C.D.B. Bridges

Vitamin a transport between retina and pigment epithelium—an interstitial protein carrying endogenous retinol (interstitial retinol-binding protein)

We have demonstrated and partially characterized an interstitial retinol-binding protein (IRBP) confined to bovine interphotoreceptor matrix (IPM). The native protein is a concanavalin A-binding glycoprotein with a mol. wt of 260 k as measured by gel-filtration and size-exclusion high-performance liquid chromatography. On SDS-gels, its mol. wt is 140-145 k. Since the protein is glycosylated, this value is probably too high. Hence, the native protein may be a dimer consisting of two identical subunits. The endogenous ligand has been analyzed by high-performance liquid chromatography--it consists mainly of all-trans retinol. Occasionally, retinal and 11-cis retinol are also associated with it. The amount of retinol bound to IRBP increases when the eyes are illuminated. The total binding capacity was estimated to represent 4-5% of the retinol released from a total rhodopsin bleach. We have established that, like serum retinol-binding protein, IRBP can be also bind retinoic acid, although it has not been established that retinoic acid is an endogenous ligand. The fluorescence emission lambda max for IRBP with its native ligand is at 470 nm and the excitation lambda max for this fluorescence is at 333 nm. Other retinoid carriers in the interphotoreceptor matrix have molecular weights of about 15 and 33 k. These probably correspond to cellular retinol- and retinal-binding proteins, respectively. Since both proteins have been identified in the pigment epithelium and retina cytosols, their presence in the IPM could be a result of cell damage. We conclude that interstitial retinol-binding protein is the best candidate for a transport protein carrying retinol between the rod outer segments and the pigment epithelium.

Vitamin A and the role of the pigment epithelium during bleaching and regeneration of rhodopsin in the frog eye

Abstract The distribution of 11- cis and all- trans isomers of retinol and retinyl ester was investigated in the frog eye during light-and dark-adaptation (LA and DA). It was also established that freshly isolated rod outer segments (ROS) could regenerate their rhodopsin almost completely from added 11- cis retinol, provided that NADP was also present. The DA frog eye stores vitamin A equivalent to 120–230 mol% of the rhodopsin in the ROS. About 96% is in the RPE and is concentrated mainly in the oil-droplets (which can be separated by centrifugal flotation), the remaining 4% being split roughly equally between ROS and the rest of the retina. The oil-droplet vitamin A is virtually 100% esterified. Vitamin A occurs mainly as ester elsewhere, with the notable exception of the ROS, where the alcohol predominates. As much as 60% of this retinol can be 11- cis , and probably constitutes a pool of prosthetic groups for rhodopsin biosynthesis in the dark. In frogs dark-adapted (DA) for more than a day, more than half of the oil-droplet retinyl ester can be 11- cis . Since ROS lack an esterifying capability, all- trans retinol accumulates during LA of the isolated retina. If contact with the retinal pigment epithelium (RPE) is maintained, however, at least 87% of this retinol leaves the ROS, is esterified in the RPE and accumulates as all- trans retinyl ester in the oil-droplets. The 11- cis isomer is gradually consumed during LA and DA. The form in which vitamin A returns to the ROS has not been established, but some lines of evidence suggest that it is as the ester, mainly in the all- trans configuration. Isomerization to 11- cis probably takes place in the ROS, and during prolonged DA this appears to “leak” back to the RPE and cause a slow rise in the proportion of 11- cis over 20–30 hr in darkness (it should be noted that this effect cannot be accounted for by an influx of prosthetic groups from phagocytized ROS particles). Since the isolated retina has only a limited regenerative capacity, it is suggested that the ROS isomerase is specific for retinyl ester, which under normal circumstances can only be provided by the RPE.

Spectroscopic properties of porphyropsins.

