Joram Heller

A specific receptor for retinol binding protein as detected by the binding of human and bovine retinol binding protein to pigment epithelial cells.

By means of autoradiographic techniques the specific plasma carrier of retinol, namely retinol binding protein (RBP) in a radioactive form (retinol-125I.RBP), bound specifically in vivo to the choroidal surface of intact, isolated bovine pigment epithelial cells. The retinol-125I.RBP did not bind to the retinal surface of the pigment epithelial cells nor did not bind to photoreceptors. Retinol is normally provided to the retina from the blood via a specific complex formation with a receptor on the chordial surface of the pigment epithelial cells. Retinol metabolism might be deranged in some diseases through a defect in the pigment epithelial receptor for RBP.

Purification and evidence for the identity of chicken plasma and egg yolk retinol-retinol binding protein-prealbumin complex☆

Abstract Retinol (vitamin A)-retinol binding protein-prealbumin complex was isolated and purified from chicken plasma and hens egg yolk by repeated chromatography on ion exchange and gel filtration columns. Chicken plasma and egg yolk retinol-retinol binding protein-prealbumin complex were similar in molecular weight, electrophoretic mobility, amino acid composition, spectroscopic properties, and dissociability in low ionic strength buffers. No retinol-retinol binding protein was found in the egg albumin. The materials isolated from chicken plasma and egg yolk, respectively, were therefore judged to be identical. The recent finding of specific cell surface receptors in target organs for retinol binding protein (Heller, J., J. Biol. Chem. 250, 3613, 1975) makes feasible a study of the ontogenesis of these receptors in the developing chick embryo.

Retinyl esterase activity of purified rat liver retinyl ester lipoprotein complex

Abstract Retinyl ester lipoprotein complex from rat liver was shown to possess a retinyl esterase activity toward its own ligand complement. In the presence of serum albumin the retinyl esterase activity at 30 °C was about fivefold larger than the activity at 4 °C, while higher temperatures than 30 °C led to some degradation of retinyl compounds. The pH optimum was 7.8. The esterase activity was markedly enhanced by serum albumin although the serum albumin as such had no retinyl esterase activity. In the presence of serum albumin and under optimal conditions, some 75 to 80% of the total retinyl ester complement of the lipoprotein was hydrolyzed in 24 h. The retinyl esterase activity was totally abolished by treatment with the serine esterase inhibitor diisopropyl fluorophosphate (1.4 × 10 −4 M), by treatment with sulfhydryl reagents, and by detergents (0.2% of Tween 80 and sodium deoxycholate). From this series of experiments it was concluded that the retinyl ester lipoprotein complex possesses the additional physiological function of hydrolyzing its own retinyl ester complement to unesterified retinol which may then combine with serum retinol-binding protein.

Characterization of rat liver cytosol retinyl ester lipoprotein complex

Abstract A soluble high molecular weight lipoprotein complex containing retinyl esters and unesterified retinol was isolated from rat liver cytosol. This material accounts for about 10% of the total liver retinyl compounds, and its protein moiety accounts for about 0.1% of the protein of the liver homogenate and about 0.7% of the cytosol protein. The lipoprotein was purified by gel filtration and hydroxyapatite chromatography. The lipoprotein complex gave a single band by electrophoresis on cellulose acetate as judged by both lipid- and proteinspecific stains. The lipoprotein complex did not dissociate into smaller subunits in low ionic strength buffer (1 m m sodium phosphate, pH 7.7). The retinyl ester lipoprotein complex has an absorption spectrum with peaks at 328 nm (retinyl chromophore) and 258 nm. Retinyl compounds in the carrier lipoprotein complex do not show an increase of the quantum yield of fluorescence and do not show energy transfer when excited at either 258 or 280 nm. There is no induced optical activity of the retinyl chromophore absorption band. The lipoprotein complex contains about 3% (by weight) of retinyl compounds, 96% of which are retinyl esters and 4% of which are unesterified retinol. The lipoprotein complex consists of about 66% (by weight) lipids, about 30% protein, and some 4% carbohydrate. There are at least 15 polypeptide chains ranging in size from about 2 × 10 4 to about 2.1 × 10 5 M r . Retinyl compounds in the lipoprotein complex are stable for at least 3 months in 0.05 m phosphate buffer, pH 7.4, at 4 °C. Stability was judged from the total amount of retinyl esters plus unesterified retinol. Retinyl compounds of the lipoprotein complex were unstable below pH 6.4 or in the presence of 1 m NaCl.

