Activation of phosphodiesterase by rhodopsin and its analogues

Activation of guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (EC 3.1.4.35.) in frog rod outer segment membrane by rhodopsin and its analogues was investigated. The Schiff-base linkage between opsin and retinal in rhodopsin was not always necessary for the phosphodiesterase activation. The binding of beta-ionone ring of retinal to a hydrophobic region of opsin was not enough to induce the enzyme activation. A striking photo-activation of the enzyme was induced by photo-isomerization of rhodopsin analogues from cis to trans form. It seems probable that an "expanded" conformation of opsin around the retinylidene chromophore induced by the cis to trans isomerization may be the trigger for the activation of phosphodiesterase. On the other hand, the phosphodiesterase in frog rod outer segment was activated by warming of bathorhodopsin to -12 degrees C and then incubating it at the same temperature. Thus, metarhodopsin II or an earlier intermediate than metarhodopsin II should be a direct intermediate for the enzyme activation.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. II

Frog (Rana catesbeiana) rod outer segment membrane contains cyclic GMP phosphodiesterase (EC 3.1.4.1). Irradiation of dark-adapted rod outer segment membrane increased the enzyme activity by 5–20-fold in the presence of GTP. The phosphodiesterase in rod outer segment membrane is also activated by mixing a photo-product of 11-cis (regenerated), 9-cis or 7-cis rhodopsin which is stable at 0°C. However, neither opsin in the membrane nor all-trans retinal activates the enzyme. The phosphodiesterase in rod outer segment membrane is also activated by irradiation at ?4°C. Thus, we conclude that the phosphodiesterase is activated by a common photolysis intermediate of these rhodopsin isomers, perhaps before metarhodopsin II decays.     

Activation of phosphodiesterase by rhodopsin and its analogues

Activation of guanosine 3,5-cyclic monophosphate (cGMP) phosphodiesterase (EC 3.1.4.35.) in frog rod outer segment membrane by rhodopsin and its analogues was investigated. The Schiff-base linkage between opsin and retinal in rhodopsin was not always necessary for the phosphodiesterase activation. The binding of -ionone ring of retinal to a hydrophobic region of opsin was not enough to induce the enzyme activation. A striking photo-activation of the enzyme was induced by photo-isomerization of rhodopsin analogues from cis to trans form. It seems probable that an expanded conformation of opsin around the retinylidene chromophore induced by the cis to trans isomerization may be the trigger for the activation of phosphodiesterase. On the other hand, the phosphodiesterase in frog rod outer segment was activated by warming of bathorhodopsin to –12 C and then incubating it at the same temperature. Thus, metarhodopsin II or an earlier intermediate than metarhodopsin II should be a direct intermediate for the enzyme activation.Based on material presented at the Fifth International Congress of Eye Research, Eindhoven, October 1982     

Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments.

Frog (Rana catesbiana) rod outer segment disc membranes contain a cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which is activated by light in the presence of ATP. This enzyme is firmly bound to the disc membrane, but can be eluted from the membrane with 10 mM Tris-HCl buffer, pH 7.4 and 2 mM EDTA. The eluted phosphodiesterase has reduced activity, but can be activated approximately 10-fold by polycations such as protamine and polylysine. The eluted phosphodiesterase can no longer be activated by light in the presence of ATP, that is, activation by light apparently depends on the native orientation of phosphodiesterase in relationship to other disc membrane components. The eluted phosphodiesterase was purified to homogeneity as judged by analytical polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The over-all purification from intact retina was approximately 925-fold. The purification of phosphodiesterase from the isolated rod outer segment preparation was about 185-fold with a 28% yield. Phosphodiesterase accounts for approximately 0.5% of the disc membrane protein. The eluted phosphodiesterase (inactive form) has a sedimentation coefficient of 12.4 S corresponding to an approximate molecular weight of 240,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates the purified phosphodiesterase into two subunits of 120,000 and 110,000 daltons. With cyclic 3':5'-GMP (cGMP) as substrate the Km for the purified phosphodiesterase is 70 muM. Protamine increases the Vmax without changing the Km for cGMP. The isoelectric point (pI) of the native dimer is 5.7. Limited exposure of the eluted phosphodiesterase (inactive form) to trypsin produces a somewhat greater activation than is obtained with 0.5 mg/ml of protamine. The trypsin-activated phosphodiesterase has a sedimentation coefficient of 7.8 S corresponding to an approximate molecular weight of 170,000. The 110,000-dalton subunit is much less sensitive to trypsin hydrolysis and the 120,000-dalton subunit is rapidly replaced by smaller fragments. On the basis of the molecular weight of the purified phosphodiesterase (240,000) and the concentrations of phosphodiesterase and rhodopsin in the rod outer segment, it is estimated that the molar ratio ophosphodiesterase to rhodopsin in the rod outer segment is approximately 1:900. Since all of the disc phosphodiesterase molecules are activated when 0.1% of the rhodopsins are bleached, we conclude that in the presence of ATP 1 molecule of bleached rhodopsin can activate 1 molecule of phosphodiesterase.     

