Protein complement of rod outer segments of frog retina

Rod outer segments (ROS) from frog retina have been purified by Percoll density gradient centrifugation, a procedure that preserves their form and intactness. One- and two-dimensional electrophoretic analysis reveals a smaller number of proteins than is observed in many cell organelles and permits quantitation of the 20 most abundant polypeptides. Rhodopsin accounts for 70% of the total protein (3 X 10(9) copies/outer segment), and approximately 70 other polypeptides are present at more than 6 X 10(4) copies/outer segment. Another 17% of the total protein is accounted for by the G-protein (3 X 10(8) copies/outer segment) that links rhodopsin bleaching and the activation of cyclic GMP phosphodiesterase (PDE). The phosphodiesterase accounts for 1.5% of the protein (1.5 X 10(7) copies/outer segment), and a 48,000-dalton component that binds to the membrane in the light accounts for a further 2.6%. The function of approximately 90% of the total protein in the outer segment is known, and two-thirds of the non-rhodopsin protein is accounted for by enzyme activities associated with cyclic GMP metabolism. The relative molar abundance of rhodopsin, G-protein, and PDE is 100:10:1. Apart from these major membrane-associated proteins, most of the other proteins are cytosolic. Thirteen other polypeptides are found at an abundance of one or more copies per 1000 rhodopsins, nine soluble and four membrane-bound, and their abundance relative to rhodopsin has been quantitated. ROS have been separated into subcellular fractions which resolve three classes of soluble, extrinsic membrane, and integral membrane proteins. A listing of the proteins that are phosphorylated and their subcellular localization is given. Approximately 25 phosphopeptides are detected, and most are in the soluble fraction. Fewer phosphorylated proteins are associated with the purified outer segments than with crude ROS. Distinct patterns of phosphorylation are associated with intact rods incubated with [32P]Pi and broken rods incubated with [gamma-32P]ATP.     

Stimulation of rhodopsin phosphorylation by guanine nucleotides in rod outer segments

Porcine rod outer segment (ROS) proteins were phosphorylated in the presence of [gamma-32P]ATP and Mg2+, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected by autoradiography. The phosphorylation of rhodopsin, the major protein-staining band (Mr approximately 34 000-38 000), was markedly and specifically increased by exposure of rod outer segments to light; various guanine nucleotides (10 microM) including GMP, GDP, and GTP also specifically increased rhodopsin phosphorylation (up to 5-fold). Adenine nucleotides (cyclic AMP, AMP, and ADP at 10 microM) and 8-bromo-GMP (10 microM) or cyclic 8-bromo-GMP (10 microM) had no detectable stimulatory effect on rhodopsin phosphorylation. GTP increased the phosphorylation of rhodopsin at concentrations as low as 100 nM, and guanosine 5'-(beta, gamma-imidotriphosphate), a relatively stable analogue of GTP, was nearly as effective as GTP. Maximal stimulation of rhodopsin phosphorylation by GTP was observed at 2 microM. GMP and GDP were less potent than GTP. Both cyclic GMP and GMP were converted to GTP during the time period of the protein phosphorylation reaction, suggestive of a GTP-specific effect. Transphosphorylation of guanine nucleotides by [32P]ATP and subsequent utilization of [32P]GTP as a more effective substrate were ruled out as an explanation for the guanine nucleotide stimulation. With increasing concentrations of ROS proteins, the phosphorylation of rhodopsin was nonlinear, whereas in the presence of GTP (2 microM) linear increases in rhodopsin phosphorylation as a function of added ROS protein were observed. These results suggest that GTP stimulates the phosphorylation of rhodopsin by ATP and that a GTP-sensitive inhibitor (or regulator) of rhodopsin phosphorylation may be present in ROS.     

