©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reduced Light-dependent Phosphorylation of an Analog Visual Pigment Containing 9-Demethylretinal as Its Chromophore (*)

(Received for publication, October 20, 1994)

Daniel F. Morrison (1) Tuow D. Ting (2) Visalakshi Vallury (1) (2) Yee-Kin Ho (2) Rosalie K. Crouch (3) D. Wesley Corson (3) (4) Nancy J. Mangini (1) David R. Pepperberg (1)(§)

From the  (1)Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences and the (2)Department of Biochemistry, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612 and the (3)Departments of Ophthalmology and (4)Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

9-Demethyl rhodopsin (9dR), an analog of vertebrate rhodopsin, consists of opsin and a covalently attached chromophore of 11-cis 9-demethylretinal. Electrophysiological evidence that photoactivated 9dR (9dR*) undergoes abnormally slow deactivation in salamander rods (Corson, D. W., Cornwall, M. C., and Pepperberg, D. R.(1994) Visual Neurosci. 11, 91-98) raises the possibility that opsin phosphorylation, a reaction involved in visual pigment deactivation, operates abnormally on 9dR*. This possibility was tested by measuring the light-dependent phosphorylation of 9dR in preparations obtained from bovine rod outer segments. Outer segment membranes containing 9dR or regenerated rhodopsin were flash-illuminated in the presence of [-P]ATP and rhodopsin kinase, further incubated in darkness, and then analyzed for opsin-bound [P]P(i). [P]P(i) incorporation by 9dR* increased with both incubation period and bleaching extent but, under all conditions tested, was less than that measured in rhodopsin controls. Results obtained with 30-s incubation periods indicated that the maximal initial rate of incorporation by 9dR* is about 25% of that by photoactivated rhodopsin. The results imply that the low incorporation of P(i) by 9dR* results from a reduced rate of phosphorylation by rhodopsin kinase and are consistent with the prolonged lifetime of 9dR* determined electrophysiologically.


INTRODUCTION

Photoisomerization of the visual pigment in vertebrate rods generates metarhodopsin II (R*), (^1)the bleaching intermediate that activates transducin and thus induces the photocurrent response (for review, see (1) ). The deactivation of metarhodopsin II involves the enzymatic phosphorylation of this species (2, 3, 4, 5, 6) and the subsequent non-covalent binding of arrestin to the phosphorylated pigment(7, 8, 9, 10, 11) .

The 11-cis isomer of 9-demethylretinal, an analog of native retinal chromophore in which a hydrogen atom replaces the methyl group at carbon-9, can combine with opsin to form 9-demethyl rhodopsin (9dR), a light-sensitive pigment(12, 13) . Evidence that light-induced properties of 9dR differ markedly from those of native rhodopsin and porphyropsin (14, 15) emphasizes the importance of retinal's 9-methyl group in visual pigment function and motivates further studies of 9dR. Particular interest in the deactivation of 9dR arises from a recent study of bright flash photocurrent responses in salamander rods (16) . In rods prepared to contain both 9dR and residual porphyropsin, it was shown that responses mediated largely by native pigment are normal, but those mediated by 9dR exhibit sluggish recovery kinetics, i.e. the lifetime of photoactivated 9dR (9dR*) is abnormally long. The present experiments on bovine rod outer segment (ROS) preparations were undertaken to test the possibility that 9dR* functions abnormally in the phosphorylation reaction. Some of our results were reported at the 1993 meetings of the Biophysical Society and the Association for Research in Vision and Ophthalmology(17, 18) .


EXPERIMENTAL PROCEDURES

Urea-washed Rod Outer Segment (ROS) Membranes

Retinas isolated from commercially obtained bovine eyes were protected from light and stored at -70 °C. Under dim red light, ROS membranes were isolated from 200 retinas(19) . The pelleted ROS membranes were resuspended to a final volume of 240 ml in urea-containing buffer (10 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP), 5 M urea, and 1 mM EDTA, pH 7.5)(20) , incubated in darkness (60 min, 4 °C), and centrifuged (194,000 times g, 20 min, 4 °C). The resulting pellet was washed three times in buffer A (20 mM BTP and 5 mM MgCl(2), pH 7.5), resuspended in 10 ml of buffer A, and stored at -70 °C. Bleaching was carried out on 1-ml aliquots that contained about 200 nmol of rhodopsin. The sample was supplemented with 3 ml of buffer that contained hydroxylamine (NH(2)OH) (buffer B: 20 mM BTP, 5 mM MgCl(2), and 10 mM NH(2)OH, pH 7.5) and exposed for 30 min (4 °C) to intense light (white light source fitted with infrared cut-off filter). The ROS membranes were then centrifuged at 121,000 times g for 10 min, washed three times with buffer A, and resuspended in buffer A.

