhibited l-[(125)I]thyroxine ([(125)I]T(4)) binding to purified transthyretin (TTR) whereas PCP was less potent inhibitor than DES and ioxynil of [(125)I]T(4) binding to diluted whole chicken serum. This result suggested that PCP interacted with serum proteins other than TTR in whole chicken serum. Following the incubation of chicken serum with each chemical (final concentrations 0.25-1.0 microM), serum proteins were fractionated by gel filtration chromatography (Cellulofine GCL-1000) and affinity chromatography (human retinol-binding protein coupled to Sepharose 4B). Although all chemicals were detected in the gel filtration chromatography 50-100 kDa fractions, DES and ioxynil, but not PCP, were co-eluted with TTR during affinity chromatography. Our results indicated that a significant proportion of DES and ioxynil, but a low proportion of PCP, interacted with TTR in whole chicken serum.     

A comparative study of vitamin D binding globulins in milk.

1. The occurrence of 25-hydroxy vitamin D binding protein in human, bovine, monkey and porcine milk was investigated. 2. Sucrose gradient ultracentrifugation revealed the presence of 4.2 S and 5.7 S binding globulins in the whey of human, monkey and porcine milk. 3. Although bovine plasma also contains a 4.2 S globulin only a 5.7 S protein was found in bovine milk. 4. The 4.2 S and 5.7 S globulins in milk could not be resolved by polyacrylamide gel electrophoresis or by isoelectric focusing. 5. Plasma and whey binding proteins of any one species had the same isoelectric point but there were small differences among species (4.5-4.8). 6. Competitive displacement studies showed that the binding proteins in milk have high affinity for 25-hydroxy-cholecalciferol and 24,25-dihydroxycholecalciferol.     

Purification and properties of retinol- and retinoic acid-binding proteins from a transplantable mouse colon tumor

Cellular retinol-binding protein and retinoic acid-binding protein, the possible mediators of the action of retinoids in epithelial differentiation and control of tumorigenesis, have been reproducibly purified from mouse colon tumor 26, and some of their properties were studied. The main steps of purification involved acid-precipitation, DEAE-Sephadex, CM-cellulose and Sephadex G-100 chromatography. About 2 mg of the binding proteins were isolated from 60 g tumor. The purified preparations showed only two protein bands on polyacrylamide gel electrophoresis. The two binding proteins were partially resolved by sedimentation equilibrium technique; but was completely separable by preparative electrophoresis in the presence of sodium dodecyl sulfate. The retinol- and retinoic acid-binding proteins are presumably monomers with molecular weights of 15,500 and 14,600, respectively, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. On gel filtration however, both the binding proteins retarded to the same molecular size of 17,800. On preparative columns, both the proteins expressed the same isoelectric pH, 4.5. Both proteins of the tumor possessed functional thiol groups. The mercurial inhibition of the binding capacity of the proteins for their ligands was reversible upon treatment with thiol compounds.     

Characterization of the human plasma binding protein for vitamin D and its metabolites synthesized by the human hepatoma-derived cell line, Hep 3B

Screening of three human hepatoma-derived cell lines revealed the presence of an immunologically similar plasma binding protein for vitamin D and its metabolites in media from Hep 3B cells. Approximately 3% of protein synthesized and secreted by these cells was immunoprecipitated by specific antiserum to the D-binding protein. Medium content of the protein increased over 11 days following cell seeding, and negligible amounts of 125I-labeled binding protein added to cultures were degraded over 48 h. The hepatoma-derived binding protein was indistinguishable from plasma binding protein or reference pure protein in gel filtration, sucrose gradient ultracentrifugation, and polyacrylamide gel electrophoresis systems. The Hep 3B cell product was found to bind mole/mol with monomeric actin, and bind vitamin D sterols with an affinity and specificity characteristic of the human plasma binding protein. The findings argue strongly for the identity of the Hep 3B cell product and the human plasma protein. The continuous availability of the Hep 3B cell line provides a reasonable model for investigations of biosynthesis and release of this important plasma protein.     

Thyroidal control of hepatic release and metabolism of vitamin A

The inverse relationship that exists between thyroxine and the vitamin A level of plasma has been examined in chicken. Thyroxine treatment leads to a decrease in the level of vitamin A carrier proteins, retinol-binding protein and prealbumin-2 in plasma and liver. There is an accumulation of vitamin A in the liver, with a greater proportion of vitamin A alcohol being present compared to that of control birds. In thyroxine treatment there is enhanced plasma turnover of retinol-binding protein and prealbumin-2, while their rates of synthesis are marginally increased. Amino acid supplementation partially counteracts effects of thyroxine treatment. Amino acid supplementation of thyroxine-treated birds does not alter the plasma turnover rates of retinol-binding protein and prealbumin-2 but increases substentially their rates of synthesis. The release of vitamin A into circulation is interfered with in hyperthyroidism due to inadequate availability of retinol-binding protein being caused by enhanced plasma turnover rate not compensated for by synthesis.     