Kinetic study of transfer of 11-cis-retinal between rod outer segment membranes using regeneration of rhodopsin

The present study demonstrates some important facts on the regeneration of rhodopsin in rod outer segment membranes. 11-cis-Retinal added to a rod outer segment membrane suspension did not react directly with opsin but was rapidly solubilized into membranes and then recombined with opsin in the membrane. It was also revealed that the regeneration of rhodopsin was perturbed by the formation of retinylidene Schiff base with phosphatidylethanolamine in rod outer segment membranes, which decreased with increasing temperature. The activation energy of rhodopsin regeneration in rod outer segment membranes was 18.7 kcal/mol, being smaller than the value of 22 kcal/mol in 1% digitonin solution. 11-cis-Retinal could be found to transfer relatively fast (tau-1/k(1) R 10(3) s) between rod outer segment membranes by using the regeneration of rhodopsin. It was demonstrated that the kinetic measurement for the transport of membrane-soluble molecules such as retinal between membranes could be perform ed with ease and precisely by the method described in this paper.     

On a soluble system for studying light activation of rod outer segment cyclic GMP phosphodiesterase

Bovine rod outer segment (ROS) cyclic GMP phosphodiesterase (PDE) could be activated about 6-fold by light, an effect that could be simulated by isolated bleached rhodopsin. About 90% of PDE activity in ROS could be extracted with 10 mM Tris-HCl, pH 7.5, but light is ineffective in activating the soluble enzyme. However, bleached rhodopsin could activate it in the presence of a very low concentration of ATP, strongly suggesting the mediation of rhodopsin in the light activation of the enzyme in ROS. Direct evidence is presented to suggest that the phosphorylation of opsin (bleached rhodopsin) is unrelated to the activation of PDE by bleached rhodopsin and ATP. The reconstitution of the light activation of PDE in a soluble system presented here opens up a new direction to future investigations on the mechanism of light regulation of cyclic GMP levels in retina and its implication in the photoreceptor function.     

Kinetics of the Hydrolysis of 8-Bromo-Cyclic GMP by the Light-Activated Phosphodiesterase of Toad Rods

