Interaction of bovine rhodopsin with calcium ions. I: the metarhodopsin I--II reaction and the regeneration of rhodopsin.

The formation of metarhodopsin II in various bovine rhodopsin preparations (rod outer segment (ROS) suspensions and rhodopsin-detergent solutions) was measured by means of flash spectrophotometry. The half-lifetime and formation of metarhodopsin II in ROS did not depend on the calcium concentration in the range of less than 10(-9) M (using EGTA ro EDTA) to 15 x 10(-3) M calcium at pH values of 5.0, 7.1, and 9.0 (Table 1). The regeneration of rhodopsin from opsin by adding 11-cis retinal to ROS-suspensions and rhodopsin digitonin solutions was measured spectrophotometrically. It was not substantially different in either saline, one containing less than 10(-7) M calcium (by adding EGTA), the other containing 10(-3) M calcium (Table 2).     

The thermal stability of rhodopsin and opsin

Rhodopsin, the red photosensitive pigment of rod vision, is composed of a specific cis isomer of retinene, neo-b (11-cis), joined as chromophore to a colorless protein, opsin. We have investigated the thermal denaturation of cattle rhodopsin and opsin in aqueous digitonin solution, and in isolated rod outer limbs. Both rhodopsin and opsin are more stable in rods than in solution. In solution as well as in rods, moreover, rhodopsin is considerably more stable than opsin. The chromophore therefore protects opsin against denaturation. This is true whether rhodopsin is extracted from dark-adapted retinas, or synthesized in vitro from neo-b retinene and opsin. Excess neo-b retinene does not protect rhodopsin against denaturation. The protection involves the specific relationship between the chromophore and opsin. Similar, though somewhat less, protection is afforded opsin by the stereoisomeric iso-a (9-cis) chromophore in isorhodopsin. The Arrhenius activation energies (E_a) and entropies of activation (ΔS‡) are much greater for thermal denaturation of rhodopsin and isorhodopsin than of opsin. Furthermore, these values differ considerably for rhodopsins from different species —frog, squid, cattle—presumably due to species differences in the opsins. Heat or light bleaches rhodopsin by different mechanisms, yielding different products. Light stereoisomerizes the retinene chromophore; heat denatures the opsin. Photochemical bleaching therefore yields all-trans retinene and native opsin; thermal bleaching, neo-b retinene and denatured opsin.     

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

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 Ellman's 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.     

Lipid composition of different preparations of brain Na+, K+-ATPase

The content and composition of phospholipids is determined in beef microsomal and synaptosomal fractions and also in these fractions preparations solubilized with triton X-100 (0.1%) and digitonin (0.2%). It is shown that the microsomal fraction is richer in phospholipids. The solubilized fragments of microsomes have less or the same amount of phospholipids per protein unit than the initial fraction of microsomes, and the solubilized fragments of synaptosomes contain a higher quantity of phospholipids than the initial fraction. The content of phospholipids in "the riton" fragments of synaptosomes is higher than in "those" of microsomes. Contrary to digitonin which solubilizes the active Na+, K+-ATPase complex of microsomes and synaptosomes, triton X-100 solubilizes the active enzyme of microsomes only. A higher total content of phospholipids in "the triton" extracts of synaptosomes does not probably correlate with the presence of Na+, K+-ATPase activity in them. But these extracts are found to contain less phosphatidylserine whose addition recovers Mg2+, Na+, K+-ATPase activity in them. The effect of phosphatidylserine is not strictly specific for "the triton" extracts of synaptosomes, this lipid activates to a definite extent the extracts of microsomes as well. It is shown that at the first stages of bull brain Na+, K+-ATPase purification the total content of phospholipids and cholesterol in the preparations increases but the composition of phospholipids remains unchanged.     

