Crystallization and preliminary X-ray data of human plasma retinol-binding protein

Crystals of human plasma retinol-binding protein have been obtained from 4.5 m-NaCl buffered at pH 6.8 with 20 mm-cacodylate. The crystals are trigonal with space group R3 and unit cell dimensions, referred to the hexagonal system. $\text{a = b = 104.2}\text{A}\text{?}\text{and}\text{c = 74.5}\text{A}\text{?}$. The crystals diffract to a resolution of 2.0 Å.     

Isolation and immunological determination of retinol-binding protein in human plasma

A retinol-binding protein and prealbumin both in the homogeneous state are isolated from human blood serum. Immunization of rabbits is used to obtain antibodies against the retinol-binding protein; the highly specific method is developed for quantitative determination of the content of retinol-binding protein in human blood. The method may be widely applied in the biochemical and clinical practice.     

Purification of human plasma retinol-binding protein by hydrophobic interaction chromatography

Human plasma retinol-binding protein has been purified to homogeneity by a simple method that requires an ammonium sulfate fractionation, a hydrophobic interaction chromatography on phenyl-Sepharose, which dissociates the complex between retinol-binding protein and its carrier, transthyretin, and a gel filtration on Sephadex G-50. The yield of pure protein is comparable or higher than that obtained with the more complex procedures previously reported.     

Structure of chicken plasma retinol-binding protein.

The crystal structure of the specific carrier of retinol (retinol-binding protein, RBP) purified from chicken plasma has been determined (space group P2(1)2(1)2(1), with a=46.06(5) A, b=53.56(6) A, c=73.41(8) A, and one protein molecule in the asymmetric unit). Despite being obtained from a species phylogenetically distant from mammals, chicken holoRBP has an overall structure that closely resembles the previously determined structures of mammalian holoRBPs. The lack in chicken RBP of eight carboxy-terminal amino acid residues characteristic of mammalian RBPs does not significantly affect the protein structure. A distinctive feature of the avian protein is a better definition of the loop 63-67, close to the opening of the beta-barrel cavity accommodating the retinol molecule, which is rather disordered in the structures of mammalian RBPs.     

A slab gel electrophoresis technique for measurement of plasma retinol-binding protein, cellular retinol-binding and retinoic-acid-binding proteins in human skin

At least four different proteins that bind retinoids could be present in a vitamin A target tissue like the skin. In order to separate cellular retinoid-binding proteins (CRBP and CRABP) from serum retinol-binding protein (RBP) and albumin, a one-step procedure was devised. The technique is based on slab polyacrylamide gel electrophoresis (PAGE) of the extracted proteins incubated with tritiated retinoids. The procedure was used to study binding proteins in the skin. The results show that epidermal extracts (the epithelial part of the skin) contain no RBP activities whereas dermal extracts (the mesenchymal part of the skin) contain 1.6 +/- 0.81 pmol/mg protein of RBP. This technique further showed higher levels of CRABP in both epidermal (9.05 +/- 1.16 pmol/mg protein) and dermal (1.5 +/- 0.54 pmol/mg protein) extracts than those previously determined by other less specific techniques. On the other hand CRBP levels were found to be lower in the two tissues (epidermis 0.2 +/- 0.1 pmol/mg and dermis 0.12 +/- 0.05 pmol/mg protein). New conditions to measure specifically CRABP with the charcoal/dextran technique could be developed and analyzed by the PAGE technique; a dissociation constant of 13.7 nM was then calculated for epidermal CRABP. This PAGE technique appears to be the most appropriate method for the study of retinoid-binding proteins including RBP in human skin.     

Liver takes up retinol-binding protein from plasma

Retinol is transported in plasma bound to a specific transport protein, retinol-binding protein. We prepared 125I-tyramine cellobiose-labeled rat retinol-binding protein and studied its tissue uptake 1, 5, and 24 h after intravenous injection into rats. The liver was the organ containing most radioactivity at all time points studied. After 5 and 24 h, 30 and 22% of the injected dose were recovered in liver, respectively. After separating the liver into parenchymal and nonparenchymal cells in the 5-h group, we found that both cell fractions contained approximately the same amount of radioactivity (per gram of liver). Most of the retinol-binding protein radioactivity in the nonparenchymal cell fraction was in the stellate cells. The implication of these results for a possible transfer mechanism for retinol between parenchymal and stellate cells is discussed.     

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.     

