F.J.M. Daemen

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.

Biochemical aspects of the visual process: XXVII. Stereospecificity of ocular retinol dehydrogenases and the visual cycle

A comparative study is made of the stereospecificity of two particulate retinol dehydrogenases from bovine eyes and of horse liver alcohol dehydrogenase. The particulate retinol dehydrogenase of outer segments reacts with the all-trans isomers of retinaldehyde and retinol but not with the 11-cis compounds. In contrast, a particulate retinol dehydrogenase present in pigment epithelium reacts preferentially with the 11-cis compounds. Horse liver alcohol dehydrogenase (EC 1.1.1.1.) can convert both isomers, but the all-trans isomers are clearly preferred. Differences with regard to cofactor preference and stability are also noted. The outer segment enzyme clearly functions in the rhodopsin cycle. It is unlikely that the 11-cis retinol dehydrogenase from pigment epithelium is directly involved in providing 11-cis retinaldehyde from rhodopsin regeneration, but it may serve to make available 11-cis retinaldehyde from rhodopdsin, digested in phagocytized rod sacs, for the synthesis of visual pigment by the visual cells.

The isolation of stable cattle rod outer segments with an intact plasma membrane

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter. Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH. The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.

Enrichment of rhodopsin in rod outer segment membrane preparations: Biochemical aspects of the visual process—XVIII

Abstract Substantial, seasonally varying amounts of opsin are present in cattle rhodopsin preparations, which cannot be converted to rhodopsin by prior dark adaptation of the excised eyes. A method is described for the quantitative conversion of the opsin to rhodopsin by treatment of a rod outer segment preparation with 11- cis retinaldehyde, followed by removal of excess retinaldehyde through reduction by endogenous retinol dehydrogenase and added NADPH and elution of the resulting retinol by sucrose gradient centrifugation. The resulting rhodopsin preparation has a high pigment content of 8 nMol/mg dry weight, which is constant within 2.5 per cent over the entire year. The preparation is of high purity, as indicated by theA 278 /A 500 ratio of 2.0–2.5 and theA 400 /A 500 ratio of 0.20–0.26. The yield is high, representing 50 per cent of the pigment present in the retina. The preparation is free of detergent, has a full regenerating capacity, and can be stored for long periods of time.

Biochemical aspects of the visual process: VI. The lipid composition of native and hexane-extracted cattle rod outer segments

Abstract Cattle rod outer segments were isolated by means of a continuous sucrose gradient technique. These preparations contained 39% total lipid and 31.5% phospholipid on a dry weight basis. The fatty acid composition of the total rod outer segment lipids was determined by means of gas-liquid chromatography. The three predominant fatty acids were: palmitic acid (19.4% (w/w) of total fatty acids), stearic acid (23.1%) and docosahexaenoic acid (34.3%). Quantitative analysis of the phospholipids by two-dimensional thin-layer chromatography showed that the three major phospholipids are: phosphatidyl ethanolamine (35.4%), phosphatidyl choline (34.7%) and phosphatidyl serine (11.2%). Half of the total amount of phospholipids was extractable with n hexane without changing the spectral and photolytic properties of the rhodopsin. Relatively more phosphatidyl ethanolamine than phosphatidyl choline was extracted by hexane, reducing the molar phosphatidyl ethanolamine: retinaldehyde ratio from 29 to 9.

[36] Isolation and purification of bovine rhodopsin

Publisher Summary This chapter describes the isolation and purification of bovine rhodopsin. Rhodopsin is the general name for the visual pigments of both the rod cells in the vertebrate retina and the invertebrate photoreceptor cells. Rhodopsin is an intrinsic membrane glycoprotein, confined to the membranous structures (photoreceptor membranes) of a special part of the rod cell, the rod outer segment (ROS), which is highly specialized for light reception. Rhodopsin is determined by measuring the decrease in absorbance at 500 nm upon illumination, using a molar absorbance of 40,500. Under some conditions, the photoproducts also absorb at 500 nm, but addition of hydroxylamine will convert all free and bound retinaldehyde into retinal oxime, which has virtually no absorption beyond 450 nm. In detergents like the quarternary ammonium derivatives (DTAB, TTAB, CTAB) or the amine oxides (C10DAO, C12DAO), addition of hydroxylamine is not required, because under these conditions retinal is completely released and hardly interferes at 500 nm. Hydroxylamine should be handled with care, because it is highly mutagenic. The assay can be performed on suspensions of photoreceptor membranes, but scattering artifacts may interfere. Most accurate results are obtained after prior solubilization of the membrane in an appropriate detergent, followed by centrifugation or filtration through a membrane filter if some turbidity remains.

