Activation of Transducin by a Xenopus Short Wavelength Visual Pigment*

(Received for publication, July 17, 1996, and in revised form, September 25, 1996)

Dorine M. Starace Dagger and Barry E. Knox §

From the Department of Biochemistry and Molecular Biology and Ophthalmology, State University of New York Health Science Center, Syracuse, New York 13210

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Phototransduction in cones differs significantly from that in rods in sensitivity, kinetics, and recovery following exposure to light. The contribution that the visual pigment makes in determining the cone response was investigated biochemically by expressing a Xenopus violet cone opsin (VCOP) cDNA in COS1 cells and assaying the light-dependent activation of transducin. Light-exposed VCOP stimulated [35S]guanosine 5'-(gamma -thio)triphosphate nucleotide exchange on bovine rod transducin in a time-dependent manner with a half-time for activation of 0.75 min, similar to that of bovine rhodopsin. In exhaustive binding assays, VCOP and rhodopsin activity showed similar concentration dependence with half-maximal activation occurring at 0.02 mol of pigment/mol of transducin. Although VCOP was able to activate as many as 12 transducins per photoisomerization, rhodopsin catalyzed significantly more. When assays were performed with lambda  > 420 nm illumination, VCOP exhibited rapid regeneration and high affinity for the photoregenerated 11-cis-retinal. Recycling of the chromophore and reactivation of the pigment resulted in multiple activations of transducin, whereas a maximum of 1 transducin per VCOP was activated under brief illumination. The decay of the active species formed following photobleaching was complete in <5 min, ~10-fold faster than that of rhodopsin. In vitro, VCOP activated rod transducin with kinetics and affinity similar to those of rhodopsin, but the active conformation decayed more rapidly and the apoprotein regenerated more efficiently with VCOP than with rhodopsin. These properties of the violet pigment may account for much of the difference in response kinetics between rods and cones.


INTRODUCTION

Photopic vision is mediated by specialized photoreceptor cone cells that function at high levels of illumination, respond to rapid changes in light, and permit color discrimination (1, 2). Each cone cell expresses a cone opsin. The cone opsins are members of a larger family of visual pigments (3, 4, 5) and share significant amino acid sequence homology with the rod pigment rhodopsin (6). Among the cone pigments, the short wavelength pigments (Group S, with wavelengths of peak absorbance (lambda max)1 ~ 415-440 nm) permit vision in the violet/blue region of the spectrum and are represented by the mammalian blue, chicken violet, and Xenopus violet pigments.2 Although cone cells expressing Group S pigments are in the minority in the vertebrate retina, they are an integral part of vision. For example, in blue monochromats, i.e. humans with red-green pigment mutations, the blue opsin is the sole mediator of photopic vision (7, 8).

The phototransduction mechanisms have been extensively investigated in rods (for reviews see Refs. 2 and 9). Following absorption of light and isomerization of 11-cis-retinal to all-trans-retinal, rhodopsin undergoes a series of conformational changes that eventually leads to a transient state, coincident with metarhodopsin(II) (MetaII), that activates the second messenger cascade (9). MetaII interacts with the heterotrimeric rod G protein, transducin, and thereby catalyzes the exchange of guanyl nucleotide, which leads to dissociation of the transducin subunits. Rods and cones express a very similar set of transduction proteins (10); however, in general, the electrical response of a cone to light is faster and less sensitive than that of a rod (11, 12). Moreover, there are differences in the properties of responses even between the various cone subtypes. In salamanders, responses of violet cones to light exhibit more rod-like behavior, with slower kinetics and increased sensitivity than long wavelength (red) cones (11). The molecular basis for the differences between cone and rod responses and among the cone subtypes has not been established.

In order to investigate the phototransduction properties of the short wavelength visual pigments, a Xenopus violet cone opsin (VCOP) was cloned and characterized.2 The protein, which was efficiently expressed in COS1 cells, formed a Schiff base with 11-cis-retinal and absorbed maximally at 425 nm. Characterization of the light-dependent interaction of VCOP with transducin was performed to identify the contribution of the pigment to the physiological responses of cones. Using a guanyl nucleotide exchange assay and purified bovine rod transducin, we found that VCOP activated rod transducin with kinetics and affinity similar to those of rhodopsin but that the active conformation decayed more rapidly and the apoprotein regenerated more efficiently than rhodopsin.