Ultrastructural localization of plasma retinol-binding protein in rat liver

Immunocytochemical studies were carried out to examine the subcellular localization of plasma retinol-binding protein (RBP) in rat liver. The studies used normal, retinol-deficient, and retinol-repleted retinol-deficient rats with or without colchicine pretreatment. Affinity-purified monomeric Fab' fragments from the IgG fraction of rabbit anti-rat RBP were conjugated to horseradish peroxidase. This conjugate effectively penetrated into tissue sections and enabled RBP to be localized by high resolution immunoelectron microscopy. In the normal liver parenchymal cell, RBP was found to be localized in the synthetic and secretory structures including endoplasmic reticulum (ER), Golgi complex (GC), and secretory vesicles. With the method used, significant localization of RBP was not observed in hepatic cells other than parenchymal cells. The distribution of RBP-positive areas within parenchymal cells changed markedly with retinol depletion. Thus, a heavy accumulation of RBP in the ER, accompanied by a marked decrease of the RBP-positive GC and secretory vesicles, was demonstrated in liver parenchymal cells from retinol-deficient rats. After repletion of deficient rats with retinol, the RBP that accumulated in the ER appeared to move rapidly from the ER through GC and secretory vesicles to the cell surface. Pretreatment with colchicine led to marked increase in RBP-positive secretory vesicles in retinol-repleted rat liver parenchymal cells. The results reported here demonstrate that the specific block in hepatic RBP secretion seen in retinol deficiency involves an inhibition of the movement of RBP from the ER to the GC in the parenchymal cell.     

Distribution of retinol-binding protein in the human digestive tract.

By employing polyclonal antibodies for retinol-binding protein (RBP), its distribution in the human pancreas and digestive tract mucosa was compared with those of transthyretin (TTR) and various peptide hormones. The materials used included surgically removed pancreas, esophagus, stomach, small and large intestines. Paraffin sections were stained by the indirect immunoenzyme method. The results indicate that RBP-containing cells are found in the pancreas and the gastrointestinal mucosa, but most frequently in the gastric antrum and duodenum. In the pancreas, RBP-containing cells are found in the islets and among acinar and ductal epithelial cells, and consistently stain for chromogranin A. RBP-containing cells in the gastrointestinal mucosa showed typical features of endocrine cells and also stained for chromogranin A. The distribution of TTR in these tissue sites resembled that of RBP, but the immunoreactive intensities of both peptides altered independently. Comparison of the distribution of RBP, TTR, and various gastrointestinal peptide hormones revealed that the distribution of RBP coincided with none of the other peptides, although some of the RBP-containing cells stained for most of the peptides examined and vice versa. These results suggest that RBP may be a consistent component of gastrointestinal endocrine cells.     

Fluorescence studies of the complex formation between the gene 5 protein of bacteriophage M13 and polynucleotides

The binding characteristics of the interaction of gene 5 protein with polynucleotides, i.e. poly(dA), poly(dT) and M13 DNA, have been determined by following the quenching of the protein fluorescence. In general, the binding is highly co-operative and for the binding of the protein to poly(dA) and M13 DNA the co-operativity parameter ω is estimated to have values between 50 and 300. Under comparable experimental conditions, the intrinsic binding constant K_int is at least two orders of magnitude higher for poly(dT) than for poly(dA), while the value for M13 DNA is intermediate. For poly(dA), the binding has been studied as a function of ionic strength and temperature. From these experiments it can be concluded that ionic interactions as well as van der Waals interactions (e.g. stacking interactions) are important for the complex formation of the protein with polynucleotides. From a comparison of the binding of the protein to poly(dA) and poly(dT), it is concluded that stacking interactions in the polynucleotide have a negative influence on protein binding. This conclusion, in conjunction with the weak temperature dependence of K_int. indicates that ionic interactions play a major role in the stabilization of the protein-poly(dA) complex. The co-operativity factor ω is little or not dependent on the ionic strength or the type of polynucleotide involved in binding. It is determined by interactions between complexed protein molecules. These interactions are primarily non-electrostatic.The binding characteristics obtained for the gene 5 protein-polynucleotide complexes are compared with those we have found for the binding to small oligonucleotides. It appears that oligonucleotide and polynucleotide binding differ in many aspects; i.e. there is a difference in K_int, ω and the number of nucleotides covered. The validity of linear lattice binding theories is discussed in this context. By comparing the binding parameters found for the gene 5 protein with those of the Escherichia coli DNA binding protein I. it is possible to explain the displacement of the E. coli protein by the gene 5 protein that occurs in vivo.