Rhodopsin and 9-Demethyl Rhodopsin

Crystalline 11-cis retinal was generously supplied by the late Paul K. Brown (University of Massachusetts, Boston) and stored under nitrogen at -20 °C. The 11-cis isomer of 9-demethylretinal was synthesized using procedures described by Kropf et al.(13) and stored under argon at -70 °C. Some experiments employed 11-cis 9-demethylretinal that was generously provided by Drs. F. Derguini and K. Nakanishi (Columbia University). Pigment formation was initiated by adding an ethanolic solution of either 11-cis retinal or 11-cis 9-demethylretinal to an aliquot of the bleached ROS membranes, at a retinoid:opsin ratio of (5, 6, 7, 8, 9, 10) :1; the concentration of carrier ethanol was 5-10% (v/v). After a 30-min incubation period in darkness (room temperature), the samples were centrifuged, resuspended in buffer B, and incubated for 5 min at room temperature in darkness. The membranes were then centrifuged and washed four times with buffer A, resuspended in buffer A, and stored in darkness at 4 °C. Pigment concentrations were determined from the difference between absorbance spectra obtained before and after exhaustive bleaching in the presence of NH(2)OH (buffer containing 0.5 M Tris, 0.5 M NH(2)OH, and 1% polyoxyethylene 10-tridecylether, pH 7.1)(19) , using molar extinction coefficients of 40,600 M cm (498 nm) for rhodopsin (21) and 41,000 M cm (465 nm) for 9-demethyl rhodopsin(13) . Pigments used within a given experiment were prepared from the same batch of bleached ROS membranes.

Rhodopsin Kinase

Rhodopsin kinase used in the phosphorylation assays was that contained in a hypotonic extract of bovine ROS unless otherwise noted. ROS membranes were isolated under dim red light from 400 freshly obtained bovine retinas (22) and homogenized in 50 ml of hypotonic buffer (buffer C: 5 mM BTP, 0.5 mM MgCl(2), 10 µM pepstatin A, 10 µM leupeptin, 10 µM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.5). The resulting suspension was centrifuged at 43,700 times g (30 min, 4 °C). The supernatant was decanted and recentrifuged (43,700 times g, 30 min, 4 °C). The pellets were resuspended in buffer C and then homogenized and centrifuged two more times as just described. The resulting supernatants were combined and concentrated to the original volume in dialysis bags (molecular weight cut-off = 14,000) using Aquacide II (Calbiochem). Solid adonitol was added to the concentrated extract to a final concentration of 20% (w/v); aliquots were stored at -70 °C.

Some experiments employed rhodopsin kinase that had been partially purified using procedures similar to those described by Buczylko et al.(23) . ROS membranes that had been isolated in the light were homogenized in 200 ml of buffer (10 mM BTP, 10 mM MgCl(2), and 0.3 mg/ml each of aprotinin, benzamidine, and leupeptin, pH 7.5) and incubated for 10 min (4 °C) in room light. The ROS membranes were pelleted by centrifugation at 46,000 times g (10 min, 4 °C). The pellet was mixed with 10 ml of Whatman DE-52 suspended in 10 mM BTP (pH 7.5), and the mixture was loaded onto a 1.6 times 10-cm column of Whatman DE-52 that had been pre-equilibrated with 10 mM BTP (pH 7.5). The column was extensively washed under room light with 10 mM BTP until the absorbance of the eluent at 280 nm (1-cm path length) was less than 0.01. The column was then protected from light, and rhodopsin kinase was eluted with buffer consisting of 10 mM BTP and 110 mM NaCl (pH 7.5). Fractions (1.5 ml) were collected, and 50-µl aliquots were analyzed for activity in light-dependent rhodopsin phosphorylation(3) . Fractions containing the highest activity were pooled, and solid adonitol was added to make a 20% (w/v) solution. The enriched rhodopsin kinase preparation was stored at -20 °C.

