Characterization of rhodopsin kinase purified from bovine rod outer segments

Rhodopsin kinase was purified by sequential chromatography on DEAE-cellulose and blue-Sepharose. Kinase activity co-purified with a 62-kDa polypeptide, which bound light-dependently in the absence of ATP to purified vesicle-reconstituted rhodopsin. Purified rhodopsin kinase is free of any detectable arrestin or the retinal G-protein. Rhodopsin kinase is autophosphorylated on serine residues which is unaffected by the presence of bleached rhodopsin and results in a transition in molecular mass to 64 kDa. Autophosphorylation of the kinase did not appear to alter the overall rate of rhodopsin phosphorylation or the apparent KM (0.6 microM) for purified reconstituted rhodopsin. Peptides corresponding to sequences within opsin loops 3-4 and 5-6 and the COOH terminus inhibited kinase phosphorylation of bleached rhodopsin, suggesting at least three potential sites to account for the stable high affinity binding of rhodopsin kinase to the bleached photoreceptor molecule that are at least in part distinct from the substrate sites for phosphorylation. These sequences are similar to those proposed for receptor recognition of G-proteins and indicate that the domains involved in light-dependent binding of rhodopsin kinase and retinal G-protein are similar or overlapping.     

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.     

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.     

Two distinct rhodopsin molecules within the disc membrane of vertebrate rod outer segments

The kinetics of the metarhodopsin I–II reaction have been measured over a wide range of temperatures (1–37°C) and pH values (4.5–8) with suspensions containing fragments of bovine rod outer segments. It was found that for all conditions the occurrence of metarhodopsin II could be described by two independent first-order processes. The fast component: slow component amplitude ratio depends upon pH and temperature.The kinetics of the lumi-metarhodopsin I reaction show the same pH dependence for the fast component: slow component amplitude ratio as the one observed for the metarhodopsin II signals.All the results observed could be described with the assumption that rhodopsin itself exists in two conformational states before bleaching which are in a pH and temperature-dependent equilibrium. This hypothesis is confirmed by its ability to explain some apparently anomalous observations in the literature.     

Two distinct rhodopsin molecules within the disc membrane of vertebrate rod outer segments.

The kinetics of the metarhodopsin I-II reaction have been measured over a wide range of temperatures (1-37C ) and pH values (4.5-8) with suspensions containing fragments of bovine rod outer segments. It was found that for all conditions the occurrence of metarhodopsin II could be described by two independent first-order processes. The fast component: slow component amplitude ratio depends upon pH and temperature. The kinetics of the lumi-metarhodopsin I reaction show the same pH dependence for the fast component: slow component amplitude ratio as the one observed for the metarhodopsin II signals. All the results observed could be described with the assumption that rhodopsin itself exists in two conformational states before bleaching which are in a pH and temperature-dependent equilibrium. This hypothesis is confirmed by its ability to explain some apparently anomalous observations in the literature.     

Two forms of intermediates of frog rhodopsin in rod outer segments

Using frog rod outer segments, we measured changes of the absorption spectrum during the conversion of rhodopsin to a photosteady-state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (440 nm) at ? 190°C and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (718 nm) at ? 190°C. The reaction kinetics was expressed by one exponential in the former case and by two exponentials in the latter. These results suggest that rhodopsin is composed of a single molecular species, while bathorhodopsin is composed of two kinds of molecular species designated as batho₁-rhodopsin and batho₂-rhodopsin. On warming the two forms of bathorhodopsin, each bathorhodopsin converted to its own lumirhodopsin, metarhodopsin I and finally a free all-trans-retinal plus opsin. The absorption spectra of the two forms of bathorhodopsin, lumirhodopsin and metarhodopsin I were measured at ? 190°C. We infer that a rhodopsin molecule in the excited state relaxes to either batho₁-rhodopsin or batho₂-rhodopsin, and then converts to its own intermediates through one of the two parallel pathways.     

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.     

Three-dimensional electron microscopical investigation of rhodopsin molecules in the disc membrane of Frog rod outer segments

Frog rod outer segment (ROS) disc membranes are investigated by three-dimensional electron microscopy of aperiodic structures. Out of the various chemical constituents of these membranes only the visual pigment rhodopsin is examined. It is suggested that rhodopsin molecules are dumb-bell shaped and it is shown that in small areas they are not statistically arranged. Most rhodopsin molecules are tilted 26±2° away from the position normal to the membrane thus showing angles α<90° or α>90°. Within the small part of the disc membrane investigated the long rhodopsin axes lie in planes parallel to the xy-plane, the y-axis being parallel with the connecting cilium of ROS. In the membrane cut-outs reconstructed there are aggregates of five to nine rhodopsin molecules either with α>90° or α<90° only. In adjacent disc membranes all the bigger aggregates appear at the same site and with the same magnitude of α.     

