Quantitative Aspects of cGMP Phosphodiesterase Activation in Carp Rods and Cones*

Background: Cones are less light-sensitive than rods. Results: The efficiency of cGMP phosphodiesterase (PDE) activation by transducin was similar in rods and cones, and PDE activity was determined by the lifetimes of activated visual pigment and transducin. Conclusion: PDE activation is less effective in cones due to lower amplification and shorter lifetimes of pigment and transducin. Significance: The lower light sensitivity of cones is partly explained. Cones are less light-sensitive than rods. We showed previously in carp that more light (>100-fold) is required in cones than in rods to activate 50% of cGMP phosphodiesterase (PDE). The lower effectiveness of PDE activation in carp cones is due partly to the fact that the activation rate of transducin (Tr) by light-activated visual pigment (R*) is 5-fold lower in carp cones than in rods. In this study, we tried to explain the remaining difference. First, we examined the efficiency of activation of PDE by activated Tr (Tr*). By activating PDE with known concentrations of the active (guanosine 5′-Ο-(γ-thio)triphosphate (GTPγS)-bound) form of Tr*, we found that Tr* activated PDE at a similar efficiency in rods and cones. Next, we examined the contribution of R* and Tr* lifetimes. In a comparison of PDE activation in the presence (with GTP) and absence (with GTPγS) of Tr* inactivation, PDE activation required more light (and was therefore less effective) when Tr* was inactivated in both rod and cone membranes. This is probably because inactivation of Tr* shortened its lifetime, thereby reducing the number of activated PDE molecules. The effect of Tr* inactivation was larger in cones, probably because the lifetime of Tr* is shorter in cones than in rods. The shorter lifetimes of Tr* and R* in cones seem to explain the remaining difference in the effectiveness of PDE activation between rods and cones.

Vertebrates have two types of visual photoreceptors, rods and cones, which convert light detection signals into electrical signals. Rods exhibit higher sensitivity to light than cones: in carp, light sensitivity is Ͼ100-fold higher in rods than in cones (1). Due to this sensitivity difference, rods function in the dark, and cones function in the light. Furthermore, light responses are much briefer in cones than in rods. As a result, the time resolution of a light stimulus is much higher in cones than in rods.
The mechanism that generates light responses is known as the phototransduction cascade, a process that has been well studied in rods (1)(2)(3). Briefly, visual pigment (R) 2 is activated by absorption of light, and activated visual pigment (R*) catalyzes the exchange of GDP for GTP in a trimeric GTP-binding protein, transducin (Tr). The GTP-bound form of Tr (Tr*) is the active form. Tr* activates cGMP phosphodiesterase (PDE), which hydrolyzes cGMP, resulting in a decrease in the cytoplasmic cGMP concentration. Consequently, cGMP dissociates from a cGMP-gated cation channel situated in the plasma membrane of a rod's outer segment; this closes the channel, thereby inducing a hyperpolarizing light response. After a light stimulus, all activated species (such as R* and Tr*) are inactivated. R* is inactivated by phosphorylation and subsequent binding of arrestin. Tr* is inactivated by hydrolysis of bound GTP to GDP via its intrinsic GTPase activity, which is accelerated by a photoreceptor-specific GTPase-accelerating protein, RGS9-1 (regulator of G-protein signaling 9-1) (4). In the rod phototransduction cascade, enormous signal amplification takes place; therefore, a rod is very sensitive to light. In cones, a similar cascade is present, but in many animal species, a different set of players in the cascade (for example, cone-type visual pigment, Tr, and PDE instead of their rod types) is known to be present. It is probable that the reactions in the cascade, including activation of R and Tr, and inactivation of R* and Tr*, are different between rods and cones, causing the sensitivity and time resolution to differ between rods and cones.
Studies of cone phototransduction mechanisms have been conducted in mice (for review, see Ref. 2). Genetic engineering, the method employed in these studies, is very effective for analyzing the cascade phenotypically. However, the expression levels and specific activities of the molecular components of the cone cascade are not known in mice. To understand the mechanisms underlying the differences between the rod and cone light responses quantitatively at the molecular level, it is necessary to understand the differences between each of the reactions in the rod and cone cascades. For this purpose, we have * This work was supported by Japan Society for the Promotion of Science established a system for examining these differences using purified rods and cones from carp (1).
