Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.     

Ultrastructural localization of rhodopsin in the vertebrate retina

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.     

Molecular mechanisms of photoreception. V. Isolation of cytoplasmic membranes of rod outer segments from the frog retina

Cytoplasmic membranes of rod outer segments from frog retina intact rods in retina were stained with fluorescent dye fluoresceinmonomercur acetate. The dye is covalently bound to proteins of cytoplasmic membrane and doesn't penetrate into the cells. Upon isolation of the purified outer segments with the labeled cytoplasmic membranes the cells were disrupted and fractionated in density sucrose gradient. Cytoplasmic membranes possess floating densities different from those of disk membranes and thus providing a mean for separating them from the latter. The main peptides of cytoplasmic membranes are 56, 53, 45, 30 and 28 kDa proteins.     

The role of cholesterol in rod outer segment membranes

The photoreceptor rod outer segment (ROS) provides a unique system in which to investigate the role of cholesterol, an essential membrane constituent of most animal cells. The ROS is responsible for the initial events of vision at low light levels. It consists of a stack of disk membranes surrounded by the plasma membrane. Light capture occurs in the outer segment disk membranes that contain the photopigment, rhodopsin. These membranes originate from evaginations of the plasma membrane at the base of the outer segment. The new disks separate from the plasma membrane and progressively move up the length of the ROS over the course of several days. Thus the role of cholesterol can be evaluated in two distinct membranes. Furthermore, because the disk membranes vary in age it can also be investigated in a membrane as a function of the membrane age. The plasma membrane is enriched in cholesterol and in saturated fatty acids species relative to the disk membrane. The newly formed disk membranes have 6-fold more cholesterol than disks at the apical tip of the ROS. The partitioning of cholesterol out of disk membranes as they age and are apically displaced is consistent with the high PE content of disk membranes relative to the plasma membrane. The cholesterol composition of membranes has profound consequences on the major protein, rhodopsin. Biophysical studies in both model membranes and in native membranes have demonstrated that cholesterol can modulate the activity of rhodopsin by altering the membrane hydrocarbon environment. These studies suggest that mature disk membranes initiate the visual signal cascade more effectively than the newly synthesized, high cholesterol basal disks. Although rhodopsin is also the major protein of the plasma membrane, the high membrane cholesterol content inhibits rhodopsin participation in the visual transduction cascade. In addition to its effect on the hydrocarbon region, cholesterol may interact directly with rhodopsin. While high cholesterol inhibits rhodopsin activation, it also stabilizes the protein to denaturation. Therefore the disk membrane must perform a balancing act providing sufficient cholesterol to confer stability but without making the membrane too restrictive to receptor activation. Within a given disk membrane, it is likely that cholesterol exhibits an asymmetric distribution between the inner and outer bilayer leaflets. Furthermore, there is some evidence of cholesterol microdomains in the disk membranes. The availability of the disk protein, rom-1 may be sensitive to membrane cholesterol. The effects exerted by cholesterol on rhodopsin function have far-reaching implications for the study of G-protein coupled receptors as a whole. These studies show that the function of a membrane receptor can be modulated by modification of the lipid bilayer, particularly cholesterol. This provides a powerful means of fine-tuning the activity of a membrane protein without resorting to turnover of the protein or protein modification.     

Rhodopsin in the rod outer segment plasma membrane

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.     

The cGMP-gated cation channel of bovine rod photoreceptor cells is associated with a 240-kDa protein exhibiting immunochemical cross-reactivity with spectrin.

A 240-kDa protein exhibiting immunochemical cross-reactivity with red blood cell spectrin has been shown to be directly associated with the 63-kDa cGMP-gated channel of bovine rod outer segments. When detergent-solubilized, chromatographically purified channel preparations were treated with Sepharose beads coupled to either an anti-240-kDa monoclonal antibody (PMs 4B2) or an anti-63-kDa channel monoclonal antibody (PMc 1D1), both the 240-kDa protein and the 63-kDa channel protein were concomitantly immunoprecipitated as analyzed by Western blotting of sodium dodecyl sulfate gels. Both of these antibody-Sepharose matrices also removed cGMP-gated channel activity as measured by functional reconstitution. In control studies anti-rhodopsin monoclonal antibody (Rho 1D4)-Sepharose beads removed residual rhodopsin, but not the 63/240-kDa complex or channel activity. Western blotting of purified rod outer segment disk and plasma membrane fractions and immunogold-dextran labeling of lysed rod outer segments indicated that the 240-kDa polypeptide, like the 63-kDa channel, is preferentially localized to the plasma membrane as visualized by electron microscopy. The 240-kDa protein does not appear to be directly involved in the cGMP-gated channel activity, but it may be part of a cytoskeletal system that serves to maintain the organization of the 63-kDa channel complex within the rod outer segment plasma membrane.     

Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration

The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.     

Glyceraldehyde-3-phosphate dehydrogenase is a major protein associated with the plasma membrane of retinal photoreceptor outer segments

A major 38-kDa protein associated with bovine rod outer segment plasma membranes, but not disk membranes, has been identified as glyceraldehyde-3-phosphate dehydrogenase on the basis of its N-terminal sequence and specific enzyme activity. This enzyme was extracted from lysed rod outer segments or isolated rod outer segment plasma membrane with 0.15 M NaCl and purified to homogeneity by affinity chromatography on a NAD(+)-agarose column. A specific activity of 90-100 units/mg of protein is within the range of activity obtained for glyceraldehyde-3-phosphate dehydrogenase isolated from other mammalian cells. Enzyme activity measurements indicate that this enzyme makes up approximately 2% of the total rod outer segment protein and over 11% of the plasma membrane protein. Protease digestion and binding studies on purified rod outer segment plasma and disk membranes suggest that glyceraldehyde-3-phosphate dehydrogenase reversibly interacts with a protease-sensitive plasma membrane-specific protein of rod outer segments. The finding that glyceraldehyde-3-phosphate dehydrogenase is present in large quantities in rod outer segments suggests that at least some of the energy required for the synthesis of ATP and GTP for phototransduction and other processes of the outer segment is derived from glycolysis which takes place within this organelle.     

Lateral diffusion of rhodopsin in photoreceptor cells measured by fluorescence photobleaching and recovery.

Frog rod outer segments were labeled with the sulfhydryl-reactive label iodoacetamido tetramethylrhodamine. The bulk of the label reacted with the major disk membrane protein, rhodopsin. Fluorescence photobleaching and recovery (FPR) experiments on labeled rods showed that the labeled proteins diffused rapidly in the disk membranes. In these FPR experiments we observed both the recovery of fluorescence in the bleached spot and the loss of fluorescence from nearby, unbleached regions of the photoreceptor. These and previous experiments show that the redistribution of the fluorescent labeled proteins after bleaching was due to diffusion. The diffusion constant, D, was (3.0 +/- 10(-9) cm2 s-1 if estimated from the rate of recovery of fluorescence in the bleached spot, and (5.3 +/- 2.4) x 10(-9) cm2 s-1 if estimated from the rate of depletion of fluorescence from nearby regions. The temperature coefficient, Q10, for diffusion was 1.7 +/- 0.5 over the range 10 degrees--29 degrees C. These values obtained by FPR are in good agreement with those previously obtained by photobleaching rhodopsin in fresh, unlabeled rods. This agreement indicates that the labeling and bleaching procedures required by the FPR method did not significantly alter the diffusion rate of rhodopsin. Moreover, the magnitude of the diffusion constant for rhodopsin is that to be expected for an object of its diameter diffusing in a bilayer with the viscosity of the disk membrane. In contrast to the case of rhodopsin, FPR methods applied to other membrane proteins have yielded much smaller diffusion constants. The present results help indicate that these smaller diffusion constants are not artifacts of the method but may instead be due to interactions the diffusing proteins have with other components of the membrane in addition to the viscous drag imposed by the lipid bilayer.     

