Rod Outer Segment Disk Membranes Research Articles

Interconversion of metarhodopsins I and II: a branched photointermediate decay model

Flash photolysis experiments designed to monitor the establishment of the metarhodopsin I to metarhodopsin II equilibrium are interpreted according to a branched model in which two spectrally indistinguishable but kinetically distinguishable forms of metarhodopsin II are postulated to exist in equilibrium with a common pool of metarhodopsin I. This interpretation arises from the consistent requirement for at least three exponentials for a valid description of the observed growth of absorbance at 380 nm following bleaching of bovine rhodopsin in rod outer segment disk membranes. Analysis of the 380-nm transient absorbance data permitted direct determination of the five physically interpretable individual rate constants of the model. This analysis represents a more explicit interpretation of kinetic data than that employed in earlier experiments of this kind, which involved estimating only apparent rates and apparent amplitudes of discrete multiexponential functions. The 380-nm absorbance contributions of all relevant species contributing to the observed dynamic absorbance change were accounted for simultaneously during nonlinear least-squares estimation of the model rate parameters. Analysis of deconvoluted equilibrium spectra acquired from samples identical with those used in the kinetics experiments confirmed the metarhodopsin I-metarhodopsin II equilibrium constants, Keq, derived from the dynamic analyses. It is shown that Keq varies from 1.28 at 10 degrees C to 7.3 at 37 degrees C and that approximately 90% of the metarhodopsin II present is in the form of metarhodopsin IIslow over the temperature range 10-37 degrees C. A physical interpretation of this decay model is discussed in the context of a distribution of metarhodopsin II structural and energetic states.

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.

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.

Molecular cloning, primary structure, and orientation of the vertebrate photoreceptor cell protein peripherin in the rod outer segment disk membrane

Peripherin, a 39-kDa membrane protein, has been previously localized to the rim region of the vertebrate rod photoreceptor disk membrane by use of monoclonal antibodies and immunocytochemical labeling techniques. As an initial step in determining the structure and function of this protein, we have cloned and sequenced cDNA containing its complete coding sequence. A bovine retinal lambda gt11 expression library was screened with the antibodies, and a 583 base pair clone was initially isolated. The remaining part of the coding sequence was obtained from subsequent rescreenings of the same library and an independent lambda gt10 library. A C-terminal CNBr fragment of peripherin was purified by immunoaffinity chromatography and reverse-phase high-performance liquid chromatography. The amino acid sequence of the isolated C-terminal peptide and the N-terminal sequence analysis of immunoaffinity-purified peripherin are in agreement with the cDNA sequence. The cDNA sequence predicts that there are possibly four transmembrane domains. On the basis of immunocytochemical studies and sequence analysis, the hydrophilic C-terminal segment containing the antigenic sites for the antiperipherin monoclonal antibodies has been localized on the cytoplasmic side of the disk membrane. There are three consensus sequences for asparagine-linked glycosylation. Deglycosylation studies have indicated that at least one of these sites is utilized. The possible function of peripherin in relation to its primary structure is discussed.

Light−induced membrane protein phosphorylation in the bovine rod outer segment: A magic angle spinning 31P-NMR study

Magic angle spinning 31P-NMR (MAS 31P-NMR) spectra of bovine rod outer segments, unphosphorylated and phosphorylated, were obtained. In the phosphorylated samples the spectra showed new resonances not assignable to phospholipids. These signals were present only, when stimulation of receptor phosphorylation occurred. These resonances were not due to exogenous, soluble phosphorus-containing compounds. Limited proteolysis to remove the carboxyl-terminal region of the photoreceptor that contains the phosphorylation sites removed these resonances. The chemical shifts were in the usual range for serine phosphate and threonine phosphate. The p K a obtained from a p H titration of the 31 P chemical shift was typical of serine phosphate. Therefore, these 31 P-NMR resonances were assigned to the phosphorylation sites on membrane proteins in the rod outer segment disk membranes. Static 31 P-NMR measurements revealed that at least some of these sites gave rise to relatively narrow resonances, indicative of considerable motional freedom of the carboxyl-terminal segment of the photoreceptor when phosphorylated. These data indicate that it is possible to study phosphorylation sites on membrane proteins using MAS 31 P-NMR, and that using in vivo 31 P “spin labelling” one can study directly and selectively regions of receptors crucial to receptor function.