Photosensitivity of 10-substituted visual pigment analogues: detection of a specific secondary opsin-retinal interaction

The photosensitivities of the bovine rhodopsin and gecko pigment 521 analogues regenerated from C-10-substituted analogues of 11-cis- and 9-cis-retinals were determined by two different methods. A similar reactivity trend was noted for both pigment systems as revealed in the photosensitivity of the gecko pigments and relative quantum yields of the bovine analogues. The 10-fluoro-11-cis photopigments had a photosensitivity less than, but approaching, that of the native (11-cis) visual pigment while the 10-fluoro-9-cis photopigments had a much lower photosensitivity than the parent 9-cis regenerated pigment. The results are interpreted in terms of recently described models of rhodopsin architecture and of the primary molecular reaction of visual pigments to light. The unusually low photoreactivity of the 10-fluoro-9-cis pigment molecule is viewed as the result of a regiospecific hydrogen-bonding interaction of the electronegative fluorine atom to the opsin.     

Interaction of bovine rhodopsin with calcium ions

The formation of metarhodopsin II in various bovine rhodopsin preparations (rod outer segment (ROS) suspensions and rhodopsin-detergent solutions) was measured by means of flash spectrophotometry. The half-lifetime and formation of metarhodopsin II in ROS did not depend on the calcium concentration in the range of less than 10^–9 M (using EGTA or EDTA) to 15×10^–3 M calcium at pH values of 5.0, 7.1, and 9.0 (Table 1).The regeneration of rhodopsin from opsin by adding 11-cis retinal to ROS-suspensions and rhodopsin digitonin solutions was measured spectrophotometrically. It was not substantially different in either saline, one containing less than 10^–7 M calcium (by adding EGTA), the other containing 10^–3 M calcium (Table 2).Abbreviations A absorption - A absorption change - CTAB N-Cetyl-N,N,N-trimethylammoniumbromide - E₇₀₀ extinction at =700 nm - EDTA ethylenediamine-NNNN-tetraacetic acid - EGTA 2,2-ethylenedioxybis [ethyliminodi (acetic acid)] - MI metarhodopsin I - MII metarhodopsin II - Rh rhodopsin - ROS rod outer segment This work is based upon a Ph. D. dissertation (Nöll, 1974) and was presented in part at the Jahrestagung der Deutschen Gesellschaft für Biophysik, Freiburg, Germany, October 1974     

The endogenous chromophore of retinal G protein-coupled receptor opsin from the pigment epithelium

The recent identification of nonvisual opsins has revealed an expanding family of vertebrate opsin genes. The retinal pigment epithelium (RPE) and Müller cells contain a blue and UV light-absorbing opsin, the RPE retinal G protein-coupled receptor (RGR, or RGR opsin). The spectral properties of RGR purified from bovine RPE suggest that RGR is conjugated in vivo to a retinal chromophore through a covalent Schiff base bond. In this study, the isomeric structure of the endogenous chromophore of RGR was identified by the hydroxylamine derivatization method. The retinaloximes derived from RGR in the dark consisted predominantly of the all-trans isomer. Irradiation of RGR with 470-nm monochromatic or near-UV light resulted in stereospecific isomerization of the bound all-trans-retinal to an 11-cis configuration. The stereospecificity of photoisomerization of the all-trans-retinal chromophore of RGR was lost by denaturation of the protein in SDS. Under the in vitro conditions, the photosensitivity of RGR is at least 34% that of bovine rhodopsin. These results provide evidence that RGR is bound in vivo primarily to all-trans-retinal and is capable of operating as a stereospecific photoisomerase that generates 11-cis-retinal in the pigment epithelium.     

Proof of oligomeric state of frog rhodopsin: Visualization of dimer and oligomers on gels after BN- and HRCN-PAGE using antibodies to rhodopsin and by retinylopsin fluorescence