Phosphorylation

[-P]ATP (Amersham Corp.) and unlabeled ATP (Sigma) were used to prepare 1 mM stock solutions of ATP (2 times 10^6 cpm/10 µl). Procedures used for the phosphorylation assay were similar to those described by Palczewski et al.(3, 4) . All preparative steps were performed under dim red light. Each reaction mixture contained 25 µl of the rhodopsin kinase preparation, 2 nmol of either regenerated rhodopsin or 9-demethyl rhodopsin in a fixed total amount of ROS membranes, and buffer A in a total volume of 90 µl. The rhodopsin and 9-demethyl rhodopsin prepared for use in a given experiment typically differed with respect to percent regeneration of the bleached ROS membranes. To afford a match of total (i.e. regenerated plus unregenerated) ROS membranes as well as regenerated pigment among reaction mixtures, the mixtures were supplemented as appropriate with ROS membranes that had undergone the preparative bleach but had not been treated with retinal or 9-demethylretinal. 10 s before illumination, the reaction mixture was supplemented with 10 µl of the 1 mM [-P]ATP stock solution. Unless otherwise indicated, illumination was provided by a Vivitar model 283 automatic electronic flash. Light from this source passed through an infrared cut-off filter, neutral density filters (Kodak), and either a Wratten 12 or Wratten 3 cut-off filter (Kodak). The Wratten 12 filter (>30% transmittance above 520 nm) and Wratten 3 filter (>30% transmittance above 465 nm) were used, respectively, for assays of rhodopsin ((max) 498 nm) and 9-demethyl rhodopsin ((max) 465 nm). Incubations continued in the dark at room temperature. At defined times the phosphorylation reaction was quenched by the addition (100 µl) of buffer that contained SDS (buffer D: 80 mM Tris, 100 mM dithiothreitol, 2% (w/v) SDS, 40% glycerol, and bromphenol blue, pH 6.8).

Analysis of Opsin-bound [P]P(i)

A 25-µl aliquot of the quenched reaction mixture was removed for analysis by SDS-polyacrylamide gel electrophoresis using a 13% gel(24) . Gels were stained with Coomassie Blue R-250, destained, and dried. [P]P(i) incorporated into the electrophoretically separated opsin band was quantitated by autoradiography using Kodak XR-5 film (cassette fitted with Lightning Plus intensifier screen). As determined by Coomassie Blue staining, neither the rhodopsin nor 9-demethyl rhodopsin samples contained detectable amounts of opsin dimer. Opsin monomer bands on the resulting autoradiogram were analyzed for integrated density of the band (after correction for film background) using a Hewlett-Packard ScanJet Plus scanning densitometer that was interfaced with a MacIntosh computer and ScanAnalysis (Biosoft) software. To convert densitometric data to molar amount of incorporated [P]P(i), known amounts of the [-P]ATP stock solution were spotted onto a strip of Whatman No. 1 filter paper. The x-ray film being used for the gel autoradiogram was simultaneously exposed to the strip, and the autoradiographic spots were analyzed by densitometry to yield a standard curve.


RESULTS

Calibration of Bleaching Irradiations

To compare the activities of rhodopsin and 9dR as phosphorylation substrates, it was necessary to determine illumination conditions required for approximately similar weak bleaching of the two pigments. These determinations employed preparations similar to those to be analyzed in the phosphorylation assay. Each sample initially contained 2 nmol of either 9dR or rhodopsin in a total volume of 100 µl. For bleaches of 9dR, the Wratten 3 filter and neutral density filters were positioned in the flash stimulator. Each sample received a single flash of intensity I/I(o), where I(o) is the unattenuated intensity; samples were then analyzed spectrophotometrically for remaining bleachable pigment. Bleaching in the rhodopsin samples was similarly examined using the Wratten 12 filter. Results obtained for 9dR and rhodopsin (Fig. 1, open and filledcircles, respectively) were analyzed in terms of the relation,