Cholesterol dynamics in membranes

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.

Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane

After neuraminidase treatment the Na+/Ca2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of Mr 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na+/Ca2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na+/Ca2+ exchange activation by sodium. We further investigated the density of the Na+/Ca2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na+/Ca2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as we have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na+/Ca2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles.

Transbilayer coupling mechanism for the formation of lipid asymmetry in biological membranes. Application to the photoreceptor disc membrane

An equilibrium transmembrane asymmetry in charged lipids is shown to arise as a result of oriented, bipolar proteins in the membrane. The basic interaction giving rise to the asymmetry is between a lipid molecule and a transbilayer potential generated by the asymmetric charge distribution in the protein. Thus, a protein can generate a lipid asymmetry without a direct binding interaction between lipid and protein. The generation of an asymmetry in charged lipid by this mechanism can also lead to a concomitant asymmetry in neutral lipids if deviations from ideality in the lipid mixture are taken into account. It is shown that regular solution theory applied to the lipid phase predicts an asymmetry in all components of a ternary mixture as long as one component is electrostatically oriented according to the mechanism mentioned above. The resulting asymmetry is not strongly salt dependent. The mechanism quantitatively accounts for the experimentally determined phospholipid asymmetry in the rod outer segment disc membrane of the vertebrate photoreceptor.

Distribution of charge on photoreceptor disc membranes and implications for charged lipid asymmetry

A novel spin labeling technique is used to determine both the inner and outer surface potentials of isolated rod outer segment disc membranes and of reconstituted membranes containing rhodopsin with defined lipid compositions. It is shown that these potentials can be accounted for in a consistent manner by the accepted model of rhodopsin, the known lipid composition, and the Gouy-Chapman theory, provided the charged lipid is asymmetric in the membrane, with approximately 75% on the external surface.

Transient response of rod outer segment cGMP phosphodiesterase to actinic light pulses: II. Detailed quantitative kinetic model

Abstract In the accompanying article (Schmidt, J.A., and Yguerabide, J. (1989) J. Biol. Chem. 264, 19790-19803), we presented a minimal quantitative kinetic model with one rate-limiting step for the transient response of rod outer segment (ROS) phosphodiesterase (PDE) to stimulating light pulses of low fractional bleach (linear response range) and showed that the model was in excellent quantitative agreement with experimental results. The model characterizes the PDE response in terms of the specific rate constant of the rate-limiting step, kL, the lifetime of photoactivated rhodopsin, tau R, and the lifetime of activated PDE, tau P, but makes no predictions on how these kinetic parameters should depend on the concentrations of the various reactive species involved in the PDE response to light and does not reveal the nature of the rate-limiting step. However, we established by curve fitting experimental data to theoretical expressions from the model that kL increases hyperbolically with [GTP], tau R decreases with [GTP], and tau P is independent of GTP. In this report we present three detailed kinetic models which make specific quantitative predictions on how the kinetic parameters of the minimal model should depend on nucleotide and G protein concentrations and test the models against experimental data. Each model consists of one rate-limiting step. The first detailed model postulates that the rate-limiting step is the dissociation of R*GT into R* and GT (T stands for GTP). The second model postulates that the rate-limiting step is the binding of GTP to R*G, and the third model postulates that the rate-limiting step is the encounter rate of R* and G on the ROS disc membrane. We find that only the first detailed model is consistent with the experimental results as characterized by the minimal model. Using this detailed model we (a) define kL and tau R in terms of more fundamental equilibrium and rate parameters, (b) develop a theory for the systematic evaluation of amplification or gain of the PDE light response from light-stimulated GTP-binding data as well as v(t) versus t graphs, and (c) clarify methods which have been used in the past to evaluate gain experimentally.