EXPERIMENTAL PROCEDURES

Expression and Purification of Visual Pigments

An expression construct, pMT-VCOP, containing the first 328 codons of the Xenopus violet cone opsin coding region and the last 14 codons of bovine rod opsin was expressed in mammalian COS1 cells by transient transfection (13). The epitope-tagged violet cone pigment (VCOP) was generated by incubation with 11-cis-retinal and purified in buffer W (50 mM HEPES, pH 6.6, 140 mM NaCl, 3 mM MgCl2, 20% (w/v) glycerol, and 0.1% dodecyl maltoside (DM)), as described elsewhere.2 Xenopus rhodopsin, in the construct pMT-XOP1 (14), was similarly expressed and purified. Bovine rhodopsin from rod outer segments (ROS) was purified in parallel with the expressed pigments. UV-visible absorption spectra of purified and solubilized visual pigments were recorded in the indicated buffers at 20 °C with a Beckman Instruments DU 640 single beam spectrophotometer using an extinction coefficient of 39,400 M-1 cm-1 at 425 nm for VCOP. Bleaching illumination was provided with light from a 300-watt projector (Eastman Kodak Co.) at a distance of 50-60 cm. Unfiltered light from the projector was termed white light, and high-pass colored glass cutoff filters (>420 nm, >440 nm, and >455 nm, Edmund Scientific, Inc., Barrington, NJ) were used as indicated. Total radiant energy of the various filtered light stimuli were measured using an optical power meter (3M, St. Paul, MN, Photodyne) equipped with a calibrated silicon photodiode (EGG, Inc., Princeton, NJ). Unfiltered light had 4.0 milliwatts, whereas the various filters had 3.4-3.6 milliwatts. Bleaching of visual pigments was monitored by recording spectra from a single sample before and several times after illumination under conditions identical to transducin assay conditions (see below). All spectra were analyzed using SigmaPlot Software (Jandel, San Rafael, CA).

Transducin Activation Assays

Bovine transducin was purified and the stimulation of the binding of [35S]GTPgamma S to bovine transducin by visual pigments was measured with the use of a nitrocellulose filter binding assay (15, 16). Unless otherwise stated, reaction volumes were 200 µl and assays were performed at 22-25 °C in 0.007-0.01% DM (Anatrace, Maumee, OH). The reaction mixtures contained 10-62.5 nM VCOP or 2.5-62.5 nM bovine rhodopsin, 2 µM GTPgamma S (with 2000-5000 cpm [35S]GTPgamma S/pmol, DuPont NEN), and 300 nM bovine transducin in 10 mM Tris acetate, pH 7.0, 100 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol. Both initial rate and extent reactions were initiated by illumination. The binding of [35S]GTPgamma S to transducin in the absence of visual pigment was subtracted from all data and was typically 5-8% of the total transducin for a 30-min reaction. Samples that were exposed to light prior to transducin assay were illuminated for 1 min, incubated for various times, and then added into the transducin assay for a 30-min reaction. Assays were performed in disposable cuvettes that were transparent in the UV region.


RESULTS

The Xenopus Violet Cone Pigment Activates Transducin

In order to examine the interaction between short wavelength visual pigments and transducin, the violet opsin was expressed in COS1 cells containing a carboxyl-terminal 1D4 epitope tag from bovine rhodopsin (VCOP). Visual pigment was generated in COS1 membranes by incubation with 11-cis-retinal, solubilized in DM, and purified by immunoaffinity chromatography on 1D4-Sepharose.2 The presence of the altered carboxyl terminus in the expressed VCOP was not expected to perturb the transducin activity significantly since the carboxyl domain does not appear to play an important role in transducin activation by rhodopsin (17, 18). The low abundance of transducin from cone cells, in either Xenopus or bovine retina, precluded the use of cone transducin in the biochemical assays. However, the high degree of homology between VCOP and bovine rhodopsin in the regions shown to be involved in the interaction of light-activated rhodopsin with rod transducin (17, 19) and the similarity of rod and cone transducin alpha  subunits in mammals (10) and in Xenopus3 suggested that VCOP would interact with rod transducin.

