Cattle Retina Research Articles

The acid mucopolysaccharides of cattle retina.

1. Two polysaccharides were isolated from the interstitial matrix surrounding the photoreceptor cells of cattle retina. They were liberated from this region of the tissue in a soluble form after agitation of whole retinas in 0.9% sodium chloride. One, which comprises two-thirds of the polysaccharides present, is a hyaluronidase-sensitive ;half-sulphated' chondroitin sulphate containing uronic acid, galactosamine and sulphate in the molar proportions 1.27:1.0:0.54. The other is a hyaluronidase-resistant non-sulphated heteropolysaccharide for which the name sialoglycan is proposed. It contains galactose, glucosamine and sialic acid in the molar proportions 2.4:1.0:0.4. Both polysaccharides contain only small amounts of nitrogen in excess of the amount calculated from their amino sugar and sialic acid content. 2. A similar combination of mucopolysaccharides is associated with the pigment epithelial-cell layer but in quantities only one-fifth of those present in the adjacent matrix area. 3. The ease with which they are released into aqueous media is consistent with the assumption that they are present in the extracellular spaces in both of these tissue layers. 4. The retinal residue left after removal of the two soluble polysaccharides is rich in amino sugar- and sialic acid-containing polymers, which appear to be firmly bound to the tissue fragments. 5. About one-third of the sialic acid and one-tenth of the amino sugar could be extracted with chloroform-methanol. The components in this fraction were tentatively identified as gangliosides. 6. Digestion of the chloroform-methanol-insoluble residue with Pronase yielded as the principal product a heteropolysaccharide containing 16.5% of glucosamine, 24.3% of neutral sugar (galactose plus fucose) and 18.1% of sialic acid. This substance has been classified as a sialoglycan of composition similar to (but not identical with) that of the soluble one isolated from the matrix area of the tissue.

Extraction of visual pigments with certain alkyl phenoxy polyethoxy ethanol surface-active compounds

Data are presented which show that certain water-soluble, nonionic, alkylphenol surfactants (Triton) are capable of extracting rhodopsins from the retinas of frog, bullfrog, albino rat and cattle. These extracts can be analyzed spectrophotometrically as readily and as precisely as extracts made with the commonly used digitonin. Moreover, the results are similar for both types of extracts. These Tritons have several practical advantages over digitonin as visual pigment extractants. The properties of these rhodopsins are similar when solubilized in the Tritons as when present in the digitonin environment.

Isolation of neutral sugar-containing mucopolysaccharides from cattle retina

Isolation of neutral sugar-containing mucopolysaccharides from cattle retina

The properties of rhodopsin particulates obtained by sonic treatment of cattle rod outer segments.

This paper deals with the best conditions for preparing rhodopsin particulates by means of sonic treatment of rod outer segments separated from the dark adapted cattle retina. Thorough washing and subsequent sonic treatment of water suspension of rod outer segments gave fairly high optical purities, namely, ca. 0.3 of E400mu/E500mu and ca.3.0 of E280mu/E500mu.As long as rhodopsin articulates were kept at 7°C, the absorption spectra remained fairly stable even 7days after preparation in spite of slightly growing turbidity of solution.Electron microscopic observation revealed that rhodopsin particulates consisted of globular particles in diameter of 50 A to 200 A. When illuminated, the diameter of particulate became larger, probably indicating the tendency of aggregation of particles upon illumination. On the basis of this observation, a model involving the rearrangement of rhodopsin lipo-protein micelle was postulated.Titration of rhodopsin particulates and the bleached products signified a decreasing tendency in the number of acid titrable groups of the latter which almost reached 15% of total titrable groups at PH 3.01. This result is not consistent with the titration data of rhodopsin digitonin complex. 87 groupswere titrable per mole of rhodopsin in the range of PH 3.75 to 10.0. Also, it seems true that the isoionic point of rhodopsin particulates (6.3-6.5) is shifted remarkably to the alkaline side than that of rhodopsin digitonin complex (5.2-5.3).This work was mainly performed at Honjo Laboratory, Department of Biology, Faculty of Science, Osaka University. The author is particularly obliged to Professor Ichijiro HONJO for his courtesy in making available the facilities of his laboratory and in preparing this manuscript.Sincere thanks are also due to Professor Sadayoshi KAMIYA, Dapartment of Ophthalmology, Medical School of Nara, for his generous help in making this accomplishment possible.The author is also indebted to Dr. Yuzo SEKOGUCHI of Honjo Laboratory for his valuable suggestion and kind help throughout this experiment, and to Dr. Akira TANAKA for his technical contribution in preparing the electron microscopic pictures.