Staining by antibodies to rhodopsin (Rh) and fluorescence of N-retinylopsin (RO) have shown that digitonin (DIG)-, dodecyl-β-D-maltoside (DM)-, and sodium dodecyl sulfate (SDS)-solubilized frog Rh after BN- and HRCN-PAGE is situated in the gradient gel in the state of dimer with a slight content of higher oligomers (trimer, tetramer, etc.). With increasing detergent harshness (DIG < DM < SDS), the proportion of higher oligomers in extracts becomes more prominent. Formation of RO in rod outer segments (ROS) in the presence of 0.7 M NaBH₃CN at pH 5.0 occurs only when Rh is simultaneously photolyzed during reduction. Dithiothreitol at the concentration of 0.005 M failed to induce RO production. Formation of a stable C-N bond between all-trans-retinal and opsin in RO is accompanied by decrease in the dimer share and increase in the share of the higher oligomers due to secondary dissociation-aggregation of solubilized opsin. The position of the Rh dimer in relation to the anode during both native electrophoreses is determined not only by its molecular mass, but probably also depends on unfolding degree (or form): the harsher the detergent, the closer to the anode the dimer is located. Treatment of ROS by agents modifying the cholesterol component of lipid membrane (MβCD, filipin III, nystatin, saponin) did not change the character of Rh oligomerization, thus showing that integrity of the cholesterol component of photoreceptor membrane is not a crucial factor for oligomerization of opsin. It is supposed that the dimer-oligomer “portrait” of frog Rh, which has been found by two methods of native electrophoresis in three detergents with different degree of harshness, corresponds to a physiological state of this protein in native photoreceptor membrane.     

Circular dichroism,optical rotatory dispersion,and absorption studies on the conformation of bovine rhodopsin in situ and solubilized with detergent

Circular dichroism, optical rotatory dispersion and absorption of rhodopsin, the visual pigment of bovine rod outer segment membranes, were studied in situ and in membranes solubilized with various detergents. The -helical content of the membrane protein is approximately 30%. The membrane protein possesses little -structure. Solubilization of the membrane by the detergents, Emulphogene BC-720 and cetyltrimethylammonium salts, results in loss of protein helical structure and perturbation of aromatic residues. These effects are not observed on digitonin solubilization.In regard to the structural stability of the membrane during bleaching, the following conclusions were reached: (1) Delocalized conformational changes of rhodopsin in situ involving secondary and/or tertiary structure are very unlikely. (2) Localized conformational changes of rhodopsin in situ involving secondary structure must be limited to the involvement of no more than three amino acid residues and localized conformational changes involving tertiary structure must be limited to very short segments of the protein chain containing, at the most, only a few aromatic residues. (3) Large changes in the interaction of lipid and protein moieties of the membrane are unlikely. (4) The detergents, Emulphogene, cetyltrimethylammonium salts, and digitonin, significantly decrease the conformational stability of rhodopsin as compared to the in situ conditions. The effect is smaller with digitonin.Evidence is presented against a proposed mechanism by which optical activity of the prosthetic group, retinal, is induced by resonance coupling of the transition dipoles of retinal and the lowest energy transitions of the aromatic groups of the apoprotein, opsin. A mechanism in which atropisomers of retinal are preferentially bound by opsin is consistent with the present results. The optical activity of the prosthetic group is markedly changed upon solubilization of the membrane by detergent. This change in optical activity is probably coupled to changes in conformation of the protein moiety induced by solubilization.This work is based in part upon a Ph.D. dissertation submitted by C.N.R. to The Ohio State University (1974). A preliminary report of this work was presented at the sixteenth annual meeting of the Biophysical Society, Toronto, Canada, February, 1972, Abstracts SaPM-H8 and SaPM-H9     

Immunoreactivity of rhodopsin and opsin

An examination by a radioimmunoassay of the relative affinity of opsin and rhodopsin for rabbit antibody raised against bovine rhodopsin revealed that opsin was the preferred antigen. About 10-fold greater amounts of rhodopsin than opsin were required to achieve 50% inhibition of binding of 125I-labeled ligand in the RIA. Opsin was more reactive when examined in the light or dark, compared to rhodopsin incubated in the dark. Mixtures of opsin and rhodopsin (prepared by partial bleaching of rhodopsin or synthetic mixtures) exhibited increased reactivity with increasing mole fraction of opsin. This response was nonlinear, with small increases in opsin producing relatively large increases in reactivity. A partial fractionation of the antibody into two groups showing differential reactivities toward opsin and rhodopsin was achieved by affinity chromatography on opsin-Sepharose. However, with both groups, opsin was still the preferred antigen. Scatchard analysis of 125I-labeled rhodopsin and opsin produced nonlinear plots, indicating the presence of multiple species of antibody. The affinities and binding capacities were similar for both labeled antigens. In competitive binding studies, the antibody showed a strong preference for either labeled ligand (rhodopsin or opsin) as compared to the unlabeled material. These latter observations indicate that altering rhodopsin either by bleaching or iodination produced changes in the relative immunoreactivity of the molecule.     

