Bovine Rod Outer Segments Research Articles

Posttranslational Modifications of the Photoreceptor-Specific ABC Transporter ABCA4

ABCA4 is a photoreceptor-specific ATP-binding cassette transporter implicated in the clearance of all-trans-retinal produced in the retina during light perception. Multiple mutations in this protein have been linked to Stargardt disease and other visual disorders. Here we report the first systematic study of posttranslational modifications in native ABCA4 purified from bovine rod outer segments. Seven N-glycosylation sites were detected in exocytoplasmic domains 1 and 2 by mass spectrometry, confirming the topological model of ABCA4 proposed previously. The modifying oligosaccharides were relatively short and homogeneous, predominantly representing a high-mannose type of N-glycosylation. Five phosphorylation sites were detected in cytoplasmic domain 1, with four of them located in the linker "regulatory-like" region conserved among ABCA subfamily members. Contrary to published results, phosphorylation of ABCA4 was found to be independent of light. Using human ABCA4 mutants heterologously expressed in mammalian cells, we showed that the Stargardt disease-associated alanine mutation in the phosphorylation site at position 901 led to protein misfolding and degradation. Furthermore, replacing the S1317 phosphorylation site reduced the basal ATPase activity of ABCA4, whereas an alanine mutation in either the S1185 or T1313 phosphorylation site resulted in a significant decrease in the all-trans-retinal-stimulated ATPase activity without affecting the basal activity, protein expression, or localization. In agreement with this observation, partial dephosphorylation of native bovine ABCA4 led to reduction of both basal and stimulated ATPase activity. Thus, we present the first evidence that phosphorylation of ABCA4 can regulate its function.

Enzymatic Properties and Regulation of the Native Isozymes of Retinal Membrane Guanylyl Cyclase (RetGC) from Mouse Photoreceptors

Mouse photoreceptor function and survival critically depend on Ca(2+)-regulated retinal membrane guanylyl cyclase (RetGC), comprised of two isozymes, RetGC1 and RetGC2. We characterized the content, catalytic constants, and regulation of native RetGC1 and RetGC2 isozymes using mice lacking guanylyl cyclase activating proteins GCAP1 and GCAP2 and deficient for either GUCY2F or GUCY2E genes, respectively. We found that the characteristics of both native RetGC isozymes were considerably different from other reported estimates made for mammalian RetGCs: the content of RetGC1 per mouse rod outer segments (ROS) was at least 3-fold lower, the molar ratio (RetGC2:RetGC1) 6-fold higher, and the catalytic constants of both GCAP-activated isozymes between 12- and 19-fold higher than previously measured in bovine ROS. The native RetGC isozymes had different basal activity and were accelerated 5-28-fold at physiological concentrations of GCAPs. RetGC2 alone was capable of contributing as much as 135-165 μM cGMP s(-1) or almost 23-28% to the maximal cGMP synthesis rate in mouse ROS. At the maximal level of activation by GCAP, this isozyme alone could provide a significantly high rate of cGMP synthesis compared to what is expected for normal recovery of a mouse rod, and this can help explain some of the unresolved paradoxes of rod physiology. GCAP-activated native RetGC1 and RetGC2 were less sensitive to inhibition by Ca(2+) in the presence of GCAP1 (EC(50Ca) ∼132-139 nM) than GCAP2 (EC(50Ca) ∼50-59 nM), thus arguing that Ca(2+) sensor properties of GCAP in a functional RetGC/GCAP complex are defined not by a particular target isozyme but the intrinsic properties of GCAPs themselves.

Catalytic properties of the retinal rod outer segment disk ADP-ribosyl cyclase

Cyclic ADP-ribose (cADPR) is a second messenger modulating intracellular calcium levels. We have previously described a cADPR-dependent calcium signaling pathway in bovine rod outer segments (ROS), where calcium ions play a pivotal role. ROS ADP-ribosyl cyclase (ADPR-cyclase) was localized in the membrane fraction. In the present work, we examined the properties of the disk ADPR-cyclase through the production of cyclic GDP-ribose from the NAD(+) analogue NGD(+). The enzyme displayed an estimated K(m) for NGD(+) of 12.5 ± 0.3 μM, a V(max) of 26.50 ± 0.70 pmol cyclic GDP-ribose synthesized/min/mg, and optimal pH of 6.5. The effect of divalent cations (Zn(2+), Cu(2+), and Ca(2+)) was also tested. Micromolar Zn(2+) and Cu(2+) inhibited the disk ADPR-cyclase activity (half maximal inhibitory concentration, IC50=1.1 and 3.6 μM, respectively). By contrast, Ca(2+) ions had no effect. Interestingly, the properties of the intracellular membrane-associated ROS disk ADPR-cyclase are more similar to those of the ADPR-cyclase found in CD38-deficient mouse brain, than to those of CD38 or CD157. The novel intracellular mammalian ADPR-cyclase would elicit Ca(2+) release from the disks at various rates in response to change in free Ca(2+) concentrations, caused by light versus dark adaptation, in fact there was no difference in disk ADPR-cyclase activity in light or dark conditions. Data suggest that disk ADPR-cyclase may be a potential target of retinal toxicity of Zn(2+) and may shed light to the role of Cu(2+) and Zn(2+) deficiency in retina.