Cholesterol movement between bovine rod outer segment disk membranes and phospholipid vesicles

The ability of cholesterol to move between bovine rod outer segment disk membranes and phospholipid membranes was examined. Disk membranes were incubated with small unilamellar phospholipid vesicles containing varying amounts of cholesterol. Aliquots were removed at specific times, and then the disks and the vesicles were separated by centrifugation and assayed for phospholipid and cholesterol content. When incubated with vesicles containing no cholesterol, the cholesterol to phospholipid ratio in the disk membrane was reduced due to migration of cholesterol from the disks into the vesicles. The cholesterol content of these cholesterol depleted disks could be readily returned to the normal disk cholesterol content by incubation of the cholesterol-depleted disks with small unilamellar vesicles containing high cholesterol. An apparent partition coefficient K was calculated as the quotient of the cholesterol/phospholipid mole ratio in the donor membranes and the cholesterol/phospholipid mole ratio in the acceptor membranes. The value of K was approximately 1 at cholesterol levels below normal disk cholesterol content, for disk membranes and phosphatidylcholine small unilamellar vesicles. Inclusion of phosphatidylethanolamine in the small unilamellar vesicle acceptor raised K, indicating that phosphatidylethanolamine creates an unfavourable environment for cholesterol. The cholesterol to phospholipid ratio of native disks could be increased by incubation with phosphatidylcholine small unilamellar vesicles (donor) which contained higher amounts of cholesterol than the disk membrane acceptor. In these experiments the distribution of cholesterol between disks and small unilamellar vesicles always favored the vesicles. The apparent partition coefficient was 1.7 at several cholesterol levels above the native disk cholesterol content. Liposomes made from lipid extracted from the disk membrane behaved in the same manner as intact disks with respect to cholesterol distribution at equilibrium. The phospholipid content of the disk membrane may be an important factor in determining the cholesterol content of the disk membrane.

Cholesterol heterogeneity in bovine rod outer segment disk membranes

Rod outer segment disk membranes have been used to study visual transduction events. Numerous studies have also focused on protein-lipid interactions in these membranes. The possible heterogeneity of the disk membrane composition has not been addressed in such studies. Freeze fracture studies (Andrews, L. D., and Cohn, A. I. (1979) J. Cell Biol. 81, 215-220; Caldwell, R., and McLaughlin, B. (1985) J. Comp. Neurol. 236, 523-537) suggest a difference in cholesterol content between newly formed and old disks. This potential heterogeneity in disk membrane composition was investigated using digitonin. Osmotically intact bovine rod outer segment disk membranes prepared by Ficoll flotation were separated based on the cholesterol content of the disks. The addition of digitonin to disk membrane suspensions in a one-to-one molar ratio with respect to cholesterol produced an increase in the density of the membranes in proportion to the amount of cholesterol present. The digitonin-treated disks were separated into subpopulations using a sucrose density gradient. Disks were shown to vary in cholesterol to phospholipid ratio from 0.30 to 0.05. The ratio of phospholipid to protein remained constant in all disk subpopulations at approximately 65 phospholipids per protein. No significant change in the fatty acid composition of the disks was observed as a function of change in cholesterol content. This work demonstrates compositional heterogeneity in disk membranes which may ultimately affect function.

Equilibrium and dynamic bilayer structural properties of unsaturated acyl chain phosphatidylcholine-cholesterol-rhodopsin recombinant vesicles and rod outer segment disk membranes as determined from higher order analysis of fluorescence anisotropy decay.