In a previous study, using membrane preparations, we compared PDE activities between carp rods and cones in response to light flashes of various intensities (5). The maximum percent PDE activity elicited by a light flash (peak PDE activity) was plotted against the flash intensity. In the presence of ATP and therefore in the presence of R* phosphorylation, the light intensity required to evoke 50% of full PDE activity was ϳ220-fold higher in cones than in rods; by contrast, in this study, the difference was found to be ϳ40-fold (see "Results and Discussion"). The molar ratio of PDE to R is similar between rods and cones (Ref. 5 and this study); therefore, the aforementioned requirement of cones for a higher intensity of light means that the efficiency of PDE activation per photon is much lower in cones. This lower effectiveness of PDE activation in cones is due partly to the fact that activation of Tr by R* is 5-fold lower in cones than in rods (6). However, the total difference in the effectiveness of PDE activation between rods and cones is not accounted for by this 5-fold difference. Therefore, in this study, we tried to determine which reactions are responsible for the much lower effectiveness of PDE activation in cones relative to rods. First, we compared the efficiency of PDE activation by Tr* in rods and cones. We then examined the contributions of Tr* and R* lifetimes to peak PDE activity elicited by a light flash to determine how their lifetimes affect the effectiveness of PDE activation in rods and cones.

EXPERIMENTAL PROCEDURES
Preparation of Rod and Cone Membranes-Purified rods and cones from carp (Cyprinus carpio; 25-30 cm in length) were obtained in the dark as described previously (7). Animal care was conducted according to the institutional guidelines. Purified rods and cones were stored at Ϫ80°C until used. Before use, stored rods and cones were thawed and centrifuged (100,000 ϫ g, 20 min) twice, and the membranes were suspended in potassium gluconate buffer (115 mM potassium gluconate, 10 mM HEPES, 2.5 mM KCl, 2 mM MgCl 2 , 0.2 mM EGTA, 0.1 mM CaCl 2 , and 1 mM dithiothreitol, pH 7.5).
Light Source-A light flash (Sunpak Auto 25SR) or a 150-watt tungsten/halogen lamp was used to bleach visual pigments. In either case, a cutoff filter was used to pass light with wavelengths greater than 410 nm.
Purity of GTP␥S-Commercially available GTP␥S is contaminated with GDP (typically, 40% of GTP␥S). To quantitate the amount of GTP␥S as accurately as possible, the purity of each sample of purchased GTP␥S was checked by HPLC using a Mono Q column (SMART system, GE Healthcare) (8). The concentration of GTP␥S indicated is its actual calibrated concentration, and that of GDP is also the actual concentration (for both GDP added to the sample and GDP present as a contaminant in the GTP␥S reagent).
Preparation of GTP␥S-bound Form of Rod Transducin-The stably active form of rod Tr with GTP␥S-bound (rTr*-GTP␥S) was used to activate PDE. (GTP␥S or GTP binds to the ␣-subunit of Tr, and the GTP␥Sor GTP-bound form of the ␣-subunit is responsible for the activation of PDE. Here, for simplicity, we use the abbreviation Tr* to indicate the molecular species that activates PDE.) rTr*-GTP␥S was prepared and purified as described previously (9), with some modifications. Rods isolated and purified from 60 -100 carp retinas were homogenized and fully bleached for 5 min on ice using a 150watt tungsten/halogen lamp. Next, the homogenized rod membranes were washed twice with potassium gluconate buffer. The membranes were further washed with a low ionic strength buffer (buffer A: 5 mM HEPES, 0.5 mM MgCl 2 , and 1 mM dithiothreitol, pH 7.5) supplemented with 0.2 mM EDTA and subsequently washed three times with buffer A. The washed membranes were suspended in buffer A containing 10 M GTP␥S to form rTr*-GTP␥S and then centrifuged to obtain rTr*-GTP␥S in the supernatant. This extraction was repeated three times. Extracted rTr*-GTP␥S was concentrated, and the buffer was replaced with potassium gluconate buffer by ultrafiltration using a Vivaspin 20 filter (M r 10,000 cutoff, GE Healthcare). The amount of rTr*-GTP␥S was quantitated by Coomassie Brilliant Blue staining after SDS-PAGE using bovine serum albumin as a standard. Only three bands (molecular masses corresponding to those of the Tr ␣-, ␤-, and ␥-subunits) were visible upon Coomassie Brilliant Blue staining.
