首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
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.  相似文献   

2.
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.  相似文献   

3.
The outer segment portion of photoreceptor rod cells is composed of a stacked array of disk membranes. Newly formed disks are found at the base of the rod outer segment (ROS) and are relatively high in membrane cholesterol. Older disks are found at the apical tip of the ROS and are low in membrane cholesterol. Disk membranes were separated based on their membrane cholesterol content and the extent of membrane protein phosphorylation determined. Light induced phosphorylation of ROS disk membrane proteins was investigated using magic angle spinning31P NMR. When intact rod outer segment preparations were stimulated by light, in the presence of endogenously available kinases, membrane proteins located in disks at the base of the ROS were more heavily phosphorylated than those at the tip. SDS-gel electrophoresis of the phosphorylated disk membranes subpopulations identified a phosphoprotein species with a molecular weight of approximately 68–72 kDa that was more heavily phosphorylated in newly formed disks than in old disks. The identity of this phosphoprotein is presently under investigation. When the phosphorylation reaction was carried out in isolated disk membrane preparations with exogenously added co-factors and kinases, there was no preferential protein phosphorylation. Taken collectively, these results suggest that within the ROS there is a protein phosphorylation gradient that maybe indicative of co-factor or kinase heterogeneity.  相似文献   

4.
The vertebrate visual pigment of rods, rhodopsin, bleaches in light and regenerates in darkness. When the bleaching and regeneration are carried out in vivo, it is found that the regeneration takes place at nonuniform rates along the rod outer segment (ROS): toads and frogs regenerate rhodopsin faster in the proximal ends of the ROS than in the distal ends. Rats do the reverse. These patterns of regeneration persist whether the bleaching is done with flashes or with steady light. They are also independent of the extent to which the retinal pigment epithelium contains melanin. Furthermore, the dichotomy of patterns (proximal faster vs. distal faster) does not seem to depend upon the presence of an excess of stored retinoid in the eye. Instead, it is suggested that the villous processes of the epithelial cells may play an important role in the regeneration patterns. These processes in amphibia extend nearly to the rod inner segment but in the rat they surround only the apical end of the outer segment. If they "funnel" the retinoids back to the ROS, their location and morphology could explain the two different kinds of patterns seen.  相似文献   

5.
Exposure of an intact vertebrate eye to light bleaches the rhodopsin in the photoreceptor outer segments in spatially nonuniform patterns. Some axial bleaching patterns produced in toad rods were determined using microspectrophotometric techniques. More rhodopsin was bleached at the base of the outer segment than at the distal tip. The shape of the bleaching gradient varied with the extent of bleach and with the spectral content of the illuminant. Monochromatic light at the lambda max of the rhodopsin gave rise to the steepest bleaching gradients and induced the greatest changes in the form of the gradient with increasing extent of bleach. These results were consistent with a mathematical model for pigment bleaching in an unstirred sample. The model did not fit bleaching patterns resulting from special lighting conditions that promoted the photoregeneration of rhodopsin from the intermediates of bleaching. Prolonged light adaptation of toads could also produce axial rhodopsin gradients that were not fit by the bleaching model. Under certain conditions the axial gradient of rhodopsin in a rod outer segment reversed with time in the light: the rhodopsin content became highest at the base. This result could be explained by an interaction between the pattern of bleaching and the intracellular topography of regeneration.  相似文献   