Figure 1: Bleaching of regenerated rhodopsin (filled circles) and 9dR (open circles). Datapoints and errorbars indicate mean values ± S.D. (n = 3). Curves fitted to the data plot text equation(1) , with k = 0.59 (9dR, dashedcurve) or k = 0.53 (rhodopsin, solidcurve). Inset, hydroxylamine difference spectra for 9dR (opencircles) and rhodopsin (filledcircles). DeltaA/DeltaA, normalized bleach-induced change in absorbance.



where r(o) = 2 nmol = the initial amount of rhodopsin or 9-de-methyl rhodopsin; r* is the measured amount, in nmol, of bleached pigment (i.e. of 9dR* or R*); and k is a photosensitivity parameter (see, e.g.(25) ). The fitting of to the 9dR and rhodopsin data yielded, respectively, the dashed (k = 0.59) and solid (k = 0.53) curves. These served as standard curves for determinations of flash bleaching in the phosphorylation assays.

Time Course of Phosphorylation

Fig. 2shows the amounts of [P]P(i) incorporated by opsin when reaction mixtures containing 9dR (opensymbols) or rhodopsin (filledsymbols) were exposed to a fixed intensity flash and then incubated for varying periods. With illumination that produced 0.033 nmol of bleached rhodopsin (filledsquares), the level of incorporated [P]P(i) increased with incubation period (0-4 min). At 4 min, the molar ratio of incorporated P(i) to bleached rhodopsin (P(i)/R*) was, on average, 0.18 ((5.9 pmol of P(i))/(33 pmol of R*)). Over the same range of incubation periods and upon similar bleaching (0.037 nmol of bleached pigment), the 9dR samples also incorporated [P]P(i) (opensquares) but at levels substantially lower than those determined for rhodopsin. The amounts of opsin-bound [P]P(i) in the 9dR samples at 1, 2, and 4 min of incubation indicated an apparent plateau of incorporation at 1.7 pmol [P]P(i), which represented an average P(i)/9dR* molar ratio of only 0.05. Additional samples of rhodopsin and 9dR were subjected to more extensive flash bleaching and were similarly analyzed for opsin-bound [P]P(i) after 0-2 min of incubation. The pattern of results obtained with 0.16 nmol of R* (filledtriangles) and 0.18 nmol of 9dR* (opentriangles) was qualitatively similar to that observed with the smaller bleaches. For example, at 2 min, average [P]P(i) incorporation for the rhodopsin samples was 6.6 pmol (average P(i)/R* of 0.041), and the average for samples of 9dR was 2.8 pmol (average P(i)/9dR* of 0.016). For both rhodopsin and 9dR, [P]P(i) incorporation in unilluminated controls that had been incubated for 4 min was <0.4 pmol (filled and opencircles).


Figure 2: Time course of light-induced [P]P(i) incorporation by 9dR and rhodopsin. Following flash illumination, reaction mixtures were incubated in darkness for the indicated period and then supplemented with SDS-containing buffer that quenched the phosphorylation reaction. Opensquares, 0.037 nmol of bleached 9dR; filledsquares, 0.033 nmol of bleached rhodopsin; opentriangles, 0.18 nmol of bleached 9dR; filledtriangles, 0.16 nmol of bleached rhodopsin. Datapoints and verticalbars indicate mean values ± S.D. (n = 3), respectively. Filled and opencircles show data obtained from unilluminated control samples containing, respectively, rhodopsin and 9dR.



Dependence on Bleaching Extent

Data obtained from both the rhodopsin and the 9dR samples in the Fig. 2experiment showed that P(i) incorporation measured at 30 s is both bleach- dependent and within the near-linear range. Accordingly, a 30-s incubation period was chosen as a standard condition, under which to compare the approximate initial rates of phosphorylation of 9dR* and R* as a function of bleaching. Fig. 3shows results obtained in an experiment of this type. The apparent initial rate of [P]P(i) incorporation increased with the extent of bleaching of both 9dR (0-3.4 µM 9dR*, opencircles) and rhodopsin (0-3.1 µM R*, filledcircles). However, over the entire range examined, [P]P(i) incorporation was substantially lower in the 9dR samples. Data obtained from each set of samples were well described by the hyperbolic (Michaelis) relation,