Isolation of plasma membranes from the bovine retinal pigment epithelium

Retinal pigment epithelium plasma membranes have been isolated by differential and density gradient centrifugation of glass-bead-bound, collagenase-treated cells. Electron microscopic evidence indicates that the glass-bead-bound cells were devoid of red blood cells, rod outer segments and other ocular cell contaminants. The plasma membranes were recovered in 4–6 μg/eye yields and purified 10-fold by 5′-nucleotidase and alkaline phosphodiesterase 1, and 6.5-fold by (Na⁺ + K⁺)-ATPase. Plasma membrane purity as measured by covalent labeling of the epithelial cell plasma membrane proteins with p-(diazonium) benzene[³²S]sulfonic acid was 8–19-fold. In purified plasma membranes contamination by mitochondria was undetectable and lysosomal contamination reduced 100-fold, while endoplasmic reticulum was 2-fold enriched. SDS-polyacrylamide gel electrophoresis of the plasma membrane proteins revealed 23–26 major bands by Coomassie blue staining and 12–16 major bands by radioactive labeling. The plasma membranes exhibited a 3-fold lower concentration of docosahexaenoic acid, a 3-fold higher cholesterol/phosphate ratio, and were 10-fold enriched in cholesterol per μg protein when compared to the whole cell fraction. Retinal epithelial plasma membranes contain an average of 1 mol cholesterol per mol of lipid phosphorus, a high palmitic acid concentration (39 mol%) and a low concentration of docosahexaenoic acid (2 mol%). The lipid profile of the retinal pigment epithelial plasma membranes indicates that they are typical of plasma membranes from many other cell types and that they appear to be less fluid than total rod outer segment membranes.     

Some quantitative characteristics of the frog retinal rod outer segments]

Retinal rod outer segments in frogs were studied by means of light microscopy, refractometry, microspectrophotometry, and electron microscopy. Analysis of the data obtained shows that an unidentified substance, which makes up about 50% of outer segment dry weight, is lost during routine biochemical investigations. The protein parts of the rhodopsin molecules make up 85% of the outer segments proteins and 25% of outer segment dry weight. Rhodopsin molecules can be arranged in a square array with a unit cell side of about 7 nm on one side of each disk membrane. Lipids in a single membrane occupy only 2 nm, and disk membranes are strongly hydrated.     

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26?000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.     

Light stimulates phosphorylation of two large membrane proteins in frog photoreceptors

By photoactivating rhodopsin, light indirectly initiates a series of biochemical reactions within photoreceptors as part of the visual process. I herein report that one of the light-stimulated reactions in bullfrog photoreceptors is the phosphorylation of two previously unreported proteins (220 and 240 kDa). Their phosphorylation by endogenous kinase(s) is readily observed in freshly isolated, fragmented rods. On subcellular fractionation, the labeled proteins copurify with the membranes of the outer segments, from which they cannot be extracted with low ionic strength. They appear to be integral membrane proteins of the disk or plasma membranes. Their light-induced phosphorylation is also observed in intact receptors when excised frog retinas are incubated under in vivo conditions with 32PO4. Thus, appropriate kinase(s) is (are) present within outer segments and presumably is (are) the one(s) responsible for phosphorylation in fragmented cells. In the presence of adenosine 5'-(gamma-[35S]thiotriphosphate) [( 35S] ATP-gamma-S), light can also stimulate thiophosphorylation, leading to preferential labeling of the 220-kDa protein. On the basis of four criteria (electroporetic mobility, membrane location, binding of concanavalin A, and mobility shifts with SH oxidation), the 220-kDa protein appears to correspond to the membrane protein previously identified at the rims of rod disks [Papermaster, D.S., Schneider, B.G., Zorn, M.A. & Kraehenbuhl, J.P. (1978) J. Cell Biol. 78, 415-425]. Identity of the other substrate protein is unknown. When fragmented cells are illuminated with a flash of 1-ms duration, the half-time for phosphorylation is about 1 min with ATP at 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Rhodopsin Regeneration in Photoreceptor Membranes is Correlated with Variations in Membrane Properties