Retinol transfer from rat liver cytosol retinyl ester lipoprotein complex to serum retinol-binding protein

Abstract Cytosol retinyl ester lipoprotein complex from rat liver was capable of transferring its unesterified retinol component to serum aporetinol-binding protein. In the presence of serum albumin and aporetinol-binding protein, 68% of retinyl ester was hydrolyzed and up to 30% of unesterified retinol was transferred from cytosol retinyl ester lipoprotein complex to serum aporetinol-binding protein in 24 h at 30 °C. The reconstituted retinol-retinol-binding protein complex showed biochemical and biophysical properties similar to native retinol-retinol-binding protein. Both native and reconstituted retinol-retinol-binding proteins had identical uv, CD, and fluorescence spectra as well as binding affinity to prealbumin. Treatment of cytosol retinyl ester lipoprotein with sulfhydryl reagent, with 1 n NaCl, or with diisopropyl fluorophosphate (0.14 m m ) abolished the hydrolysis of retinyl ester; however, the activity of retinol transfer from cytosol retinyl ester lipoprotein complex to serum retinol-binding protein was still unaffected. The activity of retinol transfer was proportional to the amount of retinol content in the complex and the amount of aporetinol-binding protein. These experiments suggest that the cytosol retinyl ester lipoprotein complex serves three major functions: (i) as a storage form of retinyl ester and retinol; (ii) as an enzyme for hydrolyzing its own retinyl ester ligand; and (iii) as a medium for transfer of unesterified retinol to serum retinol-binding protein.

Interactions of retinol-binding protein with various chromophores and with thyroxine-binding protein. A model for visual pigments

Abstract The properties of retinol-binding protein from human serum were compared with those of rhodopsin in an attempt to learn more about the role of protein in visual pigments. In the serum, retinol-binding protein (mol wt. 21 000) tightly binds one molecule of all-trans-retinol and one molecule of the tetrameric protein prealbumin (mol. wt. of tetramer, 54 000). Apo-retinol binding protein was found to be completely dissociated from prealbumin under conditions in which native retinol-retinol-binding protein and reconstituted all-trans-retinol-retinol-binding protein were tightly bound. This difference in binding to prealbumin between apo- and holo-retinol-binding protein is due most probably to different conformations of the apo- and holo-protein. By analogy, it is suggested that the incorporation of the retinal chromophore into apo-visual pigment might act as recognition marker for the incorporation of visual pigment into the photoreceptor disc membrane. Apo-retinol binding protein was shown to form stable 1 : 1 molar complexes with retinols, retinals, retinoic acid, retinyl acetate and retinyl oxime, but not with retinyl palmitate. Only the retinol isomer and retinoic acid-retinol-binding protein complexes were bound to prealbumin at physiological ionic strength, while the other chromophores-retinol·binding protein complexes were not. The various chromophores were all bound to the same site on the retinol-binding chromophore, the binding was noncovalent and irreversible under normal physiological conditions. All the chromophores-retinol-binding protein complexes showed a large induced optical activity of the chromophore absorption band upon binding to the protein. The rotatory strength of the circular dichroism bands of the various chromophore retinol-binding protein complexes was of the same order of magnitude as that of rhodopsin. The binding of retinol and retinal to bovine serum albumin did not produce optical activity in the chromophore absorption band.

Rhodopsin: conformational changes in a membrane protein.

Rhodopsin is one of the photosensitive pigments that mediate vision in vertebrates. As yet it is the only receptor molecule that can be isolated in relatively large quantities in a pure form. We have been studying rhodopsin as an example of a biological transducer that converts light into a nervous impulse, apart from its interesting complex photochemistry. An understanding of the overall function of visual pigments involves not only a detailed descnption of their molecular structure and photochemistry, but also a consideration of their site and organization in the photoreceptor cell. In this paper we will describe some of our studies on the structure and transformations of rhodopsin, both as an isolated protein and as a component of the photoreceptor cell transducer apparatus.