VCOP activated bovine rod transducin in a time-dependent manner (Fig. 1A) with a half-time of activation (t1/2) of 0.75 min. The reaction was complete in 5 min and resulted in only 24% of the available transducin being activated. Both COS1-expressed Xenopus rhodopsin and bovine ROS rhodopsin were able to completely activate transducin (Fig. 1A). Thus, although a single rhodopsin was able to catalyze the activation of multiple transducins, a single VCOP maximally stimulated only one transducin. The kinetics of activation of the rhodopsins were similar to those of VCOP (t1/2 = 0.58 and 1.0 min for Xenopus and bovine rhodopsin, respectively), suggesting that the rate of interaction between transducin and the active state of VCOP is similar to that of the rhodopsins. However, the initial rate of activation was slower for VCOP (0.46 mol of GTPgamma S bound/mol of opsin/min) than for Xenopus (1.44 mol of GTPgamma S bound/mol of opsin/min) and bovine rhodopsin (2.1 mol of GTPgamma S bound/mol of opsin/min).


Fig. 1. Activation of GTPgamma S exchange on transducin by the Xenopus violet pigment. A, kinetics of activation. 25 pmol of Xenopus violet pigment (black-triangle), Xenopus rhodopsin (black-square), or bovine ROS rhodopsin (bullet ) were incubated with 90 pmol of bovine rod transducin and 1.25 µM [35S]GTPgamma S in volumes of 400 µl containing 0.01% dodecyl maltoside. Reactions were initiated by illumination (Corning Glass filter no. 3543 for Xenopus violet pigment or lambda  > 515 nm for rhodopsin). Illumination was stopped after 1.5 or 10 min and the remaining portion of the reaction took place in the dark. The lines represent single parameter exponential fits, with half-times for activation of 0.75 min for Xenopus violet pigment, 1.0 min for Xenopus rhodopsin, and 0.58 min for bovine rhodopsin. B, titration of rod transducin activation by the Xenopus violet pigment. Various amounts of Xenopus violet pigment (black-triangle) or bovine ROS rhodopsin (bullet ) were incubated with 60 pmol of bovine rod transducin in 0.0075% dodecyl maltoside in volumes of 200 µl. Reactions proceeded for 30 min under continuous illumination (lambda  > 420 nm). The solid lines are sigmoidal fits to the data using the Hill equation with 1.2 pmol (6 nM) of pigment required to activate 50% of the transducin for both samples.
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In order to determine the total number of interactions that occur between VCOP and transducin, a second set of experiments was performed with a fixed concentration of rod transducin. VCOP caused a concentration-dependent stimulation of nucleotide exchange on transducin (Fig. 1B) with a sigmoidal behavior and half-maximal activity occurring at 1.2 pmol of pigment. Bovine ROS rhodopsin similarly stimulated transducin with a half-maximal activity also occurring at 1.2 pmol of bovine rhodopsin (Fig. 1B). Thus, the apparent affinity between VCOP and rod transducin was similar to that observed with rhodopsin. However, the maximum level of activity of VCOP occurred at 21 pmol of GTPgamma S bound, indicating that only 35% of available transducin was activated, in contrast to bovine rhodopsin, which activated all available transducin. The incomplete activation of transducin by VCOP was not due to the inability of a single VCOP molecule to activate more than one transducin, since multiple turnovers were observed at lower pigment concentrations (e.g. 1.2 pmol of pigment catalyzed the exchange of 14 pmol of GTPgamma S). By contrast, bovine rhodopsin stimulated the turnover of up to 36 transducins at its half-maximal concentration (Fig. 1B) in agreement with previous studies of the activity of bovine rhodopsin in detergent (20). The similarity of the initial rate t1/2 values of VCOP and rhodopsin and the identical concentration dependence for activation of transducin suggest that the interaction of the active state (V-MetaII or V*) of VCOP with rod transducin has a rate and an affinity comparable to those of rhodopsin.