Quantification of Activated Transducin-When necessary, we quantitated the amount of Tr* using a [ 35 S]GTP␥S filter binding assay as described previously (6). Rod or cone membranes were first suspended in potassium gluconate buffer supplemented with 150 nM GDP to reduce the binding of GTP␥S in the dark (see below). This suspension was irradiated for 1 min using a 150-watt tungsten/halogen lamp (typically, 6 -8% of rhodopsin was bleached), and the suspension was mixed with a solution containing [ 35 S]GTP␥S, GDP, cGMP, and EGTA to obtain Tr*-GTP␥S. cGMP was added to quantitate the amount of Tr* under the same conditions used in the PDE activity measurements (see below). The final sample had a volume of 12 l and contained 1.5 M visual pigment, 5 mM cGMP, 0.8 mM EGTA, and 0.6 nM to 1.8 M [ 35 S]GTP␥S. GDP was also added at a concentration four times higher than that of GTP␥S; this was necessary because, in the measurement of the dark control, GTP␥S binding increased significantly in cone membranes when GDP was not added. In our previous study (6), the GDP concentration was 0.1 mM, equal to the GTP␥S concentration. By increasing the GDP:GTP␥S ratio to 4 in this study, the rate of GTP␥S binding in the dark was reduced to ϳ25% of the rate observed at a GDP:GTP␥S ratio of 1, but the maximum binding observed in the light did not change at all. In other words, the rate of GTP␥S binding in the dark was reduced by increasing the GDP:GTP␥S ratio. GTP␥S binding in the dark was terminated 60 s (rods) or 10 s (cones) after the addition of GTP␥S by adding 100 l of potassium gluconate buffer containing both 50 mM NH 2 OH to inactivate R* and 10 mM unlabeled GTP to terminate the apparent [ 35 S]GTP␥S binding. In the light, a similar manipulation was performed to terminate the reaction 60 s (rods) or 10 s (cones) after the addition of GTP␥S. Separate studies confirmed that GTP␥S binding in the light was completed by these times in both rod and cone membranes. The samples were then filtered through a pre-rinsed nitrocellulose membrane using a vacuum manifold. The material remaining on the filter membrane was washed three times with potassium gluconate buffer containing 25 mM MgCl 2 to remove unbound GTP␥S, and the nitrocellulose membrane was dried. The radioactivity of [ 35 S]GTP␥S remaining on the membrane was quantitated using a Fuji BAS 2000 image analyzer. The manipulations described above were carried out at 20°C.
PDE Activity Measurement-PDE activity was measured by the pH assay method as described previously (7). The pH decrease caused by hydrolysis of cGMP was monitored with a Microelectrodes MI-410 combination glass microelectrode. The concentration of cGMP hydrolyzed was calibrated using the pH decreases caused by full hydrolysis of known concentrations of cGMP.
Three types of PDE activity measurements were performed under slightly different experimental conditions as described below. All of the measurements were carried out at room temperature in a V-Vial containing 100 l of sample.
In the first type of measurement, PDE was activated by exogenous rod Tr* (rTr*-GTP␥S) prepared as described above. First, rTr*-GTP␥S was added to rod or cone membranes suspended in potassium gluconate buffer. One minute after this addition, PDE activity measurement was initiated by adding cGMP (5 mM final concentration) to suspensions containing (final concentrations) visual pigment (1.5 M), EGTA (0.8 mM), and rTr*-GTP␥S (0.025-1.68 M). PDE activity was determined from the rate of cGMP hydrolysis after the addition of cGMP. Without the addition of rTr*-GTP␥S, PDE was not activated even in the light.
In the second type of measurement, PDE was activated by known concentrations of endogenous Tr* in rod and cone membranes. Rod or cone membranes suspended in potassium gluconate buffer were illuminated for 1 min with a continuous light (bleaching 6 -8% of the pigment/min), and GTP␥S was then added at various concentrations (0.6 nM to 1.8 M), ranging from a concentration much lower than that of endogenous rod or cone Tr to a concentration much higher, to produce various concentrations of the stably active form of Tr (Tr*-GTP␥S). When the GTP␥S concentration was very low and lower than the concentration of endogenous Tr, the amount of Tr*-GTP␥S was limited by the amount of added GTP␥S (10). In other words, in this case, the concentration of Tr*-GTP␥S did not exceed the concentration of added GTP␥S. The sample was kept in the light for 1 min, and the PDE activity measurement was initiated by adding a solution containing cGMP. The reaction mixture contained 1.5 M visual pigment, 5 mM cGMP, 0.8 mM EGTA, 0.6 nM to 1.8 M GTP␥S (previously added), and GDP as a contaminant at a concentration 0.67 times higher than that of GTP␥S. PDE activity was determined from the rate of cGMP hydrolysis.