Limited-frequency phase-modulation fluorometry of diphenylhexatriene (DPH) and trimethylammonium-diphenylhexatriene (TMA-DPH) was used to characterize the equilibrium and dynamic lipid structural properties of (1) reconstituted egg phosphatidylcholine (egg PC)-rhodopsin vesicles varying in rhodopsin content from 0 to approximately 1 mol %, (2) reconstituted PC-cholesterol-rhodopsin vesicles containing approximately 1 mol % rhodopsin and 0, approximately 15, or approximately 30 mol % cholesterol with egg PC, DOPC (di-18:1-PC), or PAPC (16:0,20:4-PC) as the phospholipid constituent, and (3) native bovine rod outer segment disk membranes. Experiments were conducted at 37, 25, 15, and 5 degrees C. Fluorescence lifetime analysis was performed by fitting the data to a constrained, discrete, biexponential model. Rotational depolarization properties were considered by a model requiring a single rotational diffusion coefficient and capable of producing orthogonal, bimodal orientational distributions for DPH and unimodal distributions for TMA-DPH [Straume, M., & Litman, B. J. (1987) Biochemistry 26, 5113-5120]. Unbleached rhodopsin reduced mean fluorophore lifetimes in proportion to the amount of protein present in PC vesicles as a result of probe-to-retinal energy transfer by (1) redistributing the relative lifetime contributions in favor of the short lifetime population and (2) reducing the lifetimes of each derived population. Lifetimes were increased by cholesterol and by reduction of the temperature, but the relative proportions of derived short- and long-lifetime populations were not affected. TMA-DPH lifetimes were more sensitive (in a relative manner) than were those of DPH. These observations are interpreted in terms of cholesterol and reduced temperature each inhibiting water penetrability into these bilayers, with a greater effect occurring in the headgroup and interfacial regions (probed by TMA-DPH) than in the hydrophobic bilayer interior (probed by DPH). Diunsaturated DOPC-rhodopsin recombinants were more resistant to temperature-dependent lifetime changes than were mixed-chain egg PC or PAPC vesicles. This suggests less favorable interaction of rhodopsin with diunsaturated PCs than with mixed-chain PCs. Lifetimes in disk membranes exhibited this same temperature dependence although DPH in disks had lifetimes longer than those seen in recombinant vesicles. TMA-DPH lifetimes in disks were more similar to those observed in cholesterol-containing recombinants. It would therefore appear that the large proportion of small, charged (at pH 7) phosphatidylethanolamine and phosphatidylserine headgroups present in disks reduces water penetrability into the d

Reconstitution of rhodopsin and the cGMP cascade in polymerized bilayer membranes.

The successful reconstitution of rhodopsin, the rod outer segment (ROS) G protein, and the ROS phosphodiesterase (PDE) into partially polymerized bilayer membranes is described. Purified bovine rhodopsin (Rh) was inserted into performed partially polymerized lipid vesicles. Sonicated vesicles composed of approximately equal moles of dioleoylphosphatidylcholine (DOPC) (or 1-palmitoyl-2-oleoyl-phosphatidylcholine) and 1,2-bis(octadeca-2,4-dienoyl)phosphatidylcholine (DENPC) were photolyzed with 254-nm light to polymerize the DENPC and form domains of DOPC and polyDENPC in the vesicle wall. Rh-octyl glucoside (OG) micelles were slowly added to the vesicle suspension to give 15 mM OG (below the OG critical micelle concentration). The suspension was incubated and then dialyzed and purified on a sucrose gradient. Ultracentrifugation revealed a major Rh-lipid band which was harvested and found to contain a 100 +/- 10 phosphatidylcholine to rhodopsin ratio (Rh-polyDENPC/DOPC). The orientation of Rh in the membrane was determined by limited proteolytic digestion of Rh and by competitive inhibition of monoclonal antibody binding to solubilized disk membranes. Results were compared with control membranes of Rh-DOPC (1:43) prepared by insertion and Rh-phospholipid membranes prepared by detergent dialysis. Visual inspection of thermolysin proteolytic patterns of Rh indicates one major population cleaved at the carboxy terminus, as is found in disk membranes with an asymmetric arrangement of Rh. In contrast, proteolysis of a Rh-egg PC/PE (1:50/50) membrane (detergent dialysis) produced two Rh populations, which indicates a symmetric arrangement of Rh. The Rh-polyDENPC/DOPC (1:100) membranes were allowed to compete with solubilized, immobilized disk membranes for the monoclonal antibody R2-15 (specific for the amino-terminal region of Rh). They were intermediate between the asymmetric ROS disk membranes and the symmetric dialysis membranes in their ability to bind the R2-15 monoclonal antibody. The data indicate approximately 80% of the Rh's in Rh-polyDENPC/DOPC are in the normal orientation found in disks. These Rh-containing polymerized bilayer membranes demonstrated functionality as determined by chemical regeneration, kinetic spectrophotometry, and cGMP cascade reconstitution experiments. In the latter experiments the peripheral proteins, ROS G protein and PDE, bound with comparable efficiency to both the polymerized PC bilayers and egg PC bilayers. Thus the biocompatibility of the phosphatidylcholine membrane surface was maintained after polymerization of DENPC.