The failure of VCOP to completely activate transducin was not due to a decay of the pigment during the incubation period of the transducin assay since the activity of VCOP preincubated for 30 min at room temperature before addition of transducin was equal to the activity without preincubation (data not shown). The hydrophobic media, i.e. lipid and detergent, in which transducin reactions occur play an important role in stabilizing activity. The interaction between bovine rod transducin and VCOP was measured in 0.007-0.01% DM since previous work has demonstrated that this detergent concentration range permits optimal enzymatic activity of solubilized bovine rhodopsin (20). The optimal concentration of DM for VCOP activity sharply peaked at 0.008% (Fig. 2), which is near the critical micelle concentration of the detergent (21). The detergent profiles of bovine and expressed Xenopus rhodopsin activities were the same as that of VCOP (Ref. 20 and data not shown). Thus, the incomplete activation of bovine rod transducin by VCOP at saturation cannot be attributed to nonoptimal detergent conditions.


Fig. 2. Titration of the Xenopus violet pigment activity with dodecyl maltoside. 75 pmol of bovine rod transducin and 500 pmol of [35S]GTPgamma S were incubated with (bullet ) or without (diamond ) Xenopus violet pigment (6 pmol) in the indicated concentrations of dodecyl maltoside in volumes of 250 µl. The reactions proceeded for 30 min under continuous illumination (lambda  > 420 nm) and bound nucleotide was measured.
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To directly demonstrate that the light intensity (lambda  > 420 nm) was not limiting in transducin binding assays, bleaching of VCOP was monitored spectroscopically under conditions identical to assay conditions except that transducin was omitted (Fig. 3, left panel). Photoactivation of VCOP caused a decay of the pigment (lambda max 425 nm) and a concurrent formation of a product with lambda max ~ 375 nm. This product could be free all-trans-retinal, which is released from the pigment upon bleaching, or V-MetaII, which is expected to be spectrally indistinguishable from free retinal. Acid denaturation experiments have shown that the product is most likely free all-trans-retinal (data not shown). A dark-light difference spectra (Fig. 3B) more clearly shows the progress of pigment bleaching, since the absorbances of VCOP and the photoproducts significantly overlap. After 10 s of illumination, >85% of the 425 nm absorbance has been converted. In addition, there was an increase in light scattering at shorter wavelengths, indicating that VCOP aggregates on bleaching. After 20 s of illumination, there was no additional change in the 425 nm peak, indicating that 20 s is enough time to bleach VCOP completely. The spectroscopic analysis of VCOP bleaching provides direct evidence that, under the transducin assay conditions, the VCOP was sufficiently photostimulated and cannot account for the failure of VCOP to activate all of the available transducin.


Fig. 3. Spectroscopic analysis of the time course and extent of bleaching of the Xenopus violet pigment. Left panel, Xenopus violet pigment (32 pmol) was diluted with transducin assay buffer (220 µl) containing 0.0075% dodecyl maltoside. A spectrum was recorded (1) and then the sample was illuminated (lambda  > 420 nm) under conditions identical to those used in transducin assays. After 10 s (2), 20 s (3), and 1 min (4), UV-visible absorption spectra were measured. Right panel, difference spectra were derived by subtracting spectrum 2 from 1 (Dark-Light (10")) and spectrum 3 from 2 (Light (20")-Light (10")). The solid line is fit for the light-scattering contribution.
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Decay of the Active State

The ability of VCOP to activate rod transducin suggests that illumination creates a metastable active state analogous to MetaII. To further characterize the interaction between VCOP and transducin, the lifetime of the photoexcited state (V*) was measured. V* was formed by brief illumination of VCOP and its lifetime was monitored by addition of transducin and [35S]GTPgamma S for nucleotide exchange assay at various times after illumination. The light-activated VCOP exhibited a rapid decay of its ability to stimulate GTPgamma S exchange. The half-time of decay was approximately 2 min and a complete loss of activity occurred in 5 min (Fig. 4). In contrast, light-activated rhodopsin exhibited more prolonged activity with a half-time of decay of 20 min and detectable activity even after 2 h. Thus, the reduced activity of VCOP when compared to that of rhodopsin is attributable to the relatively reduced lifetime of its photoactive state and not to a reduced rate of interaction with or affinity for rod transducin.


