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. Detergent concentrations up to 10 mM did not interfere with binding of retinol to Lipidex-1000 or binding protein. The binding capacity of Lipidex-1000 was found to exceed 400 nmol of retinol per ml of gel. Retinal pigment epithelium (RPE) cells were used as a source for cRBP (cellular retinol-binding protein). The binding protein is saturated with ligand by incubation for 60 min at room temperature at concentrations of free retinol over 180 nM. Separation of protein-bound retinol from free retinol is achieved via Lipidex-1000: protein-bound (specific and nonspecific) retinol is not retained and is eluted by buffer with the protein fraction. Free retinol is retained by Lipidex and is subsequently recovered by elution with methanol. Total recovery of ligand approaches 100%. Analysis time is about 4 hr for a maximum of ca. 50 samples. Nonspecific protein binding can be determined equally effectively either by incubation with 3 mM PCMBS or by addition of a 100-fold molar excess of nonlabeled retinol.(ABSTRACT TRUNCATED AT 250 WORDS)

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 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na+-conductance of the plasma membrane.

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. The retina then is removed carefully, after which the pigment epithelial cells are detached from the Bruch's membrane by gentle jets of buffer solution. The perfusion technique provides a high yield of intact retinal pigment epithelial cells, which show good viability in subsequent cell culture. Hence, cells isolated in this way are not only very well suited for long-term cell culture but also for direct biochemical analysis and short-term incubation studies.

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. E.G. retinoids clearly favour the PC-vesicle membrane. Apparently, rod outer segment membranes have a much lower affinity for retinoids than other artificial or natural membranes investigated so far.

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. It can be carried out rapidly, has a sensitivity of 0.01 pmol bovine opsin and gives accurate results, even in the presence of a large excess of rhodopsin. Under the conditions described, the assay can be used to measure bovine opsin and rhodopsin in each other's presence by running a sample before and after illumination, with a sensitivity 2000-times higher than with spectrophotometric methods. The opsin content of rather crude preparations such as bovine retina homogenates can be accurately determined. Rabbit and mouse opsin can also be assayed with a reasonable degree of accuracy using the same rabbit antiserum.

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. In the case of opsin this masking phenomenon is preceded by a drastic exposure of antigenic sites which in the membrane vesicles are not accessible to the antibodies. Absorption experiments show that the antigenic sites on membrane-bound rhodopsin and opsin, as well as on Emulphogene BC-720-solubilized opsin, are of the same nature. Competition experiments show that the masking effect of the detergent is due to an inhibition of the primary antigen-antibody interaction and not to the inhibition of lattice formation. The use of detergents other than Emulphogene BC-720 further demonstrates that detergents more efficiently mask the antigenicity of conformationally intact than of denatured rhodopsinoids. The balance between the masking and the denaturing efficiency of a particular detergent determines whether a detergent-induced immunological discrimination can be observed between rhodopsin and opsin. The detergent-induced masking effects described are typical for the present antiserum and are probably dependent on methodological details of the immunization procedure.

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 6.9) hydrogenation of aqueous dioleoylphosphatidylcholine dispersions, prepared by sonication, was achieved. This reaction was not affected by the presence of high salt concentrations. The rate of hydrogenation was independent of catalyst concentration. The reaction with dioleoylphosphatidylcholine dispersions, prepared by the ethanolic injection method, was preceded by a lag period of about 5 h. The reaction with dioleoylphosphatidylcholine dispersions, obtained by vigorous shaking, was rather slow, suggesting the presence of a penetration barrier. The reaction with dioleoylphosphatidylcholine proceeds via the isomerisation of the oleoyl to the elaidoyl moiety, followed by hydrogenation of the elaidoyl moiety. The possible interactions of the catalysts with the bilayer are considered and the implications of these findings are discussed.

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. The plasma membrane preparation contains 1.5% of total outer segment rhodopsin and 2.7% of total outer segment protein. it shows very little contamination with inner segment plasma membrane. Contamination with disk membranes appears to be low as well. Fatty acid analysis reveals that the plasma membranes are more saturated than the highly unsaturated disk membranes. Gel electrophoresis shows the presence of at least six additional polypeptides besides besides rhodopsin.