Figure 3: Initial rate of [P]P(i) incorporation (nmol/liter/min) by illuminated rhodopsin and 9dR. Indicated extents of flash bleaching are based on use of the curves shown in Fig. 1. Concentrations quoted in the figure and accompanying text represent molar amounts contained in the 100-µl reaction mixture. Following illumination, samples were incubated for 30 s and then analyzed for [P]P(i) incorporation. Filledcircles, rhodopsin samples; opencircles, 9-demethyl rhodopsin samples. Curves are plots of text equation(2) . Inset, representative autoradiographic data obtained at the higher bleaching levels. Upperrow (left to right), rhodopsin (rho) bleaches of 0.7, 1.6, and 3.1 µM; lowerrow (left to right), 9-demethyl rhodopsin (9dR) bleaches of 0.7, 1.8, and 3.4 µM.



where S is the measured concentration of bleached pigment, v is the measured initial rate of [P]P(i) incorporation, v(max) is the maximal value of v, and K(m) is a binding constant (curves in Fig. 3). For the rhodopsin data, the fitting of this equation yielded v(max) = 60 nmol/liter/min and K(m) = 0.20 µM; for the 9dR data, the fit yielded v(max) = 15 nmol/liter/min and K(m) = 0.61 µM. The apparent maximal rate of 9dR* phosphorylation is thus about 25% of that for the native photoactivated pigment under the present experimental conditions. The determined values of K(m) furthermore indicate that the strength of the interaction between kinase and 9dR* is considerably weaker than that between kinase and photoactivated rhodopsin.

Light-dependent phosphorylation of 9dR and rhodopsin was also examined using a partially purified preparation of rhodopsin kinase, as described under ``Experimental Procedures.'' Conditions used in these experiments for light/dark incubation of the final reaction mixtures and procedures for bleaching determinations differed from those described above (see Fig. 4legend). Shown in Fig. 4are levels of [P]P(i) incorporation for rhodopsin and 9dR samples (filled and opencircles, respectively) measured after a 10-min incubation period that included a bleaching exposure; the abscissa value of each data point indicates the spectrophotometrically determined extent of bleaching. The general pattern of the Fig. 4data is similar to that seen in the experiment of Fig. 3, which involved flash bleaching and 30-s dark incubation. That is, in Fig. 4, [P]P(i) incorporation by 9dR exhibits a near plateau over the range of 0.17-0.55 nmol of 9dR* (12 samples). Moreover, the average incorporation represented by these 12 samples, 34.5 times 10^3 ± 7.1 times 10^3 densitometric units (mean ± S.D.), is significantly less than that measured in the rhodopsin samples over a comparable bleaching range (101.8 times 10^3 ± 6.4 times 10^3 densitometric units for the 8 samples spanning 0.14-0.60 nmol of R*).


Figure 4: Bleaching dependence of [P]P(i) incorporation in rhodopsin and 9dR samples incubated with partially purified rhodopsin kinase (see ``Experimental Procedures''). Each reaction mixture initially contained 1 nmol of rhodopsin or 9dR in a total volume of 55 µl. Each sample received 5 µl of 1 mM [-P]ATP; 10 s later, phosphorylation was initiated by a period of illumination (0-300 s). The light source was a tungsten-halogen lamp fitted with an infrared cut-off filter and a broad-band gelatin filter (peak transmittance near 520 nm). Immediately after illumination, an aliquot of the reaction mixture (30 µl) was removed for spectrophotometric analysis of remaining bleachable pigment and, thus, of bleaching extent. The remainder of the sample was incubated in darkness. 10 min after the beginning of illumination, the phosphorylation reaction was quenched by the addition of SDS-containing buffer. [P]P(i) incorporation was determined by SDS-polyacrylamide gel electrophoresis and autoradiography and is expressed here in units of integrated density (D.U.) of the opsin monomer band. Ordinate values of the data points indicate the mean of results obtained from duplicate samples. Errorbars represent the range of the two determinations; this range was in some cases within the dimension of the data point.