Regulation of eye derived growth factor binding to membranes by light, ATP or GTP in photoreceptor outer segments.

Eye derived growth factor II (EDGF II), the retinal form of acidic fibroblast growth factor (aFGF) is present in rod outer segments (ROS) purified in the dark, which display higher EDGF specific activity than all other parts of the retina. EDGF binds to ROS disc membranes upon illumination. This binding is not reversible in the dark. ATP, but not GTP, readily releases EDGF from either dark-adapted or previously bleached ROS. The release of EDGF activity from ROS membranes would require a phosphorylation mechanism since AMP-PNP, an ATP analogue, is not efficient. ROS membranes compete with cellular EDGF receptors of retinal pigment epithelial cells in vitro for the binding of labelled EDGF II, suggesting that they also possess specific binding sites. These data suggest that EDGF II is involved in photoreceptor cell biology.

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.

Electron immunocytochemical localization of retinal S-antigen with a rat monoclonal antibody

This report describes the ultrastructural localization of S-antigen in pig and human retinas by means of a highly specific rat IgG monoclonal antibody, S2.4.C5, followed by a secondary antibody adsorbed to colloidal gold. This monoclonal antibody gave definitive staining with negligible background. The protein was detected in both the rod outer and inner segments. Connecting cilia and the rod outer segment disc membranes were labelled. The outer segment plasma membrane was not obviously labelled. Cones were labelled at background level. S-Antigen was not detected in any other cells of the neural retina. The fate of S-antigen was also followed to the pigment epithelial phagosomes where intracytoplasmic ROS debris was stained with the antibody. No label, however, was detected in the choroid, suggesting that trans-cellular transport of the S-antigen did not occur.

The GTP-binding protein of rod outer segments. II. An essential role for Mg2+ in signal amplification.