The ins and outs of cholesterol in the vertebrate retina

The vertebrate retina has multiple demands for utilization of cholesterol and must meet those demands either by synthesizing its own supply of cholesterol or by importing cholesterol from extraretinal sources, or both. Unlike the blood-brain barrier, the blood-retina barrier allows uptake of cholesterol from the circulation via a lipoprotein-based/receptor-mediated mechanism. Under normal conditions, cholesterol homeostasis is tightly regulated; also, cholesterol exists in the neural retina overwhelmingly in unesterified form, and sterol intermediates are present in minimal to negligible quantities. However, under certain pathological conditions, either due to an inborn error in cholesterol biosynthesis or as a consequence of exposure to selective inhibitors of enzymes in the cholesterol pathway, the ratio of sterol intermediates to cholesterol in the retina can rise dramatically and persist, in some cases resulting in progressive degeneration that significantly compromises the structure and function of the retina. Although the relative contributions of de novo synthesis versus extraretinal uptake are not yet known, herein we review what is known about these processes and the dynamics of cholesterol in the vertebrate retina and indicate some future avenues of research in this area.

Purification of a complex of transducin βγ subunits from isotonic extracts of bovine retinal rod outer segments

A method is proposed for obtaining a free complex of transducin βγ subunits from bovine retinal rod outer segments in a highly purified state.

Structural Characterization of the Rod cGMP Phosphodiesterase 6

Rod cGMP phosphodiesterase 6 (PDE6) is a key enzyme of the phototransduction cascade, consisting of PDE6α, PDE6β, and two regulatory PDE6γ subunits. PDE6 is membrane associated through isoprenyl membrane anchors attached to the C-termini of PDE6α and PDE6β and can form a complex with prenyl-binding protein δ (PrBP/δ), an isoprenyl-binding protein that is highly expressed in photoreceptors. The stoichiometry of PDE6–PrBP/δ binding and the mechanism by which the PDE6–PrBP/δ complex assembles have not been fully characterized, and the location of regulatory PDE6γ subunits within the protein assembly has not been elucidated. To clarify these questions, we have developed a rapid purification method for PDE6–PrBP/δ from bovine rod outer segments utilizing recombinant PrBP/δ. Transmission electron microscopy of negatively stained samples revealed the location of PrBP/δ and, thus, where the carboxyl-termini of PDE6α and PDE6β must be located. The three-dimensional structure of the PDE6αβγ complex was determined up to 18 Å resolution from single-particle projections and was interpreted by model building to identify the probable location of isoprenylation, PDE6γ subunits, and catalytic sites.

Lipid second messengers and related enzymes in vertebrate rod outer segments

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

A Functional Kinase Homology Domain Is Essential for the Activity of Photoreceptor Guanylate Cyclase 1

Phototransduction is carried out by a signaling pathway that links photoactivation of visual pigments in retinal photoreceptor cells to a change in their membrane potential. Upon photoactivation, the second messenger of phototransduction, cyclic GMP, is rapidly degraded and must be replenished during the recovery phase of phototransduction by photoreceptor guanylate cyclases (GCs) GC1 (or GC-E) and GC2 (or GC-F) to maintain vision. Here, we present data that address the role of the GC kinase homology (KH) domain in cyclic GMP production by GC1, the major cyclase in photoreceptors. First, experiments were done to test which GC1 residues undergo phosphorylation and whether such phosphorylation affects cyclase activity. Using mass spectrometry, we showed that GC1 residues Ser-530, Ser-532, Ser-533, and Ser-538, located within the KH domain, undergo light- and signal transduction-independent phosphorylation in vivo. Mutations in the putative Mg(2+) binding site of the KH domain abolished phosphorylation, indicating that GC1 undergoes autophosphorylation. The dramatically reduced GC activity of these mutants suggests that a functional KH domain is essential for cyclic GMP production. However, evidence is presented that autophosphorylation does not regulate GC1 activity, in contrast to phosphorylation of other members of this cyclase family.