6.
The rod outer segment (OS), comprised of tightly stacked disk membranes packed with rhodopsin, is in a dynamic equilibrium governed by a diurnal rhythm with newly synthesized membrane inserted at the OS base balancing membrane loss from the distal tip via disk shedding. Using transgenic Xenopus and live cell confocal imaging, we found OS axial variation of fluorescence intensity in cells expressing a fluorescently tagged rhodopsin transgene. There was a light synchronized fluctuation in intensity, with higher intensity in disks formed at night and lower intensity for those formed during the day. This fluctuation was absent in constant light or dark conditions. There was also a slow modulation of the overall expression level that was not synchronized with the lighting cycle or between cells in the same retina. The axial variations of other membrane-associated fluorescent proteins, eGFP-containing two geranylgeranyl acceptor sites and eGFP fused to the transmembrane domain of syntaxin, were greatly reduced or not detectable, respectively. In acutely light-adapted rods, an arrestin-eGFP fusion protein also exhibited axial variation. Both the light-sensitive Rho-eGFP and arrestin-eGFP banding were in phase with the previously characterized birefringence banding (Kaplan, Invest. Ophthalmol. Vis. Sci. 21, 395–402 1981). In contrast, endogenous rhodopsin did not exhibit such axial variation. Thus, there is an axial inhomogeneity in membrane composition or structure, detectable by the rhodopsin transgene density distribution and regulated by the light cycle, implying a light-regulated step for disk assembly in the OS. The impact of these results on the use of chimeric proteins with rhodopsin fused to fluorescent proteins at the carboxyl terminus is discussed.  相似文献   

7.
《The Journal of cell biology》1987,105(6):2589-2601
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.  相似文献   

8.
Surfaces of rod photoreceptor disk membranes: integral membrane components   总被引:8,自引:4,他引:4  
The membrane surfaces within the rod outer segment of the toad, Bufo marinus, were exposed by rapid-freezing followed by freeze-fracture and deep-etching. Platinum-carbon replicas of disk membranes prepared in this way demonstrate a distinct sidedness. The membrane surface that faces the lumen of the disk shows a fine granularity; particles of approximately 6 nm are packed at a density of approximately 30,000/micron 2. These dimensions suggest that the particles represent protrusions of the integral membrane protein, rhodopsin, into the intradisk space. In addition, when rhodopsin packing is intentionally perturbed by exhaustive digestion with phospholipase C, a concomitant change is observed in the appearance of the luminal surface granularity. The cytoplasmic surface of the disk rarely displays this rough texture; instead it exhibits a collection of much larger particles (8-12 nm) present at approximately 10% of the concentration of rhodopsin. This is about the size and concentration expected for certain light-regulated enzymes, cGMP phosphodiesterase and GTP-binding protein, which are currently thought to localize on or near the cytoplasmic surface of the disk. The molecular identity of the 8-12-nm particles will be identified in the following companion paper. A further differentiation of the cytoplasmic surface can be seen around the very edge, or rim, of each disk. This rim has relatively few 8-12- nm particles and instead displays short filamentlike structures connecting it to other membranes. These filaments extend between adjacent disks, across disk incisures, and from disk rims to the nearby plasma membrane.  相似文献   

9.
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.  相似文献   

10.
Frog retinal rod outer segments appear to contain uncharacterized chemical components whose mass is roughly equivalent to 12--51% of the rhodopsin mass. Available data suggest that such components include soluble proteins and complex polysaccharides, and that hyaluronic acid accounts for a substantial fraction of this mass. Electron microscopic histochemical staining studies suggest that these polysaccharide components are located within the ROS disks. The oligosaccharide moieties of rhodopsin also appear localized within the disks. The interdisk cytoplasm may contain carbohydrates, but their quantity and identity are uncertain. Rhodopsin oligosaccharides as well as some fraction of the intradisk polysaccharide appear to have extended saccharide chains preferentially oriented perpendicular to the surface of the disk membrane. Possible roles for these polysaccharides in disk development and photoexcitation are discussed. The immediate need for complete rod outer segment chemical composition data is emphasized.  相似文献   