In the third type of measurement, PDE activity was compared in the presence of GTP or GTP␥S in rod and cone membranes. PDE activity was measured by applying a light flash of various intensities in the presence of 0.1 mM GTP or GTP␥S and in the presence and absence of ATP. When necessary, the HEPES concentration was reduced (1.5-2.2 mM) to expand pH decrease signals. The reaction mixture contained 0.75 M visual pigment, 2.5 mM cGMP, 0.8 mM EGTA, 0.4 mM GDP, 0.1 mM GTP or GTP␥S, and (when necessary) 0.25 mM ATP in potassium gluconate buffer. In this type of measurement, the pH decrease caused by hydrolysis of ATP or GTP was corrected.
Our purified cones retained large inner segments that probably contained the hydrolytic enzyme(s) for ATP and GTP. Our HPLC analysis showed that, typically, ATP was hydrolyzed at a rate of 9.0 M s Ϫ1 in the cone sample described above. In the presence of ATP, GTP concentrations did not change significantly, but in the absence of ATP, GTP was hydrolyzed, typically at a rate of 3.3 M s Ϫ1 . In rod membranes, hydrolysis of these nucleotides was not significant. During incubation with cone membranes, hydrolysis of ATP induced a pH decrease of the sample solution. Because hydrolysis of cGMP was monitored by the pH assay method, the pH decrease caused by ATP hydrolysis recorded in a control measurement was subtracted from the pH record of the measurement of cGMP hydrolysis when ATP was present. To minimize hydrolysis, nucleotides other than cGMP were added 10 -15 s before a light flash. Our control studies showed that when the PDE activity reached its peak (1.5-3 s in cone membranes and 3-8 s in rod membranes after a light flash), ATP was present at sufficient concentrations (Ͼ0.13 mM). In the presence of GTP, PDE activity gradually increased after a light flash, reached its peak, and then declined to the dark level. The peak PDE activity obtained in the presence of GTP was a function of the flash intensity and is referred to as peak PDE activity below. When GTP␥S was used, cGMP hydrolysis increased linearly with time, so this steady PDE activity was determined 10 -20 s after the flash in cone membranes and 45-55 s after the flash in rod membranes. Because PDE activity measured in the presence of GTP␥S was also a function of the flash intensity, we also refer to this steady activity as peak PDE activity. Although the sample contained 0.4 mM GDP, we confirmed that GDP affected GTP␥S binding almost equally in rod and cone membranes: in our previous experiments conducted in the presence of 0.1 mM GDP (6), the rate of GTP␥S binding was 5-fold lower in cone membranes than in rod membranes, and a similar difference (7-fold) was observed in 0.4 mM GDP in this study.
The range of pH decreases during measurements was Ͻ0.2 pH units. Full PDE activity elicited by light was measured in a sample of the same batch used for each measurement: in the second type of measurements, full PDE activity was measured by the addition of 10 M GTP␥S in the light, and in the third type of measurements, it was measured by exposing the sample to light of saturating intensity. PDE activity measured in each sample was normalized to the corresponding full PDE activity.
In our control study (data not shown), full PDE activity elicited by a saturating light flash was dependent on the membrane concentration. At rod pigment concentrations of 0.75 and 1.5 M, PDE activities (expressed as units/visual pigment present) were 28 Ϯ 3% (mean Ϯ S.E., n ϭ 3) and 32 Ϯ 3% (n ϭ 3), respectively, of the trypsin-activated PDE activity, which was essentially constant (17.4 Ϯ 0.7 cGMP hydrolyzed per visual pigment present per second, n ϭ 12) at all membrane concentrations examined (0.75-15 M). At 15 M pigment, full PDE activity in the light was slightly lower (89 Ϯ 3%, n ϭ 3), but it was similar to the activity of trypsin-activated PDE. Our results suggest that this reduced PDE activation in the light at low pigment (membrane) concentrations was probably due to elution of Tr* (ϳ65% and ϳ50% elution at 0.75 and 15 M pigment, respectively, based on quantitation by SDS-PAGE). The reduc-tion in PDE activity at low membrane concentrations was also observed in cone membranes: 34 Ϯ 4% (n ϭ 3) and 39 Ϯ 2% (n ϭ 3) at 0.75 and 1.5 M cone pigment, respectively. The elution of Tr* from cone membranes was difficult to quantitate because our purified cones contained many inner segment proteins that hampered the identification of cone Tr by SDS-PAGE. However, because the reduction of PDE activity at low pigment concentrations was similar in rod and cone membranes, we assumed that the elution of Tr* was similar in rod and cone membranes. Thus, this study was conducted under conditions in which Tr* was partly eluted from the membranes, so PDE was maximally activated to 30 -40% of the activity of trypsin-activated PDE in both rod and cone membrane preparations.