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 (0.1-0.2 mol per mol of rhodopsin). When intact ROS resuspended in sucrose-ficoll medium were diluted in a Ringer-Krebs bicarbonate, the nucleotide ratios were markedly changed, but the total concentration remained unchanged. The concentration of cyclic nucleotides (cAMP and cGMP) was markedly enhanced when ROS were placed in Ringer buffer, whereas ATP and GTP concentration was reduced. Total nucleotide concentration is 100% higher in intact than in leaky plasma membrane ROS. Upon illumination, no change was observed in the nucleotide levels of ROS in sucrose-ficoll medium. However ATP, GTP and cAMP levels were reduced when ROS in Ringer medium were exposed to light while cGMP concentration showed no change. Relevance of relative nucleotide content and ionic concentration to the transduction phenomenon in photoreceptor is discussed.

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. After treatment with trinitrobenzenesulfonic acid or phospholipase D, the fatty acid composition of the unreacted phospholipids is the same as that before treatment, regardless of the extent of modification or hydrolysis attained. The fatty acid composition of phosphatidic acid, resulting from phospholipase D action, also remains unchanged during progressive hydrolysis. These results indicate that the fatty acyl chains of the major phospholipids have the same composition on either side of the disk membrane. Together with our previously published evidence for the distribution of the major phospholipids in rod outer segment disk membranes, this means that both the phospholipids and their fatty acyl chains have a remarkably symmetrical distribution over the two membrane faces. On the basis of literature data it is concluded that this approximate symmetry reflects the high mobility of the entire phospholipid pool of disk membranes, thus including appreciable transbilayer movements of the phospholipids.

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. It is found that enzyme activities are present, which can rapidly reshuffle the high-energy phosphate groups between the various adenine and guanine nucleotides as well as from creatine phosphate. These phosphate transfers may occur so rapidly that light-induced changes in nucleotide concentrations could easily be a delusive criterion for the enzymatic process underlying these changes.

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. Special attention is given to the early phase of enzyme treatment in order to eliminate secondary effects on the molecular organization of the membrane due to appreciable phospholipid hydrolysis. Analysis of the hydrolysis patterns for all three phospholipases in stacked disks, as compared to those in randomized control preparations, suggests a slightly asymmetrical distribution of phosphatidylcholine (40--45% at the outer face) and phosphatidylethanolamine (55--60% at the outer face) and a symmetrical distribution of phosphatidylserine in rod outer segment disk membranes. Extensive treatment with phospholipases C and A2 leads ultimately to nearly complete hydrolysis of all phospholipids, but with phospholipase D a final level of 40% phospholipid hydrolysis is observed in stacked disk preparations. This suggests that in the latter case the inner face of the membrane is inaccessible to the enzyme. Further work will be necessary in order to substantiate these conclusions.

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. All-trans, 13-cis and 9-cis-retinal all isomerize at the delta 13 double bond leading to an equilibrium with approximately 75% trans and 25% cis isomer at this bond (all-trans in equilibrium 13-cis and 9-cis in equilibrium 9,13-dicis). 11-cis-Retinal isomerizes irreversibly to a mixture of all-trans and 13-cis-retinal. 3. The active compound appears to be phosphatidylethanolamine present in the membrane. The amino group and the phosphate, as well as the hydrophobic part of the phospholipid are essential. 4. At least three factors are important for the phosphatidylethanolamine-catalyzed isomerization as studied with the 13-cis isomer: the concentration of phosphatidylethanolamine, the concentration of Schiff base between retinal and phosphatidylethanolamine and the presence of lipid aggregates. 5. Based on these observations a mechanism is proposed, which satisfactorily explains the specificity of the isomerization pattern. 6. It is suggested that reisomerization of all-trans to 11-cis retinal in vivo takes place by fixation of all-trans retinal on an adequate surface (e.g. opsin) and a localized nucleophilic attack on the C-11 atom, followed by trapping of the isomerized chromophore by opsin. 7. It is further concluded that retinal does not occur in vivo as a free intermediate. Direct transfer from one protein to another (opsin, retinol dehydrogenase, retinal binding proteins) seems to take place.