Guanosine triphosphate complex in rod outer segments of the frog retina

It is shown that nearly 70% water--soluble protein of the frog retina outer segments (ROS) consist of three polypeptides with molecular weights 39 000, 36 000 and less than 15 000 daltons. These proteins are present in equal proportions and are, apparently, the subunits of a tightly bound protein complex. The subunit of 39 000 daltons is responsible for guanyl nucleotides binding. Parameters of the investigated GTP-binding complex are similar to transducyn which transmits excitation from bleached rhodopsin to PDE molecules in the bovine retina ROS. The thermodynamic state of GTP-binding protein in frog retina ROS depends on the functional state of the photoreceptor membrane, as shown by microcalorimetric method.     

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.     

Calcium diffusion coefficient in rod photoreceptor outer segments.

Calcium (Ca(2+)) modulates several of the enzymatic pathways that mediate phototransduction in the outer segments of vertebrate rod photoreceptors. Ca(2+) enters the rod outer segment through cationic channels kept open by cyclic GMP (cGMP) and is pumped out by a Na(+)/Ca(2+),K(+) exchanger. Light initiates a biochemical cascade, which leads to closure of the cGMP-gated channels, and a concomitant decline in the concentration of Ca(2+). This decline mediates the recovery from stimulation by light and underlies the adaptation of the cell to background light. The speed with which the decline in the Ca(2+) concentration propagates through the rod outer segment depends on the Ca(2+) diffusion coefficient. We have used the fluorescent Ca(2+) indicator fluo-3 and confocal microscopy to measure the profile of the Ca(2+) concentration after stimulation of the rod photoreceptor by light. From these measurements, we have obtained a value of 15 +/- 1 microm(2)s(-1) for the radial Ca(2+) diffusion coefficient. This value is consistent with the effect of a low-affinity, immobile buffer reported to be present in the rod outer segment (L.Lagnado, L. Cervetto, and P.A. McNaughton, 1992, J. Physiol. 455:111-142) and with a buffering capacity of approximately 20 for rods in darkness(S. Nikonov, N. Engheta, and E.N. Pugh, Jr., 1998, J. Gen. Physiol. 111:7-37). This value suggests that diffusion provides a significant delay for the radial propagation of the decline in the concentration of Ca(2+). Also, because of baffling by the disks, the longitudinal Ca(2+) diffusion coefficient will be in the order of 2 microm(2)s(-1), which is much smaller than the longitudinal cGMP diffusion coefficient (30-60 microm(2)s(-1); ). Therefore, the longitudinal decline of Ca(2+) lags behind the longitudinal spread of excitation by cGMP.     

The carbohydrates in frog retinal rod outer segments

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.     

Active transducin alpha subunit carries PDE6 to detergent-resistant membranes in rod photoreceptor outer segments

cGMP-Phosphodiesterase 6 (PDE6) is the central effector enzyme in the phototransduction system of vertebrate photoreceptors. We have recently found that PDE6 accumulates in a detergent-resistant membrane (DRM) fraction in response to excitation of bovine rod phototransduction system. Here, we studied the molecular mechanism of the PDE6 translocation to DRM. Pertussis toxin inhibited the translocation of PDE6. Upon addition of AlF(4)(-) to dark-adapted ROS, PDE6 translocated to DRM along with a minor fraction of the alpha subunit of transducin (T alpha). The addition of an excess of the inhibitory subunit of PDE6 blocked its accumulation in the DRM, but did not block the translocation of the minor fraction of T alpha. These data suggested that the formation of a complex between activated T alpha and PDE6 imparted upon T alpha a high affinity for the DRM. The translocation of PDE6 to the DRM may be involved in the spatiotemporal regulation of its activity on disk membranes.     