PDE Activation by Exogenous Rod Transducin in Rod and
Cone Membranes-To examine the activation efficiency of PDE by the active form of Tr in rod and cone membranes, we compared the activation of rod and cone PDE by exogenous rod Tr*. To this end, rod PDE in rod membranes and cone PDE in cone membranes were activated by rTr*-GTP␥S at various concentrations. Fig. 1 shows the PDE activity as a function of the concentration of added rTr*-GTP␥S in rod (circles) and cone (triangles) membranes. Although cone PDE exhibited a slightly higher affinity for rTr*-GTP␥S than rod PDE, the difference was not significant. This result indicates that rod Tr activates cone PDE as effectively as it activates rod PDE. The full PDE activities determined in the presence of saturating concentrations of rTr*-GTP␥S were similar in rod and cone membranes: 15.1 Ϯ 0.6 (n ϭ 8) cGMP hydrolyzed per visual pigment present per second in rod membranes and 15.0 Ϯ 0.9 (n ϭ 6) cGMP hydrolyzed per visual pigment present per second in cone membranes. These activities were very similar to that of trypsinactivated PDE.
Because the specific activities of single molecules of rod and cone PDE are similar (11), the similar maximum PDE activities in rod and cone membranes described above indicated that the number of PDE molecules per visual pigment is similar in rod and cone membranes, as shown previously (5). Thus, the results shown in Fig. 1 indicate that the number of PDE molecules activated by a single molecule of exogenous rTr*-GTP␥S was similar in rod and cone membranes. In other words, this result strongly suggests that rod Tr* activates rod and cone PDE with similar efficiency. Therefore, we next tried to compare the efficiency of activation of rod and cone PDE by endogenous Tr*.
PDE Activation by Endogenous Transducin in Rod and Cone Membranes-To examine the efficiency of activation of PDE by Tr* in rod and cone membranes, PDE in rod and cone membranes was activated by known amounts of endogenous Tr*, which was produced by adding limiting amounts of GTP␥S in the light. In most of these measurements, the concentration of GTP␥S used was significantly lower than the concentration of endogenous Tr molecules; therefore, under this condition, the number of Tr molecules activated (Tr*-GTP␥S) was limited by the amount of added GTP␥S. Because the sample contained 1.5 M visual pigment and the Tr:R ratio was ϳ1:10 (6), the Tr concentration in the sample was estimated to be ϳ150 nM.
First, we calibrated the amount of the GTP␥S-bound form of Tr. Fig. 2A shows the relationship between the concentration of GTP␥S added and the estimated concentration of GTP␥S bound to rod Tr (black circles) and cone Tr (red triangles) in the dark (closed symbols) and in the light (open symbols). Saturation in the ordinate at high GTP␥S concentrations is most probably because all GTP␥S molecules bound to Tr. Binding in the dark (closed symbols) was very low in rod membranes (closed black circles; Ͻ1% of the binding in the light) but up to 15% in cone membranes at the highest concentration (600 nM) of GTP␥S (closed red triangles). The reason for the high GTP␥S binding in the dark in cone membranes is not known, but it is possible either that cone pigments were thermally activated to induce GTP␥S-binding to Tr in the dark or that GTP␥S bound to proteins other than Tr. However, it was evident that most of the added GTP␥S bound to rod or cone Tr in the light because the measured binding curves fit well to the theoretical curve representing 100% binding of added GTP␥S (dashed blue curve).
In Fig. 2A, we estimated the total Tr*-GTP␥S concentration at various concentrations of added GTP␥S. Bearing this estimation of the Tr*-GTP␥S concentration in mind, we measured the PDE activity by varying the concentration of GTP␥S. Fig. 2 (B and C) shows the sample traces of measurements of cGMP hydrolysis in rod and cone membranes, respectively, in the absence (0 nM) and presence (18 or 600 nM) of GTP␥S in the light. Tr*-GTP␥S was produced before the addition of cGMP (see "Experimental Procedures"). Upon the addition of cGMP (arrows), the measurement was perturbed; several seconds after the addition, the traces of cGMP hydrolysis became linear. The rate of cGMP hydrolysis (PDE activity) was determined in this linear phase.