Metabolism of the Retina: II. HETEROGENEITY AND PROPERTIES OF THE LACTIC DEHYDROGENASE OF CATTLE RETINA

Metabolism of the Retina: II. HETEROGENEITY AND PROPERTIES OF THE LACTIC DEHYDROGENASE OF CATTLE RETINA

Retinene isomerase.

Rhodopsin is formed by the condensation of opsin with a cis isomer of retinene, called neo-b. The bleaching of rhodopsin releases all-trans retinene which must be isomerized back to neo-b in order for rhodopsin to regenerate. Both retinene isomers are in equilibrium with the corresponding isomers of vitamin A, through the alcohol dehydrogenase system. An enzyme is found in cattle retinas and frog pigment layers which catalyzes the interconversion of all-trans and neo-b retinene. We call it "retinene isomerase." It is soluble in neutral phosphate buffer, and precipitates between 20 and 35 per cent saturation with ammonium sulfate. In the dark, the isomerase converts all-trans and neo-b retinene to an equilibrium mixture of 5 parts neo-b and 95 parts all-trans. With opsin present to trap neo-b, the isomerase catalyzes the synthesis of rhodopsin from all-trans retinene. This reaction, however, is too slow to account for dark adaptation. Retinene is isomerized by light, but too slowly to supply the retina with neo-b. In aqueous solution the pseudoequilibrium mixture contains about 15 per cent neo-b. When all-trans retinene is irradiated in the presence of isomerase, the rate of formation of neo-b is increased about 5 times, and the pseudoequilibrium shifted so that the mixture now contains about 32 per cent neo-b. The isomerase is specific for all-trans and neo-b retinene. It does not act on two other cis isomers of retinene, nor on all-trans or neo-b vitamin A. The role of the isomerase in vision appears to be as follows: in the light, as rhodopsin is bleached to opsin and all-trans retinene, the latter is in part converted to the neo-b isomer and stored in the pigment epithelium as neo-b vitamin A. During dark adaptation, the dominant process is the trapping by opsin of neo-b retinene supplied from stores of neo-b vitamin A, and the slow isomerase-catalyzed "dark" conversion of all-trans to neo-b retinene.

The role of sulfhydryl groups in the bleaching and synthesis of rhodopsin.

The condensation of retinene(1) with opsin to form rhodopsin is optimal at pH about 6, a pH which favors the condensation of retinene(1) with sulfhydryl rather than with amino groups. The synthesis of rhodopsin, though unaffected by the less powerful sulfhydryl reagents, monoiodoacetic acid and its amide, is inhibited completely by p-chloromercuribenzoate (PCMB). This inhibition is reversed in part by the addition of glutathione. PCMB does not attack rhodopsin itself, nor does it react with retinene(1). Its action in this system is confined to the -SH groups of opsin. Under some conditions the synthesis of rhodopsin is aided by the presence of such a sulfhydryl compound as glutathione, which helps to keep the -SH groups of opsin free and reduced. By means of the amperometric silver titration of Kolthoff and Harris, it is shown that sulfhydryl groups are liberated in the bleaching of rhodopsin, two such groups for each retinene(1) molecule that appears. This is true equally of rhodopsin from the retinas of cattle, frogs) and squid. The exposure of new sulfhydryl groups adds an important element to the growing evidence that relates the bleaching of rhodopsin to protein denaturation. The place of sulfhydryl groups in the structure of rhodopsin is still uncertain. They may be concerned directly in binding the chromophore to opsin; or alternatively they may furnish hydrogen atoms for some reductive change by which the chromophore is formed from retinene(1). In the amperometric silver titration, the bleaching of rhodopsin yields directly an electrical variation. This phenomenon may have some fundamental connection with the role of rhodopsin in visual excitation, and may provide a model of the excitation process in general.