Localization, Purification, and Functional Reconstitution of the P4-ATPase Atp8a2, a Phosphatidylserine Flippase in Photoreceptor Disc Membranes

P(4)-ATPases comprise a relatively new subfamily of P-type ATPases implicated in the energy-dependent translocation of aminophospholipids across cell membranes. In this study, we report on the localization and functional properties of Atp8a2, a member of the P(4)-ATPase subfamily that has not been studied previously. Reverse transcription-PCR revealed high expression of atp8a2 mRNA in the retina and testis. Within the retina, immunofluorescence microscopy and subcellular fractionation studies localized Atp8a2 to outer segment disc membranes of rod and cone photoreceptor cells. Atp8a2 purified from photoreceptor outer segments by immunoaffinity chromatography exhibited ATPase activity that was stimulated by phosphatidylserine and to a lesser degree phosphatidylethanolamine but not by phosphatidylcholine or other membrane lipids. Purified Atp8a2 was reconstituted into liposomes containing fluorescent-labeled phosphatidylserine to measure the ability of Atp8a2 to flip phosphatidylserine across the lipid bilayer. Fluorescence measurements showed that Atp8a2 flipped fluorescent-labeled phosphatidylserine from the inner leaflet of liposomes (equivalent to the exocytoplasmic leaflet of cell membranes) to the outer leaflet (equivalent to cytoplasmic leaflet) in an ATP-dependent manner. Our studies provide the first direct biochemical evidence that purified P(4)-ATPases can translocate aminophospholipids across membranes and further implicates Atp8a2 in the generation and maintenance of phosphatidylserine asymmetry in photoreceptor disc membranes.

Cytochrome C oxidase expression in retinal rod outer segment disks

Abstract Purpose The rod outer segment (ROS) plays a pivotal role in the phototransduction cascade, a process which requires a high level of energy consumption. In rods ATP production is confined to the mitochondria in the inner part, and the distances between sites of energy production and expenditure do not seem adequate to supply phototransduction. The goal of this study was to test the hypothesis that Cytochrome c oxydase (Cox) is located and catalytically active in ROS disk membranes. Methods Osmotically intact disks were isolated from 20 bovine retinal ROS obtained by sucrose gradient centrifugation in the presence of protease inhibitor cocktail and Ampicillin under dim red light at 4°C. Cox expression was studied by confocal laser scanning microscopy and semiquantitative Western‐immunoblotting, with a specific antibody. Cox activity in disks was investigated spectrophotometrically. Results Results from immunohystochemical confocal laser scanning microscopy, Western blot, and biochemical experiments suggest that Cox is uniformly distributed onto disks and therein catalytically active to an extent comparable to that of isolated mitochondria. Conclusion The expression and activity of Cox in bovine ROS disks is suggestive of an aerobic metabolism in ROS. This study opens new scenarios on the energy production in rods and on the possible role of its deficiency in some important retinal diseases which have not been clarified yet.

Role of the C Terminus of the Photoreceptor ABCA4 Transporter in Protein Folding, Function, and Retinal Degenerative Diseases

ABCA4 is an ATP-binding cassette transporter that is expressed in rod and cone photoreceptor cells and implicated in the removal of retinal derivatives from outer segments following photoexcitation. Mutations in the ABCA4 gene are responsible for a number of related retinal degenerative diseases, including Stargardt macular degeneration, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. In order to determine the role of the C terminus of ABCA4 in protein structure and function and understand mechanisms by which C-terminal mutations cause retinal degenerative diseases, we have expressed and purified a series of deletion and substitution mutants of ABCA4 and ABCA1 in HEK 293T cells for analysis of their cellular localization and biochemical properties. Removal of the C-terminal 30 amino acids of ABCA4, including a conserved VFVNFA motif, resulted in a loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal-stimulated ATPase activity. This mutant was also retained in the endoplasmic reticulum of cells. Replacement of the VFVNFA motif with alanine residues also resulted in loss in function and cellular mislocalization. In contrast, C-terminal deletion mutants that retain the VFVNFA motif were functionally active and localized to intracellular vesicles similar to wild-type ABCA4. Our studies indicated that the VFVNFA motif is required for the proper folding of ABCA4 into a functionally active protein. This motif also contributes to the efficient folding of ABCA1 into an active protein. Our results provide a molecular based rationale for the disease phenotype displayed by individuals with mutations in the C terminus of ABCA4.