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.     

Characterization of bovine plasma retinol-binding protein and evidence for lack of binding between it and other bovine plasma proteins.

Bovine retinol-retinol-binding protein (RBP) was isolated from serum as a free, uncomplexed protein under experimental conditions in which human, rabbit, and chicken retinol-RBP are present as tight complexes with prealbumin (thyroxine-binding protein). Purified bovine retinol-RBP formed tight complexes with purified human and chicken prealbumin in physiological ionic strength buffers as judged by gel filtration chromatography, hyperchromic effect on the absorption spectrum of retinol-RBP, and changes in the circular dichroism spectrum. Addition of purified human prealbumin to whole bovine serum shifted the elution position of the specific retinol-RBP fluorescence from a gel filtration column, indicating complex formation in the whole bovine serum. It was concluded from this series of experiments that bovine serum lacks a protein with the binding properties of prealbumin and that bovine retinol-RBP has the normal potential binding to human, chicken, and presumably other prealbumins. Bovine retinol-RBP has a molecular weight, amino acid composition, absorption, and fluorescence spectra which are indistinguishable from that of human retinol-RBP, although the magnitude of the optical rotatory strength of the induced circular dichroism signal at 330 nm was 50% larger in the bovine than in the human material (1.65 and 1.1 Debye-Bohr magnetons, respectively). About 12 liters of bovine and human urine were concentrated by pressure dialysis and a search was made for retinol-RBP using gel filtration and ion exchange chromatography. No retinol-RBP was found in either of these species. This suggested that if, indeed, bovine retinol-RBP is filtered through the kidney's glomeruli due to small molecular size (molecular weight 21,000), there are efficient mechanisms of tubular reabsorption.     

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.     

Plasma transport of 5-hydroxytryptamine-3H. II. Identification of the carrier protein of 5-hydroxyindoleacetic acid]

In the first note, we have demonstrated a 5-hydroxyindole acetic acid binding to plasma proteins. By gel filtration on Sephadex G 200 and polyacrylamide gel electrophoresis, the 5-hydroxyindole acetic acid carrier protein was identified to serum-albumin.     

Isolation and characterization of the major mannose-binding protein in chicken serum

Mannose-binding activity is abundantly present in chicken serum. The major mannose-binding protein has been isolated from chicken serum by affinity chromatography and gel filtration. The protein consists of two subunits of 75 000 and 26 500 daltons. Unlike hepatic lectins or other mannose-binding proteins, this protein does not require calcium for binding mannose-containing glycoconjugates. The chicken serum mannose-binding protein is immunochemically distinct from the chicken hepatic lectin and rabbit serum mannose-binding protein.     

Characterization of plasma triiodophenol binding proteins in vertebrates and tissue distribution of triiodophenol in Rana catesbeiana tadpoles

We investigated the interaction of 2,4,6-triiodophenol (TIP), a potent thyroid hormone disrupting chemical, with serum proteins from rainbow trout (Onchorhynchus mykiss), bullfrog (Rana catesbeiana), chicken (Gallus gallus), pig (Sus scrofa domesticus), and rat (Rattus norvegicus) using a [(125)I]TIP binding assay, gel filtration chromatography, and native polyacrylamide gel electrophoresis. [(125)I]TIP bound non-specifically to proteins in trout serum, specifically but weakly to proteins in bullfrog serum, and specifically and strongly to proteins in chicken, pig, and rat serum samples. Candidate TIP-binding proteins included lipoproteins (220-320kDa) in trout, albumin in bullfrog, albumin and transthyretin (TTR) in chicken and pig, and TTR in rat. TTR in the chicken, pig, and rat serum samples was responsible for the high-affinity, low-capacity binding sites for TIP (dissociation constant 2.2-3.5×10(-10)M). In contrast, a weak interaction of [(125)I]TIP with tadpole serum proteins accelerated [(125)I]TIP cellular uptake in vitro. Intraperitoneal injection of [(125)I]TIP in tadpoles revealed that the radioactivity was predominantly accumulated in the gallbladder and the kidney. The differences in the molecular and binding properties of TIP binding proteins among vertebrates would affect in part the cellular availability, tissue distribution and clearance of TIP.     

Proteins in the luminal fluid from the bovine oviduct.