As indicated under "Experimental Procedures", a significant proportion of Tr* was eluted from the membranes at low pigment (membrane) concentrations. However, it was not clear how the eluted Tr* contributed to the activation of PDE. On the basis of the estimation of the Tr*-GTP␥S concentration in Fig.  2A, we plotted PDE activities as a function of total Tr*-GTP␥S (Fig. 2D). (The extent of elution of Tr* might have been similar between rod and cone membranes because the reduction in PDE activity at low pigment concentrations was comparable (30 -40%) in both types of membranes.) Because we were not certain whether the GTP␥S binding in cone membranes in the dark represented binding to Tr or to other proteins, the amount of Tr*-GTP␥S in cone membranes was estimated in both ways: closed red triangles are based on the total GTP␥S binding in the light, and open red triangles are based on the binding calculated from the light-dark differences in Fig. 2A. Because GTP␥S binding in rod membranes in the dark was almost negligible, only the binding in the light is shown in Fig. 2D (rod data; open black circles). The full PDE activity elicited by a light flash varied slightly in each measurement, so the PDE activity expressed on the ordinate was normalized to the full activity in each measurement, but the average of the activity in rod membranes was similar to that in cone membranes. It is evident from Fig. 2D that the efficiency of PDE activation (and therefore, the number of PDE molecules activated by a single molecule of endogenous Tr*-GTP␥S) was almost the same in rods and cones. In other words, the photon capture signal is transmitted to PDE from Tr at a very similar efficiency between rods and cones in the carp retina.
In our previous study (6), we quantitated the amount of Tr in rod and cone membranes by subtracting the GTP␥S-binding signals in the dark from those in the light. In that study, GTP␥S binding in cone membranes in the dark was not sufficiently inhibited. For this reason, the cone Tr:R ratio reported previously (0.055 Ϯ 0.004) could be lower than the actual value. As shown in Fig. 2A, the expression level of cone Tr, estimated from the GTP␥S-binding signal in this study, was very similar to that of rod Tr (rod Tr:R ϭ 0.094 Ϯ 0.003) (6).
Contribution of Tr* Lifetime to PDE Activation-The experiments described above demonstrated that each molecule of Tr* activates PDE at a similar efficiency in rods and cones. However, those experiments were performed with a stably active form of Tr (Tr*-GTP␥S). In the actual situation in living cells, Tr* molecules accumulate with time as long as the visual pigment is active, and each Tr* molecule has its own lifetime. For this reason, the maximum number of PDE molecules activated (and thus, the peak PDE activity elicited by a weak light flash) could be a function of the lifetimes of individual Tr* molecules activated and inactivated at different times after the light flash. In our previous study (6), we found that the lifetime of Tr* was 20-fold shorter in cones than in rods, probably due to higher expression of RGS9-1 in cones. Although the activation efficiency of PDE by Tr* could be almost the same in rods and cones, as shown in Fig. 2, the number of PDE molecules activated by the same number of Tr* molecules could be lower in cones than in rods under conditions in which Tr* is inactivated.
To test this possibility, we compared peak PDE activities under two different conditions: with GTP␥S in the absence of Tr* inactivation and with GTP in the presence of Tr* inactivation. We also assumed that the amounts of Tr* remaining in and eluted from the membranes were similar between rod and cone membranes. Fig. 3 (A and B) shows sample traces of PDE activity measurements by the pH assay method in the presence of ATP in rod (A) and cone (B) membranes with GTP␥S (thin red traces) or GTP (thick red traces). In Fig. 3A, a light flash bleaching 0.024% of the pigment in rod membranes was given at time 0, and in Fig. 3B, a light flash bleaching 0.46% of the pigment in cone membranes was given. First derivatives were extracted from these traces, and the time courses of PDE activity were determined (Fig. 3, C and D; obtained from Fig. 3, A and B,  respectively). In the presence of GTP␥S, PDE activity increased monotonically to a steady level (thin red traces in Fig. 3, C and  D), whereas in the presence of GTP, PDE activity reached a peak and then declined to the dark level (thick red traces in Fig. 3, C  and D). The pH traces were noisy, especially in the case of cone membranes (Fig. 3D). Therefore, we approximated peak PDE activities by eye (horizontal dashed blue lines in Fig. 3, C and D).