The hypothesis that cyclic GMP is the internal transmitter of retinal rod phototransduction, when combined with the observations that 8-bromo-cyclic GMP opens the cyclic GMP-dependent outer segment conductance and that rods into which 8-bromo-cyclic GMP has been injected still respond to light, predicts that the light-activated phosphodiesterase (EC 3.1.4.17) must catalyze the hydrolysis of 8-bromo-cyclic GMP. This hypothesis was tested by measuring light-activated toad rod disk membrane phosphodiesterase with a pH assay technique. Phosphodiesterase-catalyzed hydrolysis of 8-bromo-cyclic GMP was confirmed: at pH 8.0, total proton production after flash activation was identical to total amount of 8-bromo-cyclic GMP added as substrate. Photoactivated phosphodiesterase was remarkably less efficient in catalyzing the hydrolysis of 8-bromo-cyclic GMP than of cyclic GMP: Vmax for 8-bromo-cyclic GMP was 0.063 M/M rhodopsin/s, whereas that for cyclic GMP was 11 M/M rhodopsin/s--170 times greater. The Km for 8-bromo-cyclic GMP was 160 microM, and for cyclic GMP, 590 microM. 8-bromo-cyclic GMP competitively inhibited phosphodiesterase-catalyzed hydrolysis of cyclic GMP with a Ki of 1.2 mM. Complete reaction progress curves were analyzed for obedience to Michaelis-Menten kinetics: cyclic GMP hydrolysis, 8-bromo-cyclic GMP hydrolysis, and cyclic GMP hydrolysis in the presence of 8-bromo-cyclic GMP as competitive inhibitor were found to follow the integrated form of the Michaelis-Menten equation over the time course of the reactions, assuming phosphodiesterase was activated as a step. The kinetic parameters extracted from reaction progress curves were consistent with those derived from analysis of the initial velocity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of transducin from bovine retinal rod outer segments. Mechanism and effects of cholera toxin-catalyzed ADP-ribosylation

Transducin, a guanine nucleotide-binding protein consisting of two subunits (T alpha and T beta gamma), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments. The T alpha subunit is an activator of the phosphodiesterase, and the function of the T beta gamma subunit is to physically link T alpha with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T alpha has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled T alpha with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) and T beta gamma were present at concentrations equal to that of T alpha and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T alpha X Gpp(NH)p, T beta gamma, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T alpha coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 X 10(-3)/s, which was 22-fold slower than the rate for the unmodified transducin.     

Calorimetric studies of bovine rod outer segment disk membranes support a monomeric unit for both rhodopsin and opsin

The photoreceptor rhodopsin is a G-protein coupled receptor that has recently been proposed to exist as a dimer or higher order oligomer, in contrast to the previously described monomer, in retinal rod outer segment disk membranes. Rhodopsin exhibits considerably greater thermal stability than opsin (the bleached form of the receptor), which is reflected in an ∼15°C difference in the thermal denaturation temperatures (T_m) of rhodopsin and opsin as measured by differential scanning calorimetry. Here we use differential scanning calorimetry to investigate the effect of partial bleaching of disk membranes on the T_m of rhodopsin and of opsin in native disk membranes, as well as in cross-linked disk membranes in which rhodopsin dimers are known to be present. The T_ms of rhodopsin and opsin are expected to be perturbed if mixed oligomers are present. The T_m remained constant for rhodopsin and opsin in native disks regardless of the level of bleaching. In contrast, the T_m of cross-linked rhodopsin in disk membranes was dependent on the extent of bleaching. The energy of activation for denaturation of rhodopsin and cross-linked rhodopsin was calculated. Cross-linking rhodopsin significantly decreased the energy of activation. We conclude that in native disk membranes, rhodopsin behaves predominantly as a monomer.     

Activation of cGMP phosphodiesterase by purified green rod pigment from frog retina

Activation of cGMP phosphodiesterase(PDE) of frog rod outer segments (ROS) by purified green rod pigment (GRP) was analyzed. GRP activated PDE in a similar manner to purified rhodopsin. This activation required illumination of the pigment and presence of GTP.     

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.     

Mechanism and specificity of rhodopsin phosphorylation.