Biochemical aspects of the visual process. XX. The molecular weight of rhodopsin.

Abstract The molecular weight of cattle rhodopsin has been reinvestigated by two independent methods. In the first method rhodopsin preparations, purified in detergent solution by gel filtration and chromatography, respectively, have been analysed for their protein content by quantitative amino acid analysis after hydrolysis and for their retinaldehyde content. This method gives a value of 39 100 ± 900 g protein per mole retinaldehyde for 2 different preparations. The second method, calibrated acrylamide gel electrophoresis in sodium dodecyl sulfate of opsin-free cattle rod outer segment membrane preparations, both before and after enzymatic delipidation, demonstrates the presence of one major band with a molecular weight of 38 600 ± 1000 for 10 preparations. Enzymatically delipidated preparations give the same value, which proves that the presence of lipids does not influence the molecular weight determination by this method. Averaging the results of the two methods, we arrive at a molecular weight of 38 850 ± 900 for the protein part of cattle rhodopsin. Over 85% of the insoluble protein part of cattle rod outer segment membranes appears to consist of rhodopsin protein. Calculation of the molar absorbance at 500 nm of cattle rhodopsin from these data and from measured absorbances, both per mole retinaldehyde and per mole rhodopsin, gives the same result; an average value of 40 300 ± 500. These results confirm that one mole retinaldehyde is present per mole rhodopsin.

Biochemical aspects of the visual process. XXV. Light-induced calcium movements in isolated frog rod outer segments

Abstract Suspensions of isolated rod outer segments are shown to have a high calcium content of up to 11 moles calcium per mole rhodopsin. Osmotic lysis of the outer segments demonstrates the presence of two calcium fractions, a soluble one and a particulate one. The particulate fraction apparently coincides with the rod disks or with disk fragments. Illumination of intact rod outer segments in calcium-free ATP-containing Ringer solution has no measurable effect upon their total caclium content, but causes a significant shift of calcium from the particulate to the soluble fraction. This light effect is retained in lysed outer segments resuspended in calcium-free ATP-containing Ringer. These results support a function of calcium as a transmitter in light transduction in rod outer segments.

Identification and characterization of syn- and anti-isomers of retinaloximes

Hydroxylamine derivatization of retinals affords two reaction products in a ratio of about 3:1. Preparative high-pressure liquid chromatography allows isolation of both products in a pure form. Mass spectroscopy and nuclear magnetic resonance spectroscopy allow identification. The main products possess the syn, the minor products the anti configuration.

Biochemical aspects of the visual process. XXIII. Sulfhydryl groups and rhodopsin photolysis

Abstract 1. The number of exposed sulfhydryl groups in cattle rod photoreceptor membranes has been determined in suspension and after solubilization in various detergents both before and after illumination. 2. In suspensions, two sulfhydryl groups are modified per mole of rhodopsin, both by Ellmans reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and N -ethylmaleimide, while no extra SH groups are uncovered upon illumination. Neither reagent affects the spectral integrity of rhodopsin at 500 nm and the recombination capacity is retained upon modification of both rhodopsin and opsin. 3. However, in detergents (digitonin, Triton X-100 and cetyltrimethylammonium bromide (CTAB)) 2–3 additional sulfhydryl groups appear upon illumination, in agreement with earlier reports. 4. A total number of six sulfhydryl groups and two disulfide bridges are found in rod photoreceptor membranes, expressed per mole of rhodopsin. 5. DTNB reacts somewhat faster with membrane suspensions after than before illumination. The less reactive sulfhydryl modifying agents O -methylisourea and methyl- p -nitrobenzene sulfonate show a similar behavior. 6. It is concluded that illumination of rhodopsin in vivo will not uncover additional SH groups, although the reactivity of one exposed SH group may increase somewhat. These findings also exclude a role of SH groups in the covalent binding of the chromophore.