The measurements were made with light flashes of various intensities, and the peak activity relative to the full activity, i.e. the percentage of PDE molecules activated in the presence of ATP, was plotted against the flash intensity (Fig. 3E). The effectiveness of light was higher in the presence of GTP␥S (closed red symbols) than in the presence of GTP (open red symbols) both in rod (red circles) and cone (red triangles) membranes. This result indicates that the Tr* lifetime affects the number of PDE molecules activated by a light flash. However, as shown in Fig. 3E, in the case of rods, this effect was not very large, and the difference was almost negligible. By contrast, in cones, the effectiveness of PDE activation by light was decreased by ϳ2-fold in the presence of GTP relative to the presence of GTP␥S. This larger difference in cones may be due to the shorter lifetime of Tr* in cones than in rods, as predicted above. Thus, the shorter Tr* lifetime in cone membranes contributes a 2-fold reduction in the effectiveness of PDE activation.
The difference in the effectiveness of PDE activation in the presence of both ATP and GTP␥S between rod and cone membranes (Fig. 3E, closed red circles and closed red triangles) reflects the difference in the number of Tr* molecules activated by R* during the R* lifetime because Tr* is not inactivated in the presence of GTP␥S. This difference could therefore be due to the multiplied effects of the differences in both the activation of Tr by R* and the lifetime of R* in the presence of ATP. The overall difference was 21-fold (closed red circles and closed red triangles). The efficiency of Tr activation is 5-fold lower in cone membranes than in rod membranes (6). When this lower activation efficiency of Tr by R* is taken into account, we can predict that, in the presence of R* phosphorylation, the lifetime of R* is shorter in cones than in rods and that the shorter lifetime of R* in cones reduces the effectiveness of PDE activation by ϳ4-fold (21/5). The effectiveness of PDE activation in the presence of both ATP and GTP was 42-fold lower in cones than in rods (open red circles and open red triangles), and this difference was somewhat smaller than we observed previously (220-fold). We believe that this difference is due to the difference in the concentration of GDP that inhibits Tr activation (see "Experimental Procedures" and below).
Contribution of R* Phosphorylation to PDE Activation-The contribution of the lifetime of R*, described above, was estimated in the presence of R* phosphorylation. Therefore, it was of interest to determine how R* phosphorylation contributes to PDE activation. To estimate this contribution, we also measured peak PDE activities in the absence of ATP in rod and cone membranes in the presence of GTP␥S and thus in the absence of Tr* inactivation (black traces in Fig. 3, A-D). The measurements were made at various intensities of light flashes. In Fig.  3F, the relationship between flash intensity and peak PDE activity was plotted for rod (black circles) and cone (black triangles) membranes, and these data were compared with the results obtained in the presence of both ATP and GTP␥S shown in Fig.  3E (dashed lines, replotted from unbroken lines in E). Because PDE peak activities were measured in the presence of GTP␥S in these studies, the difference in the effectiveness of PDE activation should be due to the difference in the amount of Tr* produced during the lifetime of R* in the presence (ϩATP) and absence (ϪATP) of R* phosphorylation. In the absence of R* phosphorylation, relative to its presence, the effectiveness of activation increased by 3.8-fold in rod membranes and by 9.5fold in cone membranes (arrows). Apart from the fact that the lifetime of a bleaching intermediate is shorter in cones than in rods in living cells (12), the effectiveness of R* phosphorylation was 2.4-fold (9.5/3.8) larger in cone membranes.