Fig. 4. Decay of the active state of the Xenopus violet pigment. Xenopus violet pigment (left panel) or bovine ROS rhodopsin (right panel) was incubated with [35S]GTPgamma S in assay buffer containing 0.0075% dodecyl maltoside. Samples were illuminated for 30 s (lambda  > 420 nm) and, after the indicated preincubation time in the dark, reaction with transducin was initiated by the addition of bovine rod transducin and proceeded for 30 min in the dark. The lines are fits to an exponential function.
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Efficient Photoregeneration of Violet Cone Opsin

Unlike rhodopsin (data not shown), transducin activation by VCOP under these in vitro conditions depended on the illumination conditions during the assay (Fig. 5). Using lambda  > 420 nm light, there was an approximately linear increase in the activity observed with the illumination time, showing an almost 10-fold increased activity with 30 min of illumination compared to 1 min. The activity of VCOP was also measured as a function of the spectral composition of the saturating illumination used during the transducin assay in reactions continuously exposed to one of the following illumination conditions: lambda  > 455 nm, lambda  > 440 nm, lambda  > 420 nm, or unfiltered projector light (white light) (Fig. 6). Maximal and equivalent activities were observed when VCOP was illuminated for 30 min with white light or with lambda  > 420 nm light. Illumination of the pigment for 30 min with lambda  > 440 nm or lambda  > 455 nm filtered light resulted in increasingly reduced activities even though VCOP was bleached by these lighting conditions.


Fig. 5. The activity of the Xenopus violet pigment is dependent on the time of illumination. Xenopus violet pigment (2 pmol) was incubated with 60 pmol of bovine rod transducin and [35S]GTPgamma S in volumes of 200 µl containing 0.0075% dodecyl maltoside. Reactions were illuminated (lambda  > 420 nm) for the indicated times and proceeded for 30 min before measurement of bound nucleotide.
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Fig. 6. Spectral profile of rod transducin activation by the Xenopus violet pigment. Xenopus violet pigment (6 pmol) was incubated with 36 pmol of bovine rod transducin and [35S]GTPgamma S in volumes of 120 µl containing 0.01% dodecyl maltoside. All reactions proceeded for 30 min with continuous illumination, except white light, with 1.5 min of illumination. Illumination was provided with high-pass filters whose cutoffs indicate the wavelengths of half-maximal transmission; white light illumination was provided with unfiltered projector light.
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The photoisomerization of all-trans-retinal to an equilibrium mixture of isomers, including 11-cis-retinal, is produced with high intensity near-UV light in aqueous solutions (22, 23). The recycling of released and photoisomerized all-trans-retinal chromophore by subsequent regeneration of VCOP apoprotein is consistent with the observed increase of activity with illumination (Fig. 5) and with the spectral profile of activity (Fig. 6). In order to determine whether pigment recycling was occurring, activation of transducin by VCOP was carried out in the presence and absence of horse liver alcohol dehydrogenase (ADH) and its cofactor, NADH (Fig. 7). In the presence of excess NADH at pH 7.0, ADH reduces retinal to retinol (24), thereby preventing the regeneration of visual pigment by recycling of the chromophore. The increase of VCOP activity with the time of illumination was eliminated when excess ADH and NADH were added, whereas the activity for the brief illumination was not changed. The sensitivity to ADH and NADH provides evidence that the illumination time-dependent VCOP activity is due to the photoisomerization and recycling of released chromophore.