Protein kinase activity of bovine retina rod outer segments

Dark-adapted pure bovine rod outer segments (ROS) (A280/A500--2.1) can be phosphorylated in the presence of [gamma-32P]ATP and [gamma-32P]GTP. The constant levels of phosphorylation, reached within 10--15 min, are 100 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP and 2--4 pmol 32P/nmol of rhodopsin for [gamma-32P]GTP. These processes are not controlled by 10(-4)--10(-8) cAMP, cGMP or Ca2+, but are inhibited at higher concentrations of these agents. In the presence of histone the constant level of phosphorylation is increased up to 200 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP, but is not changed when [gamma-32P]GTP is used. 10(-5) M cAMP is found to activate the phosphorylation in the presence of histone and [gamma-32P]ATP by 5--6 times. All this evidences that ROS contains cAMP-dependent protein kinase, which utilizes ATP, but not GTP. Moreover, ROS contains cyclic nucleotides- and Ca2+-independent protein kinase. These protein kinases are the ROS endogenous enzymes. This is shown in experiments on separation of pure ROS in a sucrose density gradient.     

Calcium content of frog rod outer segments and discs

To test the “Ca²⁺ hypothesis of visual excitation”, we measured the total Ca²⁺ content of freshly isolated bullforg rod outer segments, and have compared the total Ca²⁺ contents of fully dark-adapted discs with discs exposed to small amounts of light. Discs were prepared by hypotonically lysing outer segments under conditions expected to remove Ca²⁺ from the cytoplasm but not from the discs. Ca²⁺ was assayed by atomic absorption spectrophotometry. We find that both discs and outer segments contain a total of about 0.1–0.2 Ca²⁺ per rhodopsin molecule. Thus, each frog disc retains about $\text{2 · 10}{}^5\text{Ca}{}^{\mathrm{2+}}$. If most of this Ca²⁺ were free in the aqueous space inside the intact discs, the Ca²⁺ activity would be a few mM. Since the light-regulated Na⁺ channels have been reported to be highly sensitive to cytoplasmic Ca²⁺, this store of Ca²⁺ in the discs is far more than required by the Ca²⁺ hypothesis. However, despite several variations in experimental conditions, we did not observe any light-activated release of Ca²⁺ from discs in response to stimuli that photoactivated a small fraction of the rhodopsin, as required by the Ca²⁺ hypothesis. In the 26 experiments reported here we could have detected a release as small as 20–30% of the Ca²⁺ content of the disc.     

Cholesterol modulation of photoreceptor function in bovine retinal rod outer segments

Rhodopsin, a prototypical G protein receptor, is found both in the plasma membrane and in discs of bovine rod outer segments. The ability of each of these membranes to activate phosphodiesterase upon stimulation by light in the presence of GTP and cGMP was investigated. The plasma membrane showed little or no activity when compared with disc membranes. The plasma membrane contains approximately 28 mol% cholesterol compared to 8 mol % found in discs. Upon oxidation of at least 70 % of the cholesterol in the plasma membrane to cholestenone, the phosphodiesterase activity in the plasma membrane approached that initiated by the disc membranes. When a 50:50 mixture of disc and plasma membrane rhodopsin was tested for phosphodiesterase activity, the results were found to be additive. Therefore, cholesterol is implicated in regulation of the receptor activity.     

Light-induced dephosphorylation of two proteins in frog rod outer segments: influence of cyclic nucleotides and calcium

Two minor proteins of frog rod outer segments become phosphorylated when retinas are incubated in the dark with 32Pi. The proteins, designated component I (13,000 daltons) and component II (12,000 daltons), are dephosphorylated when retinas are illuminated. The dephosphorylation is reversible; the two proteins are rephosphorylated when illumination ceases. Each outer segment contains approximately 10(6( molecules of components I and II. These remain associated with both fragmented and intact outer segments but dissociate from the outer segment membranes under hypoosmotic conditions. The extent of the light-induced dephosphorylation increases with higher intensities of illumination and is maximal with continuous illumination which bleaches 5.0 x 10(5) rhodopsin molecules/outer segment per second. Light which bleaches 5.0 x 10(3) rhodopsin molecules/outer segment per second causes approximately half-maximal dephosphorylation. This same intermediate level of illumination causes half-suppression of the light-sensitive permeability mechanism in isolated outer segments (Brodie and Bownds. 1976. J. Gen Physiol. 68:1-11) and also induces a half-maximal decrease in their cyclic GMP content (Woodruff et al. 1977. J. Gen. Physiol. 69:667-679). The phosphorylation of components I and II is enhanced by the addition of cyclic GMP or cyclic AMP to either retinas or isolated rod outer segments maintained in the dark. Several pharmacological agents which influence cyclic GMP levels in outer segments, including calcium, cause similar effects on the phosphorylation of components I and II and outer segment permeability. Although the cyclic nucleotide-stimulated phosphorylation can be observed either in retinas or isolated rod outer segments, the light-induced dephosphorylation is observed only in intact retinas.     