The role of Mg2+ in the GTP hydrolytic cycle was investigated by using purified subunits (G alpha and G beta, gamma) of the GTP-binding protein isolated from Bufo marinus rod outer segments (ROS). Mg2+ markedly stimulated the rate of GTP and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha. This effect was especially striking in the presence of very small quantities of illuminated ROS disc membranes. GTP hydrolysis could occur in the absence of Mg2+, and Mg2+ increased the rate of GTP hydrolysis only about 50%. These data indicate that Mg2+ plays a fundamental role in amplification of the photon signal by markedly stimulating the rate of formation of GTP X G alpha complexes by very small amounts of illuminated rhodopsin while producing only a modest increase in the rate of GTP hydrolysis. Following hydrolysis of GTP, GDP X G alpha could reassociate with illuminated or unilluminated ROS disc membranes in the presence or absence of Mg2+. In the absence of guanine nucleotides, release of GDP from G alpha bound to illuminated disc membranes was detected in the presence or absence of Mg2+. Moreover, Mg2+ did not affect the rate of GDP release from membrane-bound G alpha. Illumination of B. marinus crude ROS disc membrane preparations markedly reduced pertussis toxin-mediated ADP-ribosylation of a 39,000 Mr (G alpha) protein in the presence but not in the absence, of Mg2+. Moreover, extensive dialysis of illuminated (but not unilluminated) crude ROS disc membranes against a Mg2+-containing buffer caused a marked reduction in the subsequent ADP-ribosylation of G alpha, even when Mg2+ was not present during the ADP-ribosylation step. This reduction was reversed by the addition of GDP or a GDP analogue (but not GMP or hydrolysis-resistant GTP analogues) during the ADP-ribosylation step. Dialysis of crude ROS disc membrane preparations (illuminated or unilluminated) against a Mg2+ -free buffer did not reduce the subsequent ADP-ribosylation of G alpha. These data indicate that Mg2+, in the presence of photolysed rhodopsin, can stimulate the release of GDP from crude preparations of ROS disc membranes. Four lines of evidence suggest that G alpha and G beta, gamma have Mg2+-binding site(s). When stored at 4 degrees C, in the absence of glycerol, G beta, gamma was more stable in the absence than in the presence of Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)

The GTP-binding protein of rod outer segments. I. Role of each subunit in the GTP hydrolytic cycle.

The GTP-binding protein of Bufo marinus rod outer segments (ROS) is composed of 3 subunits: G alpha, 39,000; G beta, 36,000; and G gamma, approximately 6,500. A stepwise analysis of the GTP hydrolytic cycle (GTP binding, GTP hydrolysis, and GDP release) was facilitated by using purified subunits of the GTP-binding protein. When G alpha and G beta, gamma concentrations were held constant, the initial rate of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha was dependent upon the amount of bleached rhodopsin present (as illuminated, urea-washed ROS disc membranes). When G alpha and the quantity of these membranes was held constant, the initial rate of GTP gamma-s binding to G alpha was markedly enhanced by increasing the amount of G beta, gamma. G beta preparations (free of G gamma) also stimulated the binding of GTP gamma-s to G alpha to the same extent as G beta, gamma preparations, suggesting that G gamma is not an essential component of the G beta, gamma-dependent stimulation of the rate of GTP gamma-s binding to G alpha. Nonlinear regression analysis revealed a single class of binding sites with an apparent stoichiometry of 1 mol of site/mol of G alpha under optimal binding conditions. Following GTP binding to G alpha, the GTP X G alpha complex dissociates from G beta, gamma which remains primarily bound to the ROS disc membranes. Moreover, while GTP remains in excess, the rates of GTP hydrolysis exhibited saturation in the presence of increasing amounts of G beta, gamma. Nonlinear regression analysis of these data argues against a direct role for G beta, gamma in the hydrolysis of GTP. Thus, both topologic and kinetic data support the concept that GTP hydrolysis is carried out by G alpha alone. After hydrolysis of GTP, the GDP X G alpha complex returned to the ROS disc membrane when G beta, gamma was present on the membrane surface, in the presence and absence of light. Without guanine nucleotides GDP release occurred in the presence of illuminated ROS disc membranes and G beta, gamma. Guanine nucleotides (GTP gamma-s approximately equal to GTP approximately equal to guanosine 5'-(beta, gamma-imido)triphosphate greater than GDP) could effectively displace GDP from G alpha under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural features of the terminal loop region of frog retinal rod outer segment disk membranes: I. Organization of lipid components.

We have applied thin sectioning and freeze-fracture techniques to investigate the terminal loop structure of photoreceptive disks in frog retinal rod outer segments. Our studies of this region demonstrate a highly curved terminal loop bilayer that is continuous with both lamellar bilayers of the disk, and equivalent to them in dimensions and staining properties. Rhodopsin, however, appears to be excluded from this region of high curvature.