Fig. 7. The Xenopus violet pigment activity is sensitive to horse liver alcohol dehydrogenase. Xenopus violet pigment (2 pmol) was incubated with 60 pmol of bovine rod transducin and [35S]GTPgamma S in 200 µl reactions containing 0.007% dodecyl maltoside. Reactions were performed in the presence (solid bars) or absence (open bars) of excess horse liver alcohol dehydrogenase (20 milliunits) and NADH (173 µM). Reactions were illuminated (lambda  > 420 nm) for the indicated times and terminated after 30 min for measurement of bound nucleotide.
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In another set of experiments, the regeneration of VCOP with recycled chromophore was explicitly monitored by measuring the ADH-sensitive transducin activity of bleached VCOP. Before addition of rod transducin, VCOP was illuminated (lambda  > 420 nm) for 30 s and preincubated for 30 min in the dark at room temperature to allow complete decay of the resulting active state. In addition, the dark incubation was carried out in the presence and absence of ADH and NADH and/or 11-cis-retinal. Upon addition of transducin to the bleached VCOP, the reaction proceeded for 30 min in the dark or under constant illumination (lambda  > 420 nm) to assay illumination-dependent activity (Fig. 8). The bleached VCOP showed light-sensitive activity. There was no activity when the assay was performed in the dark. The light-sensitive activity of the bleached VCOP was severely inhibited by the addition of excess ADH and NADH. This result shows that the Xenopus violet apoprotein rebinds 11-cis-retinal, formed by photoisomerization of the released chromophore, to regenerate an active pigment. The addition of 1-2 molar excess over pigment of exogenous 11-cis-retinal during the dark preincubation, immediately after preillumination, enhanced the light-sensitive activity of the bleached pigment by 1.7-fold, and this activity was also sensitive to ADH and NADH. The increase of activity caused by illumination, despite substoichiometric formation of 11-cis-retinal by photoisomerization, suggests that the affinity of Xenopus violet opsin for 11-cis-retinal is quite high.


Fig. 8. The activity of bleached violet pigment is dependent on illumination and ADH. Xenopus violet pigment (2 pmol) was incubated with 60 pmol of bovine rod transducin and [35S]GTPgamma S in 200 µl reactions containing 0.007% dodecyl maltoside. Bleached violet pigment was prepared at room temperature by preilluminating (lambda  > 420 nm) the reaction mixture without transducin for 30 s followed by a 30-min preincubation in the dark. When indicated, ADH and NADH were added. During the dark preincubation, 11-cis-retinal (2-4 pmol) was added immediately after the preillumination. Bovine rod transducin was added and the reaction proceeded for 30 min in the dark (solid bar) or with continuous illumination (lambda  > 420 nm) (open bars).
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DISCUSSION

Despite the similarities in the components of the cone and rod visual transduction cascade, the physiological responses of cone photoreceptor cells differ from those of rods in a number of ways (11, 12). First, cones are less sensitive than rods; 100 photons are required to elicit an electrical response of a cone cell, as opposed to 1 photon for a rod cell. Second, the kinetics of the electrical response of a cone cell have a rate 3-10-fold faster than that of the rod cell. Finally, the rate of regeneration of a cone is 500 times faster than that of a rod. However, the biochemical and molecular mechanisms that underlie these phenomena are unclear, largely due to the low abundance and heterogeneity of cone phototransduction components in most retinae. Using methods for mammalian cell expression of opsins, the purified violet cone visual pigment was generated in quantities amenable to a detailed study of its interaction with rod transducin, which can be purified in abundance and, is homologous to cone transducin (10). Although the rates and extents of activity measured using rod transducin may not be the same for the activation of cone transducin, the observed activity is proportional to the amount of V* formed. The characterization of the interaction of the violet pigment and rod transducin permitted the investigation of the role of the visual pigment itself in the response properties of the cone cell.

The Xenopus violet pigment was able to activate rod transducin in both a concentration- and a time-dependent manner. The amount of violet pigment required to stimulate half-maximal activity was the same as the half-maximal value for bovine rhodopsin, suggesting that the affinity of the violet pigment for rod transducin is similar to that of rhodopsin. The maximal rate of interaction between the violet pigment and transducin was also approximately equivalent to that of bovine rhodopsin. These results are consistent with both the conservation of the amino acid regions in rhodopsin and the Xenopus violet pigment that are involved in transducin binding2 and with the high homology (76%) between bovine rod and cone transducin (10) and suggest that the conformations of the violet pigment and its enzymatically active state are very similar to those of rhodopsin. These results are consistent with the similarities found between the MetaII photointermediates of chicken red cone opsin and rhodopsin using circular dichroism (25).