11.
R Wagner  N Ryba  R Uhl 《FEBS letters》1988,235(1-2):103-108
In photoreceptors of the living retina both activation and deactivation of transducin must occur in less than 1 s. In ROS preparations used for in vitro studies, however, deactivation takes minutes. This is due to the fact that activated transducin is released into the free aqueous space, whereby GTPase activity and consequent deactivation of the protein are slowed down, and due to the dilution of soluble ROS proteins involved in the quenching of rhodopsin activity. In this paper, using a convenient, non-invasive light scattering assay, we demonstrate that in an intact stack of disks, where active transducin stays membrane associated and is rapidly deactivated, the activity of rhodopsin can also be quenched in the time range of seconds when soluble ROS proteins are supplemented. Arrestin, the 48 kDa protein of the photoreceptor, is one of the proteins required for rapid recovery, however, it requires the synergistic action of other soluble proteins (besides rhodopsin kinase) in order to exert its effect: When arrestin is included in the reaction mixture without the 'helper protein(s)', it cannot speed recovery, and when a mixture of soluble proteins is added which lacks arrestin, there is also no effect. The nature and identity of this (these) helper protein(s) are still unclear.  相似文献   

12.
Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample''s copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein–lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.  相似文献   

13.
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.  相似文献   

14.
Existing hypotheses on the mode of disk formation in the photoreceptor cells of mammals appear to be incompatible: (1) plasma membranes of adjacent evaginations form a disk which, subsequently, is internalized by a disk rim; (2) pinocytotic vesicles are pinched off from the plasma membrane and fuse into a larger vesicle, which flattens and forms a disk. We have studied the development of the cone outer segment and the disk formation in Tupaia belangeri by transmission electron microscopy. During the first two postnatal weeks, the distal part of the single cilium, which is inserted apically on the inner segment, becomes balloon-shaped. Apical to the axoneme, it contains tubular and vesicular material, which, most probably, has been detached from the axonemal microtubules. These tubules and vesicles do not contribute to disks. The balloon-shaped expansion, later retained as the ciliary backbone, establishes the contact with the pigment epithelium. Formation of disks, from the 12-day-old Tupaia onwards, occurs between adjacent evaginations at the outer segment base. The initial disk rims are “hooked” to the ciliary axonemal microtubules. The axonemal microtubules are involved in the initiation and in the alignment of the disks. Disk rim formation and, thus, internalization of disks proceeds from the base to the apex of the outer segment, that is, from the younger to the older disks. In the adult Tupaia, an uneven progression of disk rim formation on both sides of the axoneme is found among consecutive disks. The seemingly incompatible hypotheses on the mode of disk formation reflect a heterochrony of the internalization of membranes and of the disk formation among different mammals and, possibly, between cones and rods. Received: 24 July 1997 / Accepted: 10 September 1997  相似文献   

15.
The development of the retinal receptor cell in the young tadpoles (Bufo raddei Strauch), from the stage 20 to the stage 25, was studied by TEM and immunohistochemical method. The morphological differentiation of the photoreceptor cell may be described as follows. The time and the degree of differentiation of the cells in the tadpole retina is asynchronous between central (posterior pole) and peripheral parts of the tadpole retina, namely, they are earlier and higher in the central than in the peripheral. The cells of the outer nuclear layer are undifferentiated at the stage 20. The cells in the posterior part of the retina elongate at the beginning of the stage 21 (Plate I, Fig. 1). This is the first sign of differentiation in the photoreceptor cell. A small hillock-like process forms the inner segment at the scleral pole of the receptor cell. The inner segment is rich in mitochondria, rough-surfaced cytomembrane, free ribosomes, and vesicles. One or two large lipid droplets are also found in the inner segment (Plate I Fig. 2-3). Later on, the connecting structure develops at the tip of the inner segment. The newly formed filaments and the plasma membrane form the outer segment. Its membrane forms some evaginations oriented perpendicularly to the longitudinal axis of the receptor cell. In this way, disks of the outer segment are formed (Plate I Fig. 4-5). The length of the outer segment gradually increases with the number of disks increasing at the base. At the same time, an axon process of receptor cell, extending vitreal, develops synapses with dendrites of the bipolar cell in the outer plexiform layer. At the beginning (the stage 22), the synaptic structure is an immature form that lacks synaptic ribbons and vesicles (Plate II Fig. 8). Later on, ribbons and vesicles are observed in the further developed synaptic structure (Plate II Fig. 9). The toad rhodopsin was prepared by a method of Dewey et al. (1969) and Papermaster & Dreyer (1974) with slight modification. A specific immune serum against the toad rhodopsin was produced in rabbits. Using the indirect Coon's antibody technique, the localization of the rhodopsin antibody and the time when the antibody was seen in the retina of the early developing tadpoles was traced.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