Oviducal fluid was collected by cannulation from four cows and by irrigation from fifteen slaughtered cows.The proteins in the fluid were examined by polyacrylamide gel electrophoresis at pH 4-5 and pH 8-9, isoelectric focusing on polyacrylamide, immunodiffusion, immunoelectrophoresis, affinity chromatography and gel filtration. The macromolecular components found were mainly serum proteins but small amounts of other proteins were detected in oestrous and dioestrous samples by electrophoresis at pH 8-9 following fractionation of the fluid by gel filtration or affinity chromatography. Small amounts of cathodically migrating proteins were detected directly by electrophoresis at pH 4-5 in dioestrous samples but not in oestrous samples. Determination of glycosidase activities revealed that the levels at oestrus were similar to the levels detected in serum. At dioestrus, the activities of B-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase were elevated.     

Isolation and partial characterization of a alpha 2-beta 1-glycoprotein from horse plasma

A alpha 2-beta 1-glycoprotein was isolated from horse plasma by classical methods. The final product appeared homogeneous by agarose gel and pore limit SDS polyacrylamide gel electrophoresis, immunoelectrophoresis and crossed immunoelectrophoresis. The protein moved in agarose gel electrophoresis just above the beta 1 region and seemed composed of a single polypeptide chain. A highly heterogenic banding pattern, focused between pH 5.1 and 6.5 was revealed by isoelectric focusing. The molecular weights determined by gel filtration on Sephadex G100 and by a pore limit polyacrylamide gel electrophoresis in presence of SDS were 65,000 and 82,300 dalton, respectively. No serological relation was found between the horse alpha 2-beta 1-glycoprotein and human and bovine plasma proteins.     

Expression in Escherichia coli of full-length and mutant rat brain calbindin D28. Comparison with the purified native protein

Studies of vitamin D-dependent 28-kilodalton calcium binding protein (calbindin D28) have been hindered by difficulties in purifying large amounts of the protein. In order to overcome this problem, we cloned and expressed a full-length rat brain calbindin D28 cDNA. In addition, we isolated and purified to homogeneity, native rat brain calbindin D28. The isolated native protein has an apparent molecular mass of 27 kDa and properties similar to those of the well-characterized chicken calbindin D28. It has an acidic isoelectric point (approximately 4.5), a high affinity for calcium, and an amino terminus blocked to Edman degradation. The properties of the native and the recombinant proteins were examined by gel electrophoresis, isoelectric focusing, protein sequencing, amino acid composition analysis, and calcium binding assays. We demonstrated that: (i) the authentic and the full-length recombinant proteins have similar molecular weights and isoelectric points; (ii) the proteins have the same amino acid composition; (iii) the proteins bind calcium in a similar manner; (iv) the absence of a blocking NH2-terminal group in the recombinant protein does not appreciably influence the binding of calcium. To further examine the calcium binding properties of this protein, we constructed deletion mutants lacking one or both of the two putative degenerated calcium binding sites (EF hand regions). These deletions resulted in smaller proteins that still bound calcium. The ability to express and purify calbindin D28 and mutants thereof should allow the systematic elucidation of structure-function relationships in this class of calcium binding proteins.     

Porcine serum cobalophilin and transcobalamin. Identification, isolation and properties including electrofocusing patterns

Pooled porcine serum was found to contain cobalophilin (also called transcobalamin I) and transcobalamin (also called transcobalamin II). The two proteins were harvested by batchwise absorption with vitamin B-12 covalently coupled to Sepharose, and then separated from each other either by gel filtration or using an immunoadsorbent. Both proteins were finally isolated as single proteins using a second vitamin B-12-Sepharose chromatography step. Cobalophilin and transcobalamin complexed with vitamin B-12 had molecular weights by gel filtration of 135 000 and 38 000 and by the formula of Svedberg 104 000 and 44 000, Stokes radii 4.97 nm and 2.65 nm, and sedimentation coefficients 5.39 S and 3.75 S, respectively. Electrofocusing resolved the cobalophilin complex into three main isoproteins isoelectric at pH 3.23, 3.42 and 3.69, and transcobalamin into only the main component isoelectric at a value as low as pH 3.47. Neither protein was capable of binding to the ileal intrinsic factor receptor.     

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.     

The transport of 25-hydroxycholecalciferol in a New World monkey.

Albumin is responsible for the transport of 25-hydroxycholecalciferol in the Capuchin monkey. This was confirmed by gel filtration, analytical polyacrylamide-disc-gel electrophoresis, polyacrylamide-gel isoelectric focusing and a competitive protein-binding assay. Association constants of the serum transport proteins of a New and an Old World monkey towards 25-hydroxycholecalciferol were calculated; the transport protein in the New World monkey has a lower affinity for the vitamin D metabolite than the transport protein in the Old World primate.