Rhodopsin in Bovine Rod Outer Segment Disk Membranes Exhibits Two Thermal Transitions

The photoreceptor, rhodopsin is a GPCR in rod outer segment disk membranes. Activation by light converts the dark-adapted form, rhodopsin is to the bleached form, opsin. Differential scanning calorimetry (DSC) studies showed that rhodopsin and opsin each exhibit an irreversible scan rate dependent endothermic transition (Tm) at approximately 72oC and 55oC respectively. We report here a scan rate dependent exothermic transition. Solubilization was used to examine the contribution of the bilayer. Freeze-thaw cycles were used to examine rhodopsin aggregation in these transitions. Disk membranes were sequentially solubilized in octylglucoside (OG) until rhodopsin was completely delipidated. DSC experiments were performed using a MicroCal VP-DSC microcalorimeter. Samples were scanned at 15, 30, 60 and 90°/hr. Because the protein transitions are irreversible, a second scan was used to determine the baseline. As the OG partitioned into the bilayer the endothermic Tm and Eact (activation energy of denaturation) rapidly decreased. Both then remained constant following rhodopsin solubilization. At low detergent concentration the exothermic Tm increased rapidly then remained constant after solubilization. Unlike the endothermic Eact, the degree of solubilization had little effect on the exothermic transition Eact. Freeze-thaw cycles generated disks in which rhodopsin was aggregated. Increasing aggregation resulted in a decrease in the Tm of the exothermic transition, but had no effect on the endothermic Tm of either rhodopsin or opsin. These results are consistent with earlier cross-linking studies suggesting the exothermic transition results from aggregation. These results indicate that the rhodopsin endothermic transition is due to weakening of the tertiary structure interactions accompanied by changes in the packing of the trans-membrane helices as well as changes in protein-lipid interactions. CD data indicate that rhodopsin secondary structure is almost unchanged.

Calorimetric Studies of Bovine Rod Outer Segment Disk Membranes Support a Monomeric Unit for Both Rhodopsin and Opsin

The photoreceptor rhodopsin is a G-protein coupled receptor that has recently been proposed to exist as a dimer or higher order oligomer, in contrast to the previously described monomer, in retinal rod outer segment disk membranes. Rhodopsin exhibits considerably greater thermal stability than opsin (the bleached form of the receptor), which is reflected in an ∼15°C difference in the thermal denaturation temperatures (Tm) of rhodopsin and opsin as measured by differential scanning calorimetry. Here we use differential scanning calorimetry to investigate the effect of partial bleaching of disk membranes on the Tm of rhodopsin and of opsin in native disk membranes, as well as in cross-linked disk membranes in which rhodopsin dimers are known to be present. The Tms of rhodopsin and opsin are expected to be perturbed if mixed oligomers are present. The Tm remained constant for rhodopsin and opsin in native disks regardless of the level of bleaching. In contrast, the Tm of cross-linked rhodopsin in disk membranes was dependent on the extent of bleaching. The energy of activation for denaturation of rhodopsin and cross-linked rhodopsin was calculated. Cross-linking rhodopsin significantly decreased the energy of activation. We conclude that in native disk membranes, rhodopsin behaves predominantly as a monomer.

Regulation of Membrane Proteins by Dietary Lipids: Effects of Cholesterol and Docosahexaenoic Acid Acyl Chain-Containing Phospholipids on Rhodopsin Stability and Function

Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol % cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature ( T m) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process ( E a) was determined from DSC thermograms by four separate methods. Both T m and E a varied with bilayer composition. Cholesterol increased the T m both the presence and absence of docosahexaenoic acid acyl chains (DHA). In contrast, cholesterol lowered E a in the absence of DHA, but raised E a in the presence of 20 mol % DHA-containing phospholipid. The relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. The T m for thermal unfolding was inversely related to acyl chain packing order. Rhodopsin kinetic stability ( E a) was reduced in highly ordered or disordered membranes. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing, such as DHA and cholesterol, may be required for maximum protein stability.

Proteomic Analysis of the Retinal Rod Outer Segment Disks

The initial events of vision at low light take place in vertebrate retinal rods. The rod outer segment consists of a stack of flattened disks surrounded by the plasma membrane. A list of the proteins that reside in disks has not been achieved yet. We present the first comprehensive proteomic analysis of purified rod disks, obtained by combining the results of two-dimensional gel electrophoresis separation of disk proteins to MALDI-TOF or nLC-ESI-MS/MS mass spectrometry techniques. Intact disks were isolated from bovine retinal rod outer segments by a method that minimizes contamination from inner segment. Out of a total of 187 excised spots, 148 proteins were unambiguously identified. An additional set of 61 proteins (partially overlapping with the previous ones) was generated by one-dimensional (1D) gel nLC-ESI-MS/MS method. Proteins involved in vision as well as in aerobic metabolism were found, among which are the five complexes of oxidative phosphorylation. Results from biochemical, Western blot, and confocal laser scanning microscopy immunochemistry experiments suggest that F 1F o-ATP synthase is located and catalytically active in ROS disk membranes. This study represents a step toward a global physiological characterization of the disk proteome and provides information necessary for future studies on energy supply for phototransduction.