Abstract The difference spectra of porphyropsins bleached at various pH as well as in the presence of hydroxylamine and sodium borohydride are described, and their relationships with absorption spectra are discussed. Since bleaching in NaBH 4 results in the formation of 3-dehydro N-retinylopsin, with an absorption band well-separated from those of the porphyropsins, difference spectra obtained in the presence of this reagent are very close to the corresponding absorption spectra over a considerable wavelength range. These difference spectra, together with the absorption spectrum of a porphyropsin extract still containing some impurities, are used to obtain an approximation to the absorption spectrum of pure porphyropsin. Bleaching in the presence of hydroxylamine also gives difference spectra which are quite close to the absorption spectrum, but over a more restricted range. Plotted against a wavenumber scale, instead of wavelength, porphyropsin difference spectra (hydroxylamine) ranging in λ max from 521 to 543 nm are identical in shape. Since we are considering only those regions unaffected by photoproduct absorption, this is applicable to the absorption spectra as well. These observations are used as a basis for tabulating a standard porphyropsin spectrum. Tables arc included which enable easy calculation of the Amax of any porphyropsin, given its difference spectrum: conversely, they may be used to predict the spectrum of any unknown porphyropsin, given its λ max . A different, narrower standard must be used in the case of rhodopsins. The spectroscopic properties of porphyropsins differ from those of rhodopsins with respect to absorption maxima, extinction coefficients and spectrum shape. Since corresponding differences are found between compounds of the retinol and 3-dehydroretinol series, it is concluded that similar features of chromophoric structure influence light-absorption in both the photopigments and these substances.

[63] Measurement of the vitamin A cycle

Publisher Summary This chapter explores the measurement of the vitamin A cycle. In the vertebrate eye, vitamin A occurs in two major forms: either in visual pigment as 11-cis-retinal attached by a Schiff base linkage to the Є-amino group of a lysine residue or stored in the pigment epithelium as the 11-cis-and all-trans-retinyl esters of long-chain fatty acids. In the dark-adapted eye, only a small proportion is found as the free alcohol, some probably attached to cytosol binding proteins, some occurring in the rod outer segment membranes. This situation is critically dependent on the state of adaptation. In the light, all-trans-retinol is formed when the visual pigment bleaches and flows from the retina into the pigment epithelium, where it is esterified. A number of colorimetric techniques for measurement of vitamin A are described in this chapter. However, in relation to the visual system the Carr–Price reaction is still one of the most useful in that it permits measurement of vitamins A1 and A2 simultaneously and is independent of the isomeric configuration and state of esterification.

Rhodopsin, 11-cis vitamin A, and interstitial retinol-binding protein (IRBP) during retinal development in normal and rd mutant mice

Biochemical and immunological techniques were used to determine the emergence of interstitial retinol binding protein (IRBP), rhodopsin, and stored retinyl esters (all-trans and 11-cis) during retinal development in normal and rd mice. IRBP could be demonstrated at embryonic Day 17 (E17), corresponding to an early stage of inner segment development. Although all-trans retinyl esters were present earlier, 11-cis retinyl esters did not appear until postnatal Days 6-7 (P6-P7), corresponding to rod outer segment (ROS) disc formation. Rhodopsin was detected at the same developmental stage. The proportion of 11-cis retinyl esters reached a maximum of 40-50% at P15-P20. Thereafter, the proportion dropped, due to more rapid accumulation of the all-trans isomer. Rhodopsin and IRBP increased in parallel with ROS elongation up to P25, when the ROS had reached their mature lengths. The increases then continued up to P40-P50. In rd (retinal degeneration) mice, IRBP and rhodopsin were identical with the controls until P12, but then dropped as the photoreceptors degenerated. Synthesis and secretion of IRBP in vitro was less than 10% of the controls in rd retinas at P26, when only 4-5% of the photoreceptors survived. The quantities of retinyl esters (mainly stearate and palmitate in the ratio of 6:1, respectively) stored in dark-adapted mouse eyes progressively increased as the animals aged, representing 0.5 mole eq. of the rhodopsin at 8 months. Although retinyl esters (11-cis and all-trans) also accumulated in rd mouse eyes up to P12, little further increase occurred. At P93, the retinyl esters (0.01 nmole X eye-1) were only 4% of the controls at P91. A peak in the proportion of 11-cis isomer occurred at P10-P20, but it averaged only 15% of the total ester and declined to 5% at P93. These findings support the hypothesis that IRBP is synthesized by the rods and cones, and suggest that its synthesis and secretion are initiated when the photoreceptor inner segments start to differentiate. 11-cis Retinoids and rhodopsin do not appear until the outer segments start to form. It is suggested that in the rd mouse the absence of photoreceptors, perhaps coupled with lack of normal interphotoreceptor matrix, leads to a loss in the ability of the pigment epithelium to store retinyl esters.