Methylation of the active-site lysine of rhodopsin

Purified bovine rhodopsin was reductively methylated with formaldehyde and pyridine/borane with the incorporation of approximately 20 methyl groups in the protein. Rhodopsin contains 10 non-active-site lysines, which account for the uptake of the 20 methyl groups. The permethylated rhodopsin thus formed is active toward bleaching, regeneration with 11-cis-retinal, and the activation of the GTPase (G protein) when photolyzed. The critical active-site lysine of permethylated rhodopsin can be liberated by photolysis. This lysine can be reductively methylated at 4 degrees C. Methylation under these conditions leads to the incorporations of approximately 1.5 methyl groups per opsin molecule using radioactive formaldehyde, with the ratio of epsilon-dimethyllysine:epsilon-monomethyllysine:lysine being approximately 5:4:1. The modified opsin(s) can regenerate with 11-cis-retinal to produce a mixture of active-site methylated and unmethylated rhodopsins having a lambda max = 512 nm. Using [14C]formaldehyde and [3H]retinal followed by reduction of the Schiff base, digestion, and chromatography showed that the active-site N-methyllysine was bound to the retinal. Treatment of the methylated opsin mixture (containing 1.5 active-site methyl groups) with o-phthalaldehyde/mercaptoethanol to functionalize the opsin bearing unreacted lysine, followed by regeneration with 11-cis-retinal and chromatographic separation, led to the preparation of the pure active-site epsilon-lysine monomethylated rhodopsin with a lambda max = 520 nm, significantly shifted bathochromically from rhodopsin or permethylated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variability of the Characteristics of the First Scale Annulus and Length of Year-Old Fish in the Bering Sea Walleye Pollock Theragra chalcogramma

Geographic and interannual variability of the number of annuli and the radius of the first scale annulus, as well as the retrospective length of year-old walleye pollock from the western, northern, and eastern parts of the Bering Sea, was examined using data for the years 1995, 1996, 1998, and 1999. The characteristics of the first scale annulus were varying. By these parameters, walleye pollock from the western Bering Sea significantly differs statistically from walleye pollock of the eastern Bering Sea. With respect to number of annuli, the radius of the first ring, and the retrospective length of year-old specimens, the walleye pollock from the Navarinskii region occupies an intermediate position between fish from the western and eastern parts of the Bering Sea. Interannual variability of the three parameters was found for walleye pollock from the Navarinskii region.     

The amino- and carboxyl-terminal sequence of bovine rhodopsin

The amino terminus of bovine rhodopsin is blocked and has the sequence x-Met-Asn(CHO)-Gly-Thr-Glu-Gly-Pro-Asn-Phe-Tyr-Val-Pro-Phe-Ser-Asn(CHO)-Lys-Thr-Gly-Val-Val-Arg, where CHO represents sites of carbohydrate attachment. The carboxyl-terminal sequence of rhodopsin is Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala. Upon short-term digestion of rod outer segment (ROS) membranes with thermolysin, opsin (～ 35,000 daltons) is converted to a membrane-bound fragment O′ (～ 30,500 daltons) and 2 peptides containing 12 amino acids are released from the carboxyl terminus of rhodopsin into the supernatant. Upon long-term digestion of ROS with thermolysin, opsin and O′ are replaced by the membrane-bound fragments F₁ (～25,000 daltons), and F₂ (～9,500 daltons). When ³²P-ROS are digested, F₂ carries the ³²P. Both O′ and F₁ contain the amino-terminal glycopeptide.     

Isolation and identification of phospholipids of bovine rhodopsin

Phospholipids present in digitonin solutions of bovine rhodopsin have been identified and assayed. Digitonin interferes with extraction of lipids by the usual methods; digitonin was therefore removed from the preparation as an ergosterol digitonide, soluble in absolute ethanol but precipitated in 80% ethanol. The supernatant 80% ethanol contained one portion of the phospholipid, mainly choline and ethanolamine phosphoglycerides with traces of serine phosphoglyceride and sphingomyelin. The rhodopsin residue (free from digitonin) was extracted with chloroform-methanol 2:1; this extract contained the rest of the phospholipid, which consisted only of choline and ethanolamine phosphoglycerides. Plasmalogens were not found, but could have decomposed during the procedures.     