Partial separation of protein kinase activity from rhodopsin in isolated bovine retinal photoreceptor outer segments was accomplished by mild ultrasonic treatment followed by ultracentrifugation. Residual kinase activity in the rhodopsin-rich sediment was destroyed by chemical denaturation which did not affect the spectral properties of the rhodopsin. The retinal outer segment kinase was found to be specific for rhodopsin, since in these preparations it alone of several bovine protein kinases was capable of phosphorylating rhodopsin in the light. The phosphorylation reaction apparently requires a specific conformation of the rhodopsin molecule since it is abolished by heat denaturation of rhodopsin, and it is greatly reduced or abolished by treatment of the visual pigment protein with potassium alum after the rhodopsin has been "bleached" by light. When kinase and rhodopsin or opsin fractions were prepared from dark-adapted and bleached outer segments and the resultant fractions were mixed in various combinations of bleached and unbleached preparations, the observed pattern of light-activated phosphorylation was consistent only with the interpretation that a conformational change in the rhodopsin molecule in the light exposes a site on the visual pigment protein to the kinase and ATP. These results rule out the possibility of a direct or indirect (rhodopsin-mediated) light activation of the kinase. Finally, phosphorylation of retinal outer segment protein in monochromatic lights of various wavelengths followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both rhodopsin and the higher molecular weight visual pigment protein reported by several laboratories have the same action spectrum for phosphorylation. This result is consistent with the suggestion that the higher molecular weight species is a rhodopsin dimer.     

Subspecies of arrestin from bovine retina. Equal functional binding to photoexcited rhodopsin but various isoelectric focusing phenotypes in individuals

Arrestin (also named 48-kDa protein or S-antigen) binds to photoexcited and phosphorylated rhodopsin and thereby prevents activation of cGMP phosphodiesterase (EC 3.1.4.35) by transducin in retinal rods. We report here that retinal arrestin consists of several subspecies (isoelectric points between pH 5.5-6.2), which can be separated by FPLC anion-exchange chromatography and by FPLC chromatofocusing resulting in highly enriched individual subspecies. The entire heterogeneity pattern of arrestin is present in rod outer segments, independently of whether arrestin orginated from the outer or mostly from the inner segment of rod cells. The different subspecies show a similar binding behavior to photoexcited rhodopsin phosphorylated to various degrees and they quench the cGMP phosphodiesterase activity equally well. In the presence of rod outer segment membranes, arrestin is phosphorylated light-dependently by protein kinase C (0.2 mol phosphate/mol arrestin). This implies that the heterogeneity of arrestin is not primarily due to phosphorylation. Arrestin from different individuals exists as four isoelectric focusing patterns which occur with remarkably different frequencies in calf and cattle. The complexity of the IEF pattern does not increase with aging. Distinct subspecies of arrestin may reflect differences in their primary structure, or may result from differentially regulated post-translational modifications in individuals.     

A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern

To study rhodopsin biosynthesis and transport in vivo, we engineered a fusion protein (rho-GFP) of bovine rhodopsin (rho) and green fluorescent protein (GFP). rho-GFP expressed in COS-1 cells bound 11-cis retinal, generating a pigment with spectral properties of rhodopsin (A(max) at 500 nm) and GFP (A(max) at 488 nm). rho-GFP activated transducin at 50% of the wild-type activity, whereas phosphorylation of rho-GFP by rhodopsin kinase was 10% of wild-type levels. We expressed rho-GFP in the rod photoreceptors of Xenopus laevis using the X. laevis principal opsin promoter. Like rhodopsin, rho-GFP localized to rod outer segments, indicating that rho-GFP was recognized by membrane transport mechanisms. In contrast, a rho-GFP variant lacking the C-terminal outer segment localization signal distributed to both outer and inner segment membranes. Confocal microscopy of transgenic retinas revealed that transgene expression levels varied between cells, an effect that is probably analogous to position-effect variegation. Furthermore, rho-GFP concentrations varied along the length of individual rods, indicating that expression levels varied within single cells on a daily or hourly basis. These results have implications for transgenic models of retinal degeneration and mechanisms of position-effect variegation and demonstrate the utility of rho-GFP as a probe for rhodopsin transport and temporal regulation of promoter function.     

A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin

Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation.     

Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis

Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and alpha adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.     