The membrane bound retinol dehydrogenase from bovine rod outer segments

Retinol dehydrogenase from bovine rod outer segments was solubilized in detergent and partially purified 25-fold through a combination of hydroxyapatite and retinyl-Sepharose chromatography. Alltrans retinol solubilized in protein solutions of bovine serum albumin or β-lactalbumin was a better substrate for the enzyme than retinol solubilized in detergents or suspended in buffer. Retinol dehydrogenase was sensitive to the carbonyl reagent pyridoxal-5′-phosphate but was not inhibited by retinal followed by reduction with NaBH₄. The solubilized enzyme requires phospholipids to maintain enzymatic activity, as was evidenced by the inactivating effect of phospholipase A₂ on the partially purified enzyme.     

Light- and guanosine 5'-3-O-(thio)triphosphate-sensitive localization of a G protein and its effector on detergent-resistant membrane rafts in rod photoreceptor outer segments

Detergent-resistant membrane microdomains in the plasma membrane, known as lipid rafts, have been implicated in various cellular processes. We report here that a low-density Triton X-100-insoluble membrane (detergent-resistant membrane; DRM) fraction is present in bovine rod photoreceptor outer segments (ROS). In dark-adapted ROS, transducin and most of cGMP-phosphodiesterase (PDE) were detergent-soluble. When ROS membranes were exposed to light, however, a large portion of transducin localized in the DRM fraction. Furthermore, on addition of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) to light-bleached ROS, transducin became detergent-soluble again. PDE was not recruited to the DRM fraction after light stimulus alone, but simultaneous stimulation by light and GTPgammaS induced a massive translocation of all PDE subunits to the DRM. A cholesterol-removing reagent, methyl-beta-cyclodextrin, selectively but partially solubilized PDE from the DRM, suggesting that cholesterol contributes, at least in part, to the association of PDE with the DRM. By contrast, transducin was not extracted by the depletion of cholesterol. These data suggest that transducin and PDE are likely to perform their functions in phototransduction by changing their localization between two distinct lipid phases, rafts and surrounding fluid membrane, on disc membranes in an activation-dependent manner.     

Hindered diffusion in excised membrane patches from retinal rod outer segments.

Excised inside-out membrane patches are useful for studying the cGMP-activated ion channels that generate the electrical response to light in retinal rod cells. We show that strong ionic current across a patch changes the driving force on the current by altering the ionic concentration near the surface membrane, an effect somewhat like that first described by Frankenhaeuser and Hodgkin (1956) in squid axons. The dominant concentration change occurs in the solution adjacent to the cytoplasmic (inner) surface of the membrane, where diffusion is impaired by intracellular material that adheres to the patch during excision. The magnitude and time course of the ionic changes are consistent with the expected volume of this material and with an effective diffusion coefficient about an order of magnitude less than that in free solution. Methods are described for correcting current transients observed in voltage clamp experiments, so that channel gating kinetics can be obtained without contamination by changes in driving force. We suggest that restricted diffusion may occur in patches excised from other types of cells and influence rapid kinetic measurements.     

Protein kinases of retinal rod outer segments: identification and partial characterization of cyclic nucleotide dependent protein kinase and rhodopsin kinase

Protein kinase activity of dark-adapted bovine rod outer segments is partitioned by centrifugation into soluble and membrane-bound fractions. The soluble kinases are separated by DEAE-cellulose chromatography into three peaks of activity, which can be classified by substrate specificity and cyclic nucleotide dependence into two categories. One peak of protein kinase activity has the characteristics reported for rhodopsin kinase (category one); it phosphorylates only bleached rhodopsin, and its activity is not affected by light, exogenous adenosine cyclic 3',5'--monophosphate (cAMP), guanosine cyclic 3',5'-monophosphate (cGMP), or a protein kinase inhibitor from skeletal muscle. Rhodopsin kinase has an apparent molecular weight of 68 000. The second category of kinase includes two peaks of activity which are stimulated severalfold by cAMP or cGMP but not by light. These protein kinases phosphorylate soluble proteins including histones and a protein kinase substrate prepared from rat intestine but not rhodopsin. The two peaks elute from DEAE-cellulose with 0.09 and 0.20 M KCl, suggesting that they are similar respectively to type I and type II cyclic nucleotide dependent protein kinases that have been characterized in other tissues. The activity of type I kinase is variable and much less than that of the type II enzyme; its molecular weight was not determined. The type II protein kinase has an apparent molecular weight of 165 000. This study confirms that different protein kinase enzymes catalyze selectively the phosphorylation of bleached rhodopsin and soluble proteins, and it repudiates the speculation in a previous publication [Farber, D. B., Brown, B. M., & Lolley, R. N. (1979) Biochemistry 18, 370-378] that a single protein kinase might catalyze both phosphorylation reactions.     