The interconversion of the retinenes and vitamins a in vitro

The retinene 1 which results from the bleaching of rhodopsin now appears to be vitamin A 1 aldehyde. Morton et al. have given the best evidence for this, and have shown that retinene 1 can be prepared by the mild oxidation of vitamin A 1. A simple procedure is described for performing this process chromatographically on a column of manganese dioxide. In the retina, retinene 1 is converted irreversibly to vitamin A 1 by an enzyme system in which reduced cozymase (reduced Coenzyme I, DPNH 2 serves as coenzyme. The essential process is the transfer of two hydrogen atoms from DPNH 2 to retinene 1, reducing its aldehyde group to the primary alcohol group of vitamin A 1. The enzyme system which performs this reduction can be assembled in solution from the following components: the coenzyme, DPNH 2; as substrate, synthetic retinene 1; and the apoenzyme extracted with dilute salt solutions from homogenized frog or cattle retinas. The apoenzyme is non-dialysable, is precipitated by half-saturated ammonium sulphate, and is destroyed by heating at 100°C within 30 seconds. Its p H optimum lies at about 6.5. In the rods of freshwater fishes, a parallel enzyme system reduces retinene 2 to vitamin A 2. This can be assembled from the following components, all in true solution: the coenzyme, DPNH 2; as substrate, synthetic retinene 2, prepared by the chromatographic oxidation of vitamin A 2 on manganese dioxide; and the apoenzyme extracted with dilute salt solutions from freshwater fish retinas (sunfish, yellow perch). The apoenzyme from frog retinas reduces retinene 2 as effectively as retinene 1. Similarly the fish apoenzyme acts equally well upon both retinenes. One need consider only one apoenzyme, retinene reductase, which together with one coenzyme, DPNH 2, reduces either of the retinenes to the corresponding vitamin A. The retinene reductase system brings a second vitamin into the chemistry of rod vision. It presents the novel phenomenon of one vitamin regenerating another, for the central component of DPNH 2 is nicotinamide, the anti-pellagra factor of the vitamin B complex. Rhodopsin solutions and retinal homogenates rapidly lose their power to reduce the retinenes, through destruction of their DPN by a nucleotidase. Rhodopsin solutions which have lost their activity in this way are re-activated by the addition of new DPNH 2. The coenzyme can also be protected by the presence of free nicotinamide and of α-tocopheryl phosphate. On addition to the enzyme system, the synthetic retinenes rapidly couple with other molecules, and primarily with protein. The normal state of the retinenes in retinas and retinal extracts is a labile equilibrium between the free and the coupled condition. The retinenes couple with a variety of retinal molecules, and migrate freely from one to the other.

THE REDUCTION OF RETINENE1 TO VITAMIN A1 IN VITRO

In the surviving vertebrate retina the retinene(1) liberated by bleaching rhodopsin is converted quantitatively to vitamin A(1). Recent chemical studies have indicated that in this process the aldehyde group of retinene(1) is reduced to the primary alcohol group of vitamin A(1) (Morton; Wald). Some time ago we brought this reaction into a cell-free brei prepared from cattle retinas. The retinas were frozen, desiccated, ground, and exhaustively extracted with petroleum ether; the resulting powder, stirred in neutral buffer solution and exposed to light, converted its retinene(1) completely to vitamin A(1). Some time ago also we observed that fresh rhodopsin solutions exhibit a special type of fading in darkness following exposure to light, which is absent from the same solutions after aging. We have confirmed Bliss's identification of this reaction as the conversion of retinene(1) to vitamin A(1). The system which reduces retinene(1) is fractionated anatomically in the retinal rods. The outer segments of the rods, broken off from the underlying retinal tissue, are unable to convert their retinene(1) to vitamin A(1). In the presence of a water extract of crushed retina they do perform this conversion. On the other hand the retinal tissue from which a water extract was taken has lost this capacity. Such washed retinal tissue is reactivated by returning the washings to the solid material. The activating effect of retinal washings on isolated outer limbs or washed retina is duplicated by a boiled muscle juice. This in turn can be replaced by reduced cozymase (reduced coenzyme I; DPN-H(2)); or by a mixture of DPN and fructosediphosphate. The conversion of retinene(1) to vitamin A(1) is therefore a reduction in which two atoms of hydrogen are transferred to retinene(1) from reduced cozymase. It is assumed that this reaction is catalyzed by an apoenzyme, retinene(1) reductase, present in the rod outer limb. This process is coupled with a second system in the outer segment which reduces DPN, using hexosediphosphate or one of its derivatives as hydrogen donor. This action of DPN brings a member of the vitamin B complex, nicotinic acid amide, into an auxiliary position in the rhodopsin system. In the isolated retina or in vitro systems the reduction of retinene(1) proceeds irreversibly. Yet this reduction must be balanced by an oxidative process elsewhere in the rhodopsin cycle, since through rhodopsin as intermediate vitamin A(1) regenerates retinene(1).