Involvement of Lysophosphatidic Acid, Sphingosine 1-Phosphate and Ceramide 1-Phosphate in the Metabolization of Phosphatidic Acid by Lipid Phosphate Phosphatases in Bovine Rod Outer Segments

The aim of the present research was to evaluate the generation of [2-3H]diacylglycerol ([2-3H]DAG) from [2-3H]-Phosphatidic acid ([2-3H]PA) by lipid phosphate phosphatases (LPPs) at different concentrations of lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and ceramide 1-phosphate (C1P) in purified ROS obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. Western blot analysis revealed the presence of LPP3 exclusively in all membrane preparations. Immunoblots of entire ROS and depleted ROS did not show dark-light differences in LPP3 levels. LPPs activities were diminished by 53% in BLROS with respect to DROS. The major competitive effect on PA hydrolysis was exerted by LPA and S1P in DROS and by C1P in BLROS. LPPs activities in depleted ROS were similar to the activity observed in entire DROS and BLROS, respectively. LPA, S1P and C1P competed at different extent in depleted DROS and BLROS. Sphingosine and ceramide inhibited LPPs activities in entire and depleted DROS. Ceramide also inhibited LPPs activities in entire and in depleted BLROS. Our findings are indicative of a different degree of competition between PA and LPA, S1P and C1P by LPPs depending on the illumination state of the retina.

The G protein cascade of visual transduction: kinetics and regulation.

In retinal rods photoexcited rhodopsin (R*) catalyses the activation of transducin (T) by GTP, which in turn activates the cGMP phosphodiesterase (PDE). The ensuing decrease in cGMP concentration reduces the cell membrane's channel conductance. To account for the kinetics of the response to light, all underlying biochemical reactions must reach maximum speed and be turned off within a second. Kinetic analysis of transducin activation suggests that because of the fast lateral diffusion of T, the rate-limiting step is not the collision between R* and T but the entry of GTP after the release fo GDP from the R*-bound T alpha. T alpha-GTP dissociates from both R* and T beta gamma and diffuses through the cytoplasm to activate PDE. In suspensions of bovine rod outer segments, time-resolved microcalorimetry yields rates of approximately 1-2 s-1 for the GTPase of T alpha and the correlated deactivation of PDE. But for isolated T alpha-GTP the single turnover GTPase rate measured by a stopped-flow technique is only 0.05 s-1. To activate PDE, T alpha-GTP binds tightly to the PDE gamma subunit. In vitro the soluble T alpha-GTP.PDE gamma complex dissociates from activated PDE alpha beta. Thus PDE gamma might be the GTPase activator of T alpha, but no GTPase acceleration was observed in isolated T alpha-GTP.PDE gamma. The GTPase activation must depend on the interaction of T alpha-GTP.PDE gamma with membrane-bound PDE alpha beta.

Rhodopsin palmitoylation influences the redistribution of phospholipids in bovine rod outer segment disc membranes

Rhodopsin palmitoylation influences the redistribution of phospholipids in bovine rod outer segment disc membranes

Very Long-chain Polyunsaturated Fatty Acids Are the Major Acyl Groups of Sphingomyelins and Ceramides in the Head of Mammalian Spermatozoa

Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups ( approximately 70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 degrees C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.

X-Ray Crystallographic Analysis of 9-cis-Rhodopsin, a Model Analogue Visual Pigment†

Recent progress in high-resolution structural study of rhodopsin has been enabled by a novel selective extraction procedure with rod photoreceptor cells. In this study, we applied the method for rapid and efficient preparation of a purified analogue pigment using bovine rod outer segment membranes with 9-cis-retinal. After complete bleaching of the membranes and subsequent regeneration with the exogenous retinal, 9-cis-rhodopsin is selectively extracted from the membranes using combination of zinc and heptylthioglucoside. The solubilized sample, even with a small amount of contaminating retinal oximes, is shown to be pure enough for three-dimensional crystallization. The X-ray diffraction from 9-cis-rhodopsin crystals was examined and the electron density map at 2.9 angstroms resolution in the chromophore region can be fitted well with the model of 9-cis-retinal Schiff base.