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.     

Purification and properties of guanylate kinase from bovine retinas and rod outer segments

The presence of three soluble nucleotide phosphotransferases in bovine rod outer segments was demonstrated: guanylate kinase (EC 2.7.4.8), nucleoside-diphosphate kinase (EC 2.7.4.6) and adenylate kinase (EC 2.7.4.3). The enzyme guanylate kinase, which catalyzes the reaction GMP + ATP in equilibrium GDP + ADP, was purified to homogeneity from isolated bovine rod outer segments as well as from bovine retinas. The enzyme preparations obtained from both sources are identical in their chromatographic properties, molecular mass (20-23 kDa for both native enzyme and dodecylsulfate-denatured polypeptide), Km values (13 microM for GMP and 430 microM for ATP), specific activities, and nucleotide specificities. The enzyme's turnover number was estimated to be 130 s-1. The minimum amount of enzyme found in rod outer segments is about 1 copy per 800 rhodopsin molecules. The role of the enzyme in the cyclic GMP cycle in rod outer segments is discussed.     

Light inhibits tyrosine kinase in rod outer segments in vitro]

It was shown that short-term (10 min) light exposure of dark-adapted retinal rod outer segments (ROS) leads to a threefold inhibition of the tyrosine kinase activity. Tyrosine kinase activity in the ROS from bleached retinas is by 30% lower than in the dark-adapted ROS. Prolonged illumination (60 min) of the dark-adapted ROS restores the tyrosine kinase activity to the level of ROS from the bleached retinas.     

The effect of rhodopsin phosphorylation on the light-dependent activation of phosphodiesterase from bovine rod outer segments

ATP quenches light-dependent phosphodiesterase (PDE) activation in rod outer segments presumably due to rhodopsin phosphorylation. Here we compared the efficiency of phosphorylated and non-phosphorylated rhodopsins as PDE activators in a reconstituted cell-free system. It is shown that the ability of phosphorylated membranes to activate this enzyme is suppressed compared with non-phosphorylated ones.     

ATP-dependent calcium uptake activity associated with a disk membrane fraction isolated from bovine retinal rod outer segments

Ca2+ sequestration and release from disks of rod outer segments may play a critical role in visual transduction. An ATP-dependent Ca2+ uptake activity has been identified in association with purified disks of bovine rod outer segments. A crude preparation of osmotically active disks was obtained from rod outer segments by hypoosmotic shock and subsequent flotation on a 5% Ficoll 400 solution. These "crude" disks were further purified by separation into two distinct components by centrifugation in a linear Ficoll gradient. Disks comprised the major component; at least 60% of the protein was rhodopsin. This fraction also contained a Ca2+ uptake activity stimulated approximately 4-fold by ATP. The initial rate was approximately 0.21 nmol of Ca2+ (mg of protein)-1 min-1. Most of the ATP-dependent accumulation of 45Ca2+ was released by the calcium ionophore A23187. The uptake activity was sensitive to vanadate (Ki approximately 20 microM) and insensitive to the mitochondrial Ca2+ uptake inhibitor ruthenium red (10 microM). The ATP-dependent Ca2+ uptake exhibited Michaelis-Menten activation kinetics with respect to [Ca2+] (Km approximately 6 microM). The osmotic properties of the sealed disk membranes were exploited to determine whether the association of Ca2+ transport activity with the disks was merely coincidental. The sedimentation properties of these disks, upon centrifugation on a second Ficoll linear density gradient, varied with the osmolarity of the gradient solution. In several separate gradient solutions of differing osmotic and ionic strengths, the Ca2+ uptake activity always comigrated with the disks. These results indicate that the ATP-dependent Ca2+ uptake activity was physically associated with sealed native disk membranes. The characteristics of the Ca2+ uptake activity suggest that it may play a major role in the regulation of cytosolic Ca2+ levels in rod cells in vivo.