DISCUSSION

The results show that in bovine ROS preparations, conditions that support the phosphorylation of photoactivated rhodopsin also support the phosphorylation of illuminated 9-demethyl rhodopsin. The level of 9dR* phosphorylation increases with both incubation period (Fig. 2) and bleaching extent (Fig. 3Fig. 4). However, by comparison with phosphorylation levels in the rhodopsin samples, those in the samples of 9dR are lower under all conditions examined.

What property of 9dR underlies its low activity in light-dependent phosphorylation? The facts that metarhodopsin II is the principal substrate of rhodopsin kinase and that a metarhodopsin II-like conformation is not observed spectrally upon illumination of 9dR (14) imply that the low extent of phosphorylation of 9dR* is linked with the absence of a species that resembles metarhodopsin II. Precisely how conformational properties of 9dR* affect the course of the phosphorylation reaction is not resolved by the present study. The complex formed by 9dR* and rhodopsin kinase may exhibit reduced phosphorylation rate; alternatively, the 9dR* may exhibit an altered interaction with the kinase. Some information on this issue comes from the present Fig. 3data, which indicate a remarkably weak dependence of [P]P(i) incorporation rate on bleaching extent over the range of 0.7-3.0 µM 9dR*. This weak dependence appears not to reflect either the saturation of kinase activity or the exhaustion of ATP, based on the high levels of [P]P(i) incorporation observed in the rhodopsin controls. The near-plateau character of the rate data obtained over this range of 9dR bleaching appears, rather, to reflect a low maximal velocity of phosphorylation of 9dR*. One possibility consistent with this finding and with the relatively low phosphorylation of the complex formed between opsin and all-trans-9-demethylretinal (26) is that 9dR* forms a non-productive complex with rhodopsin kinase and thereby reduces maximal kinase activity. Such a possibility is compatible with the evidence that the binding of kinase by illuminated rhodopsin does not strictly depend on attainment of the metarhodopsin II conformation (27, 28, 29) .

Available information on the properties of 9dR within intact rods comes from electrophysiological experiments on rods of the salamander(15, 16) . It was not possible in the present study to investigate phosphorylation in salamander ROS preparations. However, both biochemical and electrophysiological studies suggest a generally similar operation of phototransduction reactions in amphibian and mammalian rods, including that of R* phosphorylation(1, 30, 31, 32, 33) . Furthermore, the deactivation of R*, i.e. the process thought to be mediated by R* phosphorylation and arrestin binding (2, 3, 4, 5, 6, 7, 8, 9, 10, 11) , proceeds with apparently similar kinetics in bovine and salamander rods at similar temperature. That is, light-scattering data obtained from intact rods of the bovine retina following flash illumination (fractional bleaches of <10) imply an exponential, i.e. first order, decline of R* with a time constant of 3-5 s(34, 35, 36) ; in salamander rods, the decline of R* inferred from electrophysiological data is also exponential, with a time constant of about 2 s(16, 36) . Moreover, the finding that 9dR* in salamander rods mediates quantal photocurrent responses of low peak amplitude (15) is compatible with the observation, in bovine ROS membranes, that 9dR* exhibits a low efficiency of transducin activation (14) . Based on these considerations, the present data appear consistent with the notion that the abnormally long 9dR* lifetime determined electrophysiologically in salamander rods (16) reflects, at least in part, a reduced efficiency of phosphorylation of the illuminated pigment.


FOOTNOTES

*
This research was supported by Grants EY-06038, EY-05494, EY-01792, EY-05788, EY-04939, EY-07543, and EY-07586 from NEI, National Institutes of Health; by unrestricted grants from Research to Prevent Blindness, Inc.; by the Lions of Illinois Foundation, Maywood, Illinois; and by the Illinois Eye Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor St., Chicago, IL 60612, Tel.: 312-996-4262.

(^1)
The abbreviations used are: R*, photoactivated rhodopsin; 9dR, 9-demethyl rhodopsin; 9dR*, photoactivated 9-demethyl rhodopsin; ROS, rod outer segments; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane.


ACKNOWLEDGEMENTS

We are grateful to Dr. F. Derguini, Dr. K. Nakanishi, and the late P. K. Brown for gifts of 11-cis 9-demethylretinal and 11-cis retinal. We also thank Drs. K. P. Hofmann, K. Palczewski, and T.-I. L. Okajima for helpful discussions.


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