Superoxide dismutase of bovine and frog rod outer segments.

Bovine rod outer segments (ROS) contain soluble superoxide dismutase (SOD) which from cyanide sensitivity and electrophoretic mobility appears identical to CuZn SOD of erythrocytes. Enzyme activity of ROS extracts is 200–400 times as much as remainder of retina. Frog ROS also contains a cyanide-sensitive SOD which is not due to erythrocyte contamination since the retina is avascular. SOD in ROS may inhibit free radical oxidation of polyunsaturated fatty acids. In light, high oxygen concentrations in developing retina may activate lipid peroxidation leading to retrolental fibroplasia. High concentrations of ascorbic acid in the retina may act as a protective mechanism against superoxide.     

Phosphorylation of endogenous proteins of bovine retina rod outer segments

The components of bovine rod outer segments (ROS) and water-soluble extracts of ROS were separated by SDS-electrophoresis after incubation with [gamma-32P]ATP or [gamma-32P]GTP at different experimental conditions. After that gels were autoradiographed to reveal the phosphorylated intermediates. Our results suggest, that ROS contains the following protein kinase systems: 1) water-soluble cAMP-dependent protein kinases, that uses ATP, but not GTP, and phosphorylates the water-soluble 30 000 molecular weight protein; 2) protein kinase that uses GTP (probably, ATP also) and phosphorylates the 20 000 molecular weight protein in light-adapted ROS; 3) water-soluble cyclic nucleotide- and Ca2+-independent protein kinase that uses ATP rather than GTP and phosphorylates the water-soluble 70 000 molecular weight protein. The concentrations of phosphorylated intermediates in bovine ROS are estimated.     

Adenylate kinase activity in rod outer segments of bovine retina

The rod outer segments of bovine retina contain two different adenylate kinases: a soluble activity, which is not sensitive to calcium ion, and an activity bound to disk membranes, which is dependent on the calcium levels. In fact, the maximal activity associated to the disks is reached at Ca(2+) concentrations between 10(-6) and 10(-7) M, which is the range of calcium level actually present in the rod cell. The Michaelis-Menten kinetics of the enzyme activity on disk membranes was determined and the actual concentrations of ATP, AMP and ADP were measured in the photoreceptor outer segment. Therefore, the physiological relevance of the adenylate kinase activity was discussed considering the above results. The formation of ATP catalyzed by the enzyme seems appropriate to supply at least some of the reactions necessary for phototransduction, indicating that ATP could be regenerated from ADP directly on the disk membranes where the photoreception events take place.     

Morphological changes induced by -SH reagents in rod photoreceptor outer segments

Summary Rat retinas were treated in vitro with -SH reagents and stained with zinc iodide-osmium tetroxide (ZIO). Dithioerythritol (DTE), an -S-S-reducing agent, increased the electron opaque deposits observed after ZIO staining in the intraand extradiskal spaces of the rods. N-ethyl-maleimide (NEM), an -SH blocking agent, applied directly or after DTE, blocks the ZIO reaction. Furthermore, after treatment with NEM, distorted tubular and vesicular structures are substituted for the stacks of disks. These results strongly suggest that ZIO reacts with -SH groups in rod outer segments. They also indicate that SH-groups play an important role in the structural organization of rod outer segments.Supported by Grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina and Fight for Sight, Inc. N.Y. United StatesI am grateful to Miss Margarita López for her skilful technical assistance and to Mr. Alberto Saenz for the electron micrographs