Rhodopsin, the major transmembrane protein in both the plasma membrane and the disk membranes of photoreceptor rod outer segments (ROS) forms the apo-protein opsin upon the absorption of light. In vivo the regeneration of rhodopsin is necessary for subsequent receptor activation and for adaptation, in vitro this regeneration can be followed after the addition of 11-cis retinal. In this study we investigated the ability of bleached rhodopsin to regenerate in the compositionally different membrane environments found in photoreceptor rod cells. When 11-cis retinal was added to bleached ROS plasma membrane preparations, rhodopsin did not regenerate within the same time course or to the same extent as bleached rhodopsin in disk membranes. Over 80% of the rhodopsin in newly formed disks regenerated within 90 minutes while only 40% regenerated in older disks. Since disk membrane cholesterol content increases as disks are displaced from the base to the apical tip of the outer segment, we looked at the affect of membrane cholesterol content on the regeneration process. Enrichment or depletion of disk membrane cholesterol did not alter the % rhodopsin that regenerated. Bulk membrane properties measured with a sterol analog, cholestatrienol and a fatty acid analog, cis parinaric acid, showed a more ordered, less fluid, lipid environment within plasma membrane relative to the disks. Collectively these results show that the same membrane receptor, rhodopsin, functions differently as monitored by regeneration in the different lipid environments within photoreceptor rod cells. These differences may be due to the bulk properties of the various membranes.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Solubilization, purification, and reconstitution of the sodium-calcium exchanger from bovine retinal rod outer segments

We have previously described a method for the solubilization and reconstitution of the cGMP-gated cation channel from the membranes of bovine rod outer segments (Cook, N. J., Zeilinger, C., Koch, K.-W., and Kaupp, U. B. (1986) J. Biol. Chem. 261, 17033-17039). Here we report that not only cGMP but also sodium is capable of releasing entrapped calcium from liposomes reconstituted with total rod outer segment membrane proteins. Other alkali cations tested were unable to induce calcium efflux; therefore, we concluded that the sodium-induced calcium efflux was due to the sodium-calcium exchanger. Sodium was found to activate calcium efflux from these liposomes with an EC50 of approximately equal to 35 mM, comparable to values reported for the sodium-calcium exchanger in native rod outer segment membranes. We found that reconstitution of the sodium-calcium exchanger is quantitative and used this method to assay the exchange protein during purification using conventional protein chromatographic techniques. In this way, we were able to purify and identify as the rod outer segment sodium-calcium exchanger a glycoprotein of apparent Mr = 220,000 to greater than 90% homogeneity. The specific activity of the purified protein at room temperature was 8.2 mumol of Ca2+ exchanged min-1 mg-1 of protein at 50 mM Na+, corresponding to a turnover number of approximately equal to 30 Ca2+ (or 90 Na+) s-1 exchanger-1. The Mr = 220,000 protein reported here appears to be distinct from another protein ("rim protein") with an identical Mr known to exist in these membranes.     

Fusion between disk membranes and plasma membrane of bovine photoreceptor cells is calcium dependent.

Disk membranes and plasma membrane vesicles were prepared from bovine retinal rod outer segments (ROS). The plasma membrane vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C and in the presence of micromolar calcium, an increase in R18 fluorescence with time was observed when R18-labeled plasma membrane vesicles were introduced to a suspension of disks. This result was interpreted as fusion between the disk membranes and the plasma membranes, the fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes as a result of lipid mixing during membrane fusion. While the disk membranes exposed exclusively their cytoplasmic surface, plasma membrane vesicles were found with both possible orientations. These vesicles were fractionated into subpopulations with homogeneous orientation. Plasma membrane vesicles that were oriented with the cytoplasmic surface exposed were able to fuse with the disk membranes in a Ca(2+)-dependent manner. Fusion was not detected between disk membranes and plasma membrane vesicles oriented such that the cytoplasmic surface was on the interior of the vesicles. ROS plasma membrane-disk membrane fusion was stimulated by calcium, inhibited by EGTA, and unaffected by magnesium. Rod photoreceptor cells of vertebrate retinas undergo diurnal shedding of disk membranes containing the photopigment rhodopsin. Membrane fusion is required for the shedding process.     

Glycoproteins specific for the retinal rod outer segment plasma membrane

Two ricin-specific glycoproteins have been identified on neuraminidase-treated rod outer segment plasma membranes of bovine retinal photoreceptor cells. Ricin-gold-dextran particles were observed by electron microscopy to densely label the surface of neuraminidase-treated rod outer segments. Western blotting of proteins separated by SDS-gel electrophoresis indicated that two ricin-binding glycoproteins of Mr 230,000 and 110,000 are specific for the plasma membrane and are not found in disk membranes. These glycoproteins can serve as specific probes for the purification of the rod outer segment plasma membrane.     

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.