[20] Detergents for extraction of visual pigments: Types, solubilization, and stability

Publisher Summary This chapter discusses the detergents for extraction of types, solubilization, and stability of visual pigments. Visual pigments are intrinsic membrane proteins. They are insoluble in water; hence their characterization can only be accomplished with the aid of appropriate solubilizing agents, usually detergents. The choice of detergent is critically dependent on the needs of each individual investigator. Factors to be considered include detergent solubility, efficiency of membrane solubilization, visual pigment stability, regenerability, acceptable UV transmission, ease of removal, and defined chemical structure and purity. The majority of comparative studies on visual pigments from various species, however, have been carried out with digitonin. Commercial-quality digitonin usually precipitates in 1–2% aqueous solutions. The problem has been substantially overcome by isolating the soluble component, but this material has not been defined chemically. Although visual pigments are very stable in digitonin extracts, the agent is a very inefficient extractant.

Storage, distribution and utilization of vitamins A in the eyes of adult amphibians and their tadpoles.

Abstract This study has investigated the quantities, isomeric configuration and ocular distribution of the two retinols (vitamins A 1 and A 2 ) in the eyes of goldfish. Xenopus . frogs and tadpoles, with particular emphasis on the shifting relationships of rhodopsin, porphyropsin. retinol and 3-dehydroretinol in tadpoles kept in light and darkness. The amounts of retinols (mainly esterified) stored in the dark-adapted eyes range from 41 moles per mole of visual pigment in the goldfish down to 0.4 mole in the Xenopus . The cleanly isolated frog retina has 0.07 mole of 11- cis and all- trans vitamin A (50 per cent esterified) per mole of ROS rhodopsin, the remainder (1 per cent esterified) being found in the pigment epithelium, 80 per cent in the oil-droplets separated by centrifugal flotation. A maximum of 3 per cent of the rhodopsin in the ROS is apparently in the pigment epithelium. During light-adaptation, retinol formed by rhodopsin bleaching in the ROS leaves the retina, enters the pigment epithelium where most of it passes into the oil-droplets. The dark-adapted stores always contain 11- cis isomer, which is present in least amount in eyes that have undergone one cycle of light- and dark-adaptation. During prolonged periods in the dark, there was an increase of 11- cis at the expense of all- trans retinyl ester. In illuminated tadpoles retinol and 3-dehydroretinol are utilized for visual pigment regeneration in the proportions present in the pigment epithelium. This is not observed in the dark, where the situation is dominated by the visual pigment renewal process. The prosthetic group used for visual pigment biosynthesis in the tadpole retina is mainly retinol, even though there is an overwhelming preponderance of 3-dehydroretinol in the pigment epithelium.

Cones in the retina of the Lemon Shark (Negaprion brevirostris)

Abstract Sections of the retinae from six Lemon Sharks, Negaprion brevirostris, have been examined. These show that the Lemon Shark retina contains rods and cones, the former preponderating in the ratio of about 12:1.

