Sidney Futterman

Metabolism of glucose and reduction of retinaldehyde in retinal photoreceptors.

—The capacity of a variety of substrates to support reduction of retinaldehyde to retinol by preparations of outer segments from photoreceptor cells of bovine retina, the requirement for supplements, the effect of iodoacetate, and the stoichiometry of the metabolism of glucose and the reduction of retinaldehyde have been studied. Metabolism of glucose supported reduction of retinaldehyde by preparations of outer segments at approximately the rate observed for the intact retina. Throughout the physiological range of pH, the dehydrogenase reactions of the pentose cycle in outer segments provided virtually all of the reduced pyridine nucleotides that were utilized for reduction of retinaldehyde. It was concluded that reduction of retinaldehyde during light adaptation is exclusively dependent upon NADPH.

Retinol and retinoic acid-binding proteins in bovine retina: aspects of binding specificity.

Bovine serum contains no apoprotein of low molecular weight with the capacity to bind either [3H]retinol or [3H]retinoic acid, and the intact retinol-binding protein complex of bovine serum is resistant to exchange of ligand. Acetone-extracted serum bound both ligands, but no evidence of the presence of any low molecular weight protein showing preferential binding of retinoic acid could be found. Bovine retina contains low molecular weight proteins capable of binding and exchanging [3H]retinol and [3H]retinoic acid, one displaying specificity toward retinol and another showing greater specificity toward retinoic acid.

Retinol-binding protein in bovine retina: isolation and partial characterization.

Abstract A supernatant fraction prepared from bovine retina was examined for the presence of protein-bound forms of vitamin A. Five fractions displaying a retinol-like fluorescence were resolved by DEAE-cellulose ion exchange chromatography. Two of the fractions were examined in more detail. Two retinol-binding proteins of apparent molecular weight 17 500 (a doublet band) were isolated from fraction V, the only fraction to bind exogenous [ 3 H]retinol. Interaction of retinol with the protein components of this fraction results in a considerable stabilization of retinol fluorescence. Two retinol-binding components (a doublet band) of similar apparent molecular weight were isolated from fluorescent fraction IV. Interaction of retinol with the components of this fraction was, however, weaker, based on fluorescence loss during gel filtration. The components of both fractions can be distinguished from the bovine serum retinol-binding protein by differences in size and electrophoretic mobility.

Separable binding proteins for retinoic acid and retinol in bovine retina

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [3H] retinoic acid which could not be effectively displaced by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [3H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.

The stability of 11-cis-retinal and reactivity toward nucleophiles

Abstract The prosthetic group of visual pigments, 11-cis--retinal, is the least resistant of the mono-cis isomers to isomerization and is converted to all-trans-retinal, about 20% in 16 h in aqueous solution at 37 °C, 5 % in the absence of solvent under the same conditions and virtually none at −22 °C in the absence of solvent or in solution in light petroleum. Several nucleophiles, such as dihydroFMN, dithiothreitol, dehydroascorbate, dihydrofolate, tetrahydrofolate, and dihydroriboflavin, promote the conversion of 11-cis-retinal to all-trans-retinal. In the dihydroFMN-catalyzed geometrical isomerization of 11-cis-retinal at 37 °C, the isomeric mixture at equilibrium consists of 61% all-trans-retinal, 15% 9-cis-retinal and 24%13-cis--retinal.

A simple biuret method for the estimation of protein in samples containing detergents

Abstract A biuret procedure suitable for the determination of protein in samples containing detergents has been developed. In the procedure, 1,2-propandiol is employed to solubilize the copper-protein complex. The method is applicable to samples in which protein is accompanied by phospholipid, urea, guanidine·HCl, ammonium sulfate and retinal, but not hydroxylamine.

Recent studies on a possible mechanism for visual pigment regeneration

Photoreceptor outer segments can couple retinol oxidation to flavin reduction. Dihydroflavins catalyze the interconversion of all-trans-retinal, 9-cis-retinal and 13-cis-retinal. Outer segments that have been rendered essentially free from mitochondrial flavoprotein contamination appear to contain bound flavin that after reduction by SnCl3 can function as an efficient catalyst for the geometrical isomerization of endogenous or exogenous all-trans-retinal. The photosensitive pigment regenerated in each case is isorhodopsin.

[35] Cellular retinol-, retinal-, and retinoic acid-binding proteins from bovine retina

Publisher Summary This chapter discusses cellular retinol-, retinal-, and retinoic acid-binding proteins from bovine retina. An aqueous extract of retina contains three separable retinoid-binding proteins with specificities directed toward retinol, retinoic acid, and retinal. The latter binding protein may be unique to retina, whereas binding proteins specific for retinol and retinoic acid have been reported in many other tissue extracts. The presence of retinoid-binding proteins in tissue extracts may be detected by incubation with radiolabeled ligand (retinol, retinal, or retinoic acid) followed by gel filtration chromatography. Liquid scintillation counting of the column fractions typically reveals peaks at both the void, and included volume of the column in addition to the binding protein peak. The cellular retinol-, retinoic acid-, and retinal-binding proteins are acidic proteins with apparent molecular weights of 16,600, 16,300, and 33,000, respectively, as determined by polyacrylamide gel electrophoresis in the presence of SDS. Each appears to form a one-to-one complex with its respective ligand. Interaction of retinol with the cellular retinol-binding protein results in the appearance of fine structure in the absorption spectrum of the ligand and a red shift in the observed absorption maximum.

A Catalytic Role of Dihydroriboflavin in the Geometrical Isomerization of all-trans Retinal

The mechanism of visual pigment regeneration during dark adaptation has remained obscure despite sporadic study in a number of laboratories (1–7). It has, however, been clearly established (8) that the apoprotein of rhodopsin reacts spontaneously with 11-cis retinal to regenerate the visual pigment. If 11-cis retinal arises through the direct geometrical isomerization of all-trans retinal, it would then seem reasonable to look for the occurence of such a reaction in photoreceptor outer segments where the Schiff base, metarhodopsin II, and possibly free all-trans retinal occur transiently as a result of visual pigment bleaching. Our recent studies (9, 10) indicate some of the metabolic requirements for rapid complete regeneration of photosensitive pigment in preparations of bleached bovine photoreceptors incubated with all-trans retinal.

Inadequacy of [15-3H]all-trans-retinal as a tracer for 11-cis-retinal formation

Ever since its synthesis and application to the identigcation of the retinal-opsin linkage by Akhtar et al. (1967). [1S3H]retinal has proved valuable in visual pigment research. It has been used to demonstrate the occurrence of 1 i-cis-retinaI-binding protein in the retina (Futterman et nl, 1977) and to provide evidence for the existence of a physiological mechanism for the formation of ll-&retinal from all-transretinal in slightly bleached rat retinae (Amer and Akhtar, 1973). In the use of tritium labeled all-nonsretinal to study visual pigment regeneration there is an implicit assumption that no difference occurs in the reactivities of labeled and unlabeled molecules of retinal. We examined this assumption to evaluate the suitability of [lS-3HJall-~~~s-retinal for studies on the possible participation of the I!-cis-retinal-binding protein in the conversion of all-trans-retinal to the 1 l-&s co~gu~tion and have observed a discrepancy attributabie to an isotope effect, a problem that was not encountered with [1S-13C]retinal.