Synthesis of 11-cis-locked bicyclo[5.1.0]octanyl retinal and an enantioselective binding to bovine opsin

Both enantiomers of 13-(E) and 13-(Z) isomers of 11-cis-locked bicyclo[5.1.0]octanyl retinal were prepared by an improved synthesis and incubated with bovine opsin. The synthesis also establishes the absolute configuration of the enantiomers. Only one of the enantiomers binds to opsin, thus showing the steric restrictions regarding the middle polyene moiety of the retinoid molecule; this is in sharp contrast to the known leniency of the ring moiety binding site of retinoids. However, although one enantiomer is incorporated into the pigment, the circular dichroic spectrum of the pigment incorporating the bound enantiomer yields only a very weak Cotton effect, showing that, once incorporated, the bicyclo[5.1.0]octanyl chromophore is flattened by the opsin binding site. The titled retinoid was synthesized for study of the absolute conformation of the retinal pigment in rhodopsin.     

Interplay of phosphorylation and dephosphorylation in vision: protein phosphatases of bovine rod outer segments

Two types of protein phosphatases were identified in carefully prepared bovine rod outer segments (ROS). Extraction of the ROS with a medium-salt buffer solubilized protein phosphatase activity that was mainly type 2A, since it was active toward phosphorylase a in the absence of divalent cations, was not retained by heparin-Sepharose, dephosphorylated the alpha-subunit of phosphorylase kinase faster that the beta-subunit, and was unaffected by inhibitor 2. Further extraction of the resulting membranes with a high-salt buffer solubilized additional phosphatase activity which was predominantly type 1, since it was retained by heparin-Sepharose and was blocked by inhibitor 2. The molecular mass of the type 2A phosphatase estimated by gel permeation chromatography on Superose 12 was 100 kDa, suggesting it may be the 2A2 form. Only the ROS type 2A phosphatase dephosphorylated opsin and rhodopsin efficiently. Concordant with this finding, the purified catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle dephosphorylated opsin efficiently, while the type 1 catalytic subunit isolated from this tissue was inactive. Together, the results suggest that the ROS type 2A protein phosphatase plays an important role in regenerating rhodopsin from the various phosphorylated species in vivo. The activity of the enzyme per retina (approximately 85 pmol of Pi released/min) is comparable to that of rhodopsin kinase (100 pmol of phosphate transferred/min).     

Regeneration of bovine and octopus opsins in situ with natural and artificial retinals

We consider the problem of color regulation in visual pigments for both bovine rhodopsin (lambda max = 500 nm) and octopus rhodopsin (lambda max = 475 nm). Both pigments have 11-cis-retinal (lambda max = 379 nm, in ethanol) as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 +/- 3000 M-1 cm-1 at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.     

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.     

Effect of phospholipid removal on the kinetics of the metarhodopsin I to metarhodopsin II reaction.

The kinetics of the metarhodopsin (meta) I → metarhodopsin II reaction have been studied by flash photolysis in two different types of preparations of bovine rhodopsin: (i) digitonin-solubilized rod outer segment (ROS) membranes with a molar ratio of phospholipid to rhodopsin of approximately 90, and (ii) digitonin-solubilized phospholipid-free rhodopsin with a molar ratio of phospholipid to rhodopsin of less than 0.2. At 20 °C the kinetics in both preparations are multiexponential, but four terms are required to fit the data with the solubilized membranes, whereas only two are required with the phospholipid-free preparation. Thus, phospholipid removal simplifies the kinetics of the meta I → meta II reaction, but the resulting preparation still does not show first-order kinetics. The ratio of the time constants of these two components with detergent-solubilized phospholipid-free rhodopsin was nearly equal to the values found with ROS particles, rhodopsin-phospholipid recombinants and intact rabbit eyes. This suggests a common origin for these two components in all these preparations and appears to exclude heterogeneity in bound phospholipid as the basis of these two-component kinetics.