A rapid versatile microassay for cellular retinol-binding protein using Lipidex-1000 microcolumns.

A new, rapid and versatile microassay for cellular retinol-binding protein has been developed based on separation of bound and free ligand by means of Lipidex-1000, a hydrophobic Sephadex derivative. This requires quantitative manipulation of retinol in aqueous solution. The tendency of retinol to adhere to glass and plastic surfaces was overcome by addition of the detergent Ammonyx LO, which yields a micellar dispersion.

Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina.

The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylation of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed.

A new isolation procedure for retinal pigment epithelium.

A new isolation procedure for bovine retinal pigment epithelial cells has been developed. It is based on perfusion of the whole bovine eye via the central ophthalmic artery with a cold, buffered isotonic salt solution free of divalent cations for 15 min. The perfusion both weakens the association of the pigment epithelial cells with Bruch's membrane and the adhesion between retina and pigment epithelium.

Rod outer segment membranes: good acceptors for retinoids?

The exchange of all-trans retinoids (retinal, retinol, retinylpalmitate) between PC-vesicles, PC-vesicles and liver microsomes or PC-vesicles and rod outer segment membranes is investigated using 11,12(3)H labeled compounds. In the first two systems, retinal and retinol exchange rapidly, retinyl acetate slowly and retinyl palmitate not at all. Rod outer segment membranes however take up relatively small amounts of retinoids (retinylpalmitate less than retinol less than retinal) and rapidly lose 60-90% of their label in the presence of PC-vesicles.

Detergent-induced specificity of an antirhodopsin serum for opsin. Micro-complement fixation studies.

An antiserum elicited in rabbit against dark-adapted rod outer segment membranes has been characterized by means of the micro-complement fixation technique. Both particulate rhodopsin and opsin, either biochemically intact or denatured and either membrane-bound or in the absence of lipids, are able to interact with the antiserum. Solubilization of the antigens in increasing concentrations of Emulphogene BC-720 leads to complete loss of complement fixation with both rhodopsin and opsin, but in the case of opsin this requires almost 10-times more detergent.

A radioimmunoassay specific for opsin.

A radioimmunoassay is developed for bovine opsin using a rabbit antiserum against bovine rod outer segment membranes. The assay is specific for opsin. Rhodopsin, bacteriorhodopsin and hemoglobin do not show cross-reaction.

Studies on the catalytic hydrogenation of biomembranes. Hydrogenation of phosphatidylcholine liposomes by two water-soluble rhodium-phosphine catalysts.

The reactions of two water-soluble amphiphiolic rhodium-phosphine hydrogenation catalysts, chlorotris(sodium diphenylphosphinobenzene-m-sulfonate)rhodium(I) and chlorotris(bissodium diphenylphosphinoundecylphosphate)rhodium(I), with aqueous dispersions of unsaturated phosphatidylcholines have been studied. Under mild reaction conditions (pH2 = 1.2 atm, 37 degrees C, pH 6.

Use of a density modification technique for isolation of the plasma membrane of rod outer segments.

The external surface of rod outer segments contains receptors for the Jack Bean lectin, concanavalin A. Using isolated intact bovine rod outer segments, we have modified the density of the outer segment plasma membranes by means of polystyrene beads carrying covalently linked concanavalin A. After hypotonic lysis the bulk of the disk membranes can be removed and a plasma membrane fraction is isolated.

Nucleotide content of isolated bovine rod outer segments.

Endogenous nucleotide levels of isolated intact ROS were analyzed by high pressure liquid chromatography. Intact bovine ROS showed a total nucleotide concentration of 1.0 mM, ATP and GTP being the major components (0.

Transbilayer distribution of phospholipid fatty acyl chains in photoreceptor membrane.

The transverse distribution of the fatty acyl chains of the major phospholipids over the two faces of the photoreceptor membranes has been determined in bovine rod outer segment (stacked disk) preparations. For this purpose, the fatty acid composition of the phospholipids has been analyzed before and after treatment with trinitrobenzenesulfonic acid and phospholipase D. The latter agents are used under conditions in which they have been demonstrated to attack only the outer (cytoplasmic) face of the membrane.

Transfer of high-energy phosphate in bovine rod outer segments. A nucleotide buffer system.

The nucleotide requirement for the recycling of NADPH, necessary in rod outer segment cytoplasm for the reduction of the chromophore upon bleaching, has been investigated. It is found that the latter process specifically requires ATP. Using this specificity the pathways have been investigated, by which in the rod outer segment cytoplasm ATP is resynthesized from other high-energy phosphate donors.

Transbilayer distribution of phospholipids in photoreceptor membrane studied with various phospholipases.

The distribution of the three major phospholipids of bovine rod outer segment disk membranes over the two faces of the membrane has been studied by means of treatment with phospholipase C, phospholipase A2 and phospholipase D. Two different preparations of rod outer segment disk membranes have been used, which are called 'stacked disks' and 'disk vesicles' on account of their morphological appearance. The hydrolysis patterns obtained by phospholipase treatment of these preparations have been compared to those of a retinal lipid suspension or detergent-solubilized disk membranes, which serve as control preparations with a similar phospholipid composition but a random availability of the phospholipids.

Dark isomerization of retinals in the presence of phosphatidylethanolamine.

1. Dark incubation of retinoids (retinyl ester, retinol, retinal, retinaloxime) in suspensions of rod outer segment membranes leads to substantial isomerization (and partial degradation) in the case of retinals only. 2.

Quantitative determination of retinals with complete retention of their geometric configuration.

A method is described for the quantitative extraction of retinal in its original isomeric configuration from retinal-containing pigments. Using excess of hydroxylamine under denaturing conditions, the chromophore of retinal bearing natural products is converted into the corresponding retinaloxime with complete retention of geometric configuration. The retinaloximes can be quantitatively extracted with dichloromethane and analyzed by high-performance liquid chromatography.

The disk membrane as model for an excitable membrane.

Excitable membranes appear to be biochemically characterized by the presence of a highly specific receptor protein, which undergoes conformational changes upon stimulation and returns to its original conformation for renewed excitability. Our present knowledge of the disk membrane and its receptor protein rhodopsin is reviewed in the light of this model. The influence of the membrane environment on rhodopsin has been determined in bovine rod outer segment membranes, in rhodopsin preparations which are depleted of the membrane phospholipids, and in reconstituted phospholipid-rhodopsin vesicles.

Transverse distribution of phospholipids in the vertebrate photoreceptor membrane.

A study has been made of the distribution of the three major phospholipids of the bovine photoreceptor membrane over the two faces of the lipid bilayer. Three different approaches have been used: a . Treatment of isolated intact outer segments and disk vesicles (lysed outer segments) with three different phospholipases and determination of the degradation pattern.