Visual cycle in the mammalian eye. Retinoid-binding proteins and the distribution of 11-cis retinoids

This work was designed to provide an insight into the mammalian visual cycle by investigating the possible function of retinoid-binding proteins in this system, and the distribution and type of 11-cis retinoids present in the interphotoreceptor matrix and the cytosols of the retinal pigment epithelium and retina. The total retinol and retinal in the soluble fractions from these three compartments was 8% (3.31 nmol/eye) of the retinyl palmitate and stearate stored in the pigment epithelium membrane fractions (39 nmol/eye). Only small amounts of retinoids were detected in the rod outer segment cytosol. The insoluble fractions also contained retinol, nearly all of which was found in the retina. The retinoids in the soluble fractions appeared to be bound to cellular retinol-binding protein (CRBP), cellular retinal-binding protein (CRA1BP) and interstitial retinol-binding protein (IRBP, a high-Mr glycoprotein). Using immunospecific precipitation, immunoblot and immunocytochemical techniques it was demonstrated that IRBP was localized in the interphotoreceptor matrix and was synthesized and secreted by the retina, a process that did not require the protein to be glycosylated. The amount of retinol bound to IRBP increased if the eyes were exposed to light, when it was estimated that the protein carried up to 30% of its full capacity for all-trans retinol. In addition to all-trans retinol, IRBP carried smaller amounts of 11-cis retinol. The proportion of 11-cis retinol was frequently higher in eyes that had been protected from illumination, suggesting that IRBP plays a role in rhodopsin regeneration during dark-adaptation. Additionally, endogenous 11-cis retinoids in the retina and RPE cytosols were bound to an Mr 33,000 protein tentatively identified as CRA1BP. The 11-cis retinoid in the retina cytosol was mainly in the form of retinol, while in the RPE cytosol it was mainly in the form of retinal. Substantial amounts of 11-cis retinol were also found in the insoluble (membrane) fraction from the retina. It is suggested that in the mammalian retina 11-cis retinol is generated from all-trans retinol (possibly in the Muller cells). Lack of an 11-cis retinol oxidoreductase in the retina prevents it from being utilized for rhodopsin regeneration until it has been transported to the pigment epithelium, where it is converted to 11-cis retinal and returned to the rod outer segments. It is also suggested that IRBP may be implicated in the transport of retinoids between the rod outer segments, the Muller cells and the pigment epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

The grouping of fish visual pigments about preferred positions in the spectrum

Abstract 1. The absorption maxima and vitamin A basis are reported for visual pigments from twenty species (eleven families) of fishes inhabiting fresh, brackish and marine waters of Florida and fresh waters of Wisconsin U.S.A. Nineteen of these species have pigments based on vitamin A2 with λmax between 521 and 536 nm. 2. In one-half of the species examined the vitamin A2 pigment was “paired” with another based on vitamin A1 with λmax between 498 and 505 nm. Five of these species were freshwater cyprinids whilst the others passed between marine and freshwatcrs or spent their entire lives in brackish waters. 3. If these results are considered in conjunction with those of other authors, it is seen that the λmax of vitamin A2 pigments in fishes tend to form a series of groups of clusters about certain preferred points in the spectrum, viz. 543, 534, 524 and 512 nm. Some pigments in each of these groups are paired with vitamin A1 pigments, probably using the same opsins, which have λmax grouped at 510, 506, 500 and 492 nm. 4. The central wavelength, λ, of a group is empirically expressed by the equation λ = nx + 462, where n is an integer (or series number) and x is best given by 6.3 for the vitamin A1 series and 10.1 for the vitamin A2 series. 5. It is suggested that these observations may be accounted for by two hypotheses. (a) The grouping of λmax is the result of adaptation to a corresponding series of lightenvironments. (b) The structures of visual pigment molecules only permit discontinuous variation of λmax. It is proposed that there exists a unique series of discrete opsins (differing from each other in a regular and stepwise manner) and that these combine with retinene1 or retinene2 chromophores to yield two visual pigment series.