Binding and activation of rod outer segment phosphodiesterase and guanosine triphosphate binding protein by disc membranes: influence of reassociation method and divalent cations

Attempts to optimize the recovery of light-stimulated phosphodiesterase activity following reassociation of the hypotonically extractable proteins derived from retinal rod segments with hypotonically stripped disc membranes lead to the following observations: the best reassociations were obtained by mixing proteins and stripped disc membranes under hypotonic conditions and slowly increasing the salt concentration; the binding of G-protein and phosphodiesterase to stripped disc membrane occurs in less than 5 minutes and the recovery of light-stimulated phosphodiesterase activation in response to subsaturating stimulus levels requires 2-3 h to plateau. Stripped disc membranes and proteins were reassociated in 'isotonic' buffers containing KCl/NaCl, KCl/NaCl plus Mg2+, or KCl/NaCl plus Ca2+. Large fractional rhodopsin bleaches produced nearly identical light-stimulated phosphodiesterase activities in each of these samples and in the control rod outer segment membranes. Rod outer segment membranes and reassociated stripped disc membrane samples containing divalent cations showed similar phosphodiesterase activities in response to low fractional rhodopsin bleaches (e.g. less than or equal to 0.1%), however, samples devoid of divalent cations during reassociation required rhodopsin bleaches up to 10-fold larger to elicit comparable phosphodiesterase activities. These results suggest that not all phosphodiesterase and/or G-protein molecules bound to the disc membrane surface are equivalent with regard to their efficiency of activation by bleached rhodopsin and that divalent cations can modulate the distribution of G-protein and/or phosphodiesterase between these populations.     

Light-induced conformational change in rhodopsin detected by modification of G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation

CNBr treatment of rod outer segments was performed in dark and in light conditions. With the subsequent modified rhodopsin and opsin the cGMP phosphodiesterase activation system was reconstituted. The recombination systems exhibited greatly reduced G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation. The reduction in activity of these three steps of the PDE activation cascade is most significant with modified opsin and is shown to be due to its inability to bind the G alpha subunit. The correlation between the localization of CNBr cleavage in dark and light conditions and these results is strongly indicative that a light-induced conformational change occurs in two extradiscal regions of rhodopsin.     

A link between rhodopsin and disc membrane cyclic nucleotide phosphodiesterase. Action spectrum and sensitivity to illumination.

Frog (Rana pipiens) rod outer segment disc membranes contain guanosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.1.c) which, in the presence of ATP, is stimulated 5- to 20-fold by illumination. The effectiveness of monochromatic light of different wavelengths in activating phosphodiesterase was examined. The action spectrum has a maximum of 500 nm, and the entire spectrum from 350 to 800 nm closely matches the absorption spectrum of rhodopsin, which is apparently the pigment which mediates the effects of light on phosphodiesterase activity. trans-Retinal alone does not mimic light. Half-maximal activation of the phosphodiesterase occurs with a light exposure which bleaches 1/2000 of the rhodopsins. Half-maximal activation can also be achieved by mixing 1 part of illuminated disc membranes in which the rhodopsin is bleached with 99 parts of unilluminated membranes. Regeneration of bleached rhodopsin by addition of 11-cis-retinal is illuminated disc membranes reverses the ability of these membranes to activate phosphodiesterase in unilluminated membranes. If the rhodopsin regenerated by 11-cis-retinal is illuminated again, it regains the ability to activate phosphodiesterase. These studies show that the levels of cyclic nucleotides in vetebrate rod outer segments are regulated by minute amounts of light and clearly indicate that rhodopsin is the photopigment whose state of illumination is closely linked to the enzymatic activity of disc membrane phosphodiesterase.     

Purification method of bovine rhodopsin kinase using regeneration of rhodopsin

We report a rapid and high-yield purification method of bovine retinal rhodopsin kinase. According to our method, 500 micrograms of rhodopsin kinase was purified from 100 bovine retinae within 12 h. Rhodopsin kinase bound to bleached rhodopsin was extracted effectively from rod outer segment membranes after regeneration of rhodopsin by the incubation with exogenous 11-cis-retinal. Subsequent DE52 column chromatography further purified the protein to homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified rhodopsin kinase had an apparent molecular weight of 68,000 and phosphorylated rhodopsin at the rate of 10 nmol phosphate/min/mg of the enzyme.