16.
A rhodopsin phosphorylation reaction that occurs with high-gain is observed if measurements are made in electropermeabilized frog rod outer segments (ROS) stimulated by a dim flash of light in the operating range of the photoreceptor. Flashes of light exciting 1000 or fewer of the 3 x 10(9) rhodopsins present/ROS results in the incorporation of 1400 phosphates from ATP into the rhodopsin pool for each excited rhodopsin (Rho*). This amplification decreases with increasing light intensity, falling most sharply after each disk has absorbed one photon. The high-gain reaction is lost if the ROS are broken into vesicles by shearing, leaving a low-gain rhodopsin phosphorylation characterized in previous studies using brighter illumination. The high-gain but not the low-gain phosphorylation appears to be regulated by G-protein and by calcium levels in the range over which intracellular calcium changes when rod photoreceptors are illuminated. Kinetic measurements made on the phosphorylation observed at higher light intensities shows that it initially occurs rapidly enough for a role in terminating the photoresponse. The high-gain phosphorylation observed at lower light intensities may play a global role in regulating light-adaptation of the rod photoreceptor, and its existence suggests that a search for a similar high-gain modification in systems using the homologous beta-adrenergic or muscarinic acetylcholine receptors might be rewarding.  相似文献   

17.
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.  相似文献   

18.
R Wagner  N Ryba  R Uhl 《FEBS letters》1988,234(1):44-48
A fast, regenerative light scattering signal from bovine ROS, the PA-signal, reflects the light-induced, transient activation of transducin. Its rate of recovery depends on the number of photolysed rhodopsin molecules, indicating that rhodopsin deactivation and not GTPase activity is rate limiting in our in vitro system. When rhodopsin deactivation is accelerated (in the presence of NH2OH), PA-signal recovery is also accelerated. A GTPase turnover number of more than 2 s-1 (at 37 degrees C) can be derived from these experiments. This is more than one order of magnitude faster than the GTPase rates so far described in the literature and is rapid enough for a physiological shut-off mechanism. The fast GTPase is attributed to a highly intact disk stack, which never releases transducin into the free aqueous space.  相似文献   

19.
A B Fawzi  J K Northup 《Biochemistry》1990,29(15):3804-3812
Transducin (Gt) is a member of a family of receptor-coupled signal-transducing guanine nucleotide (GN) binding proteins (G-proteins). Light-activated rhodopsin is known to catalyze GN exchange on Gt, resulting in the formation of the active state of the Gt alpha-GTP complex. However, purified preparations of Gt have been shown to exchange GN in the absence of activated receptors [Wessling-Resnick, M., & Johnson, G. L. (1987) Biochemistry 26, 4316-4323]. To evaluate the role of rhodopsin in the activation of Gt, we studied GN-binding characteristics of different preparations of Gt. Gt preparations obtained rom the supernate of GTP-treated bovine rod outer segment (ROS) disks, followed by removal of free GTP on a Sephadex G-25 column, bound GTP gamma S at 30 degrees C in the absence of added exogenous rhodopsin with an activity of 1 mol of GTP gamma S bound/mol of Gt (Gt-I preparations). Binding of GTP gamma S to Gt-I preparations closely correlated with the activation of ROS disk cGMP phosphodiesterase. GN-binding activity of Gt-I preparations was dependent on reaction temperature, and no binding was observed at 4 degrees C. In the presence of 10 microM bleached rhodopsin, Gt-I preparations bound GTP gamma S at 4 degrees C. However, hexylagarose chromatography of Gt-I preparations led to a preparation of Gt that showed less than 0.1 mol/mol binding activity following 60-min incubation at 30 degrees C in the absence of rhodopsin (Gt-II preparations).(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

20.
The interaction of bovine rhodopsin with poly- and monounsaturated lipids was studied by (1)H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号