The transducin activation catalyzed by the Xenopus violet pigment differed markedly from that of bovine rhodopsin in the number of molecules activated by transducin. The lifetime of the enzymatically active state, V*, was about 10 times shorter than the active state of rhodopsin and thus may account for the fewer turnovers and hence the reduced sensitivity to light of cones compared to rods. It was also demonstrated that photolysis of the violet pigment was accompanied by a light-driven isomerization of all-trans-retinal, which was able to recombine with the violet opsin to regenerate a photosensitive pigment and further stimulate transducin. This system thus exhibited efficient pigment recycling and directly implicated the effective regeneration of the cone pigment in mediating the enhanced dark adaptability of the cones. A summary of these findings is illustrated in Fig. 9. Our experiments have lent direct support to the hypothesis that the rates of pigment regeneration and V* decay play an important role in determining cone responsiveness (4, 26). In addition, the rapid decay of the active species raises the question of whether a rhodopsin kinase/arrestin pathway (9) is required in cone cells to inactivate responses to light.


Fig. 9. A summary of the violet cone pigment activity. Upon absorption of light (open arrows), VCOP is photoconverted to the enzymatically active state, V* or V-MetaII, analogous to metarhodopsin(II). The retinal chromophore is isomerized from 11-cis- to all-trans-retinal. V-MetaII activates transducin (GT) until it thermally decays to an inactive state and releases all-trans-retinal. Recycling of VCOP occurs as a consequence of the photoconversion of all-trans- to 11-cis-retinal. Subsequent reactivation of the VCOP leads to increased turnovers of transducin. The recycling can be blocked by the addition of alcohol dehydrogenase, which reduces free retinal to retinol (-OH).
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These experiments demonstrate the effectiveness of using expressed Xenopus violet cone opsin as a model system for exploring cone visual pigment biochemistry and structure-function relationships. Previously, several studies on the chicken red cone pigment have demonstrated that it activates transducin (4, 25, 27) and the cGMP phosphodiesterase (28) and is a substrate for rhodopsin kinase (29). Although these studies have demonstrated the basic interactions, kinetic comparisons relevant to cone-rod physiological differences were not carried out. The experiments reported here have extended the biochemistry of phototransduction to the cone pigments and are novel for the short wavelength group of cone pigments. Recent spectroscopic work on chicken green opsin and its photobleaching intermediates (26, 30) have postulated differences in signal transduction properties between rod and cone pigments arising from differences in the MetaII lifetimes. The results presented in this report have confirmed this idea. Future work will be directed toward identifying the molecular basis for the different phototransduction properties exhibited by the short wavelength visual pigment and rhodopsin.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant EY09409, a Basil O'Connor March of Dimes award, and Central New York Children's Miracle Network. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Present address: Dept. of Anesthesiology, University of California, Los Angeles, Los Angeles, CA 90095.
§   To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, SUNY Health Science Center, 750 East Adams St., Syracuse, NY, 13210. Tel.: 315-464-8719; Fax: 315-464-8750; E-mail: knoxb{at}vax.cs.hscsyr.edu.
1    The abbreviations used are: lambda max, wavelengths of peak absorbance; MetaII, metarhodopsin(II); VCOP, violet cone pigment; V*, photoexcited violet cone pigment; V-MetaII, violet cone metarhodopsin(II)-like photointermediate; DM, dodecyl maltoside; GTPgamma S, guanosine 5'(gamma -thio)triphosphate; ROS, rod outer segment; ADH, alcohol dehydrogenase.
2    D. M. Starace and B. E. Knox, submitted for publication.
3    B. E. Knox, unpublished data.

Acknowledgments

We gratefully acknowledge Dr. Arjun Surya for the transducin used in this work and for many helpful suggestions, Drs. Marianne Max and Ed Pugh for critical reading of the manuscript, and Dr. Jack Sullivan for help measuring light intensities.


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