In summary, under a pseudo-intracellular condition in which R* phosphorylation is present, the difference in the effectiveness of PDE activation between rods and cones, determined by FIGURE 3. Contributions of Tr* and R* lifetimes to PDE activity. A, sample traces of PDE activity measurement by the pH assay method in rod membranes containing 0.75 M pigment. A light flash bleaching 0.024% of the pigment was given at time 0 in the presence of ATP (red traces) and either GTP (thick red trace) or GTP␥S (thin red trace). Measurements were also made in the absence of ATP with GTP␥S present (black trace). B, PDE activity measurement in cone membranes containing 0.75 M pigment. As in A, a light flash bleaching 0.46% of the pigment was given at time 0 in the presence of ATP (red traces) with either GTP (thick red trace) or GTP␥S (thin red trace) or in the absence of ATP with GTP␥S (black trace). C and D, PDE activation and inactivation time courses. PDE activities in rod membranes (C) were determined from the first derivatives of the traces in A, and those in cone membranes (D) were determined from B. Peak PDE activity was determined by eye (dashed blue lines). E, contribution of Tr* inactivation (GTP hydrolysis) to peak PDE activity in the presence of ATP in rod and cone membranes. PDE activity measurements in the presence of ATP (see sample red traces in A-D) were made at various intensities of light flash, and the relationship between flash intensity and peak PDE activity was plotted for the results obtained in the presence of GTP␥S (closed symbols) or GTP (open symbols) in rod (red circles) and cone (red triangles) membranes. F, PDE activity measurements in the absence of ATP with GTP␥S present (see sample black traces in A-D) were also made at various intensities of light flash, and the relationship between flash intensity and peak PDE activity was plotted for rod (black circles) and cone (black triangles) membranes. The results of PDE activity measurements in the presence of ATP and GTP␥S are replotted from E (dashed lines): to avoid crowding, only the unbroken lines are shown. Arrows show the increase in effectiveness of PDE activation in the absence of R* phosphorylation. In E and F, the results are the mean Ϯ S.E. (n ϭ 3). peak activities, was found to originate from three steps in the phototransduction cascade. First, at the stage when Tr* is generated by R*, the signal amplification is 5-fold lower in cones than in rods (6). Second, in the presence of R* phosphorylation, the shorter lifetime of R* in cones decreases the effectiveness of PDE activation by 4-fold. Third, the shorter lifetime of Tr* in cones decreases the effectiveness by 2-fold. Overall, due to lower amplification and shorter lifetimes of R* and Tr*, the effectiveness of PDE activation was lowered in cones to ϳ1/40 (1/5 ϫ 1/4 ϫ 1/2, multiplied effect of lower Tr* activation, faster R* phosphorylation, and faster Tr* inactivation) of the effectiveness in rods.
Signal amplification is comparable between mammalian rods and cones, although this similarity does not seem to hold in amphibian rods and cones (3). In mice, signal amplification may be lower in cones than in rods (13). It may be the case that the rate of the reaction in the phototransduction cascade is different between rods and cones and that the extent of this difference depends on the species.

Mechanism of PDE Activation by Transducin in Rods and
Cones-PDE is activated by displacement of the inhibitory ␥-subunit of PDE (PDE␥) from its catalytic subunit (14). The stoichiometry of removal of PDE␥ by Tr* is 1:1 (15). Because Tr*-GTP␥S activated PDE at similar efficiencies in rods and cones (Fig. 2D), cone Tr* probably removes PDE␥ of cone PDE at 1:1 stoichiometry.
However, this does not indicate that all Tr* molecules bind to PDE␥. As shown in Fig. 2D, maximum PDE activation was observed at ϳ100 nM Tr*-GTP␥S. In this measurement, 66% of Tr*-GTP␥S could have been eluted from the membrane, and 34% remained in the membrane (see "Experimental Procedures"). Then, the Tr*-GTP␥S concentration remaining in the membrane at 1.5 M visual pigment was ϳ30 nM. In case the eluted Tr*-GTP␥S does not have activity to activate PDE, ϳ30 nM is the lower limit of the concentration of Tr*-GTP␥S that contributes to activate all of the PDE molecules present in the membrane. This concentration is higher than the concentration of PDE in the membrane: assuming that the molar ratio of holo-PDE to visual pigment is 1:270 in rods (16), at 1.5 M visual pigment, the holo-PDE concentration is estimated to be 5.6 nM, or the concentration of the catalytic subunit of PDE is estimated to be 11.2 nM. This calculation shows that a molar excess of Tr*-GTP␥S is required for activation of PDE. A similar observation was reported in a previous study of rod membranes (10).
In this study, the effectiveness of PDE activation was 42-fold lower in cone membranes than in rod membranes in the presence of both ATP and GTP. This difference in the effectiveness of PDE activation was lower than the value we reported previously (220-fold) (5). The reason for this inconsistency is not clear, but it is possible that, in our previous study, GTP hydrolysis in cone membranes was much higher than in rod membranes; the resultant GDP, which inhibits Tr activation (see "Experimental Procedures"), may have reduced the effectiveness of Tr* production in cone membranes.
In this study, we showed that the efficiency of PDE activation by Tr* is similar between rods and cones. However, due to the shorter lifetimes of R* and Tr* in cones, PDE activation becomes less effective in cones. Although the quantitative contribution of the reduced effectiveness of PDE activation to the reduced light sensitivity in cones needs to be determined, it is evident that the reduced rate of cGMP hydrolysis tends to shorten the light response time to peak, i.e. the time at which the hydrolysis and the synthesis of cGMP are balanced. Because photoreceptor light sensitivity is defined as the amplitude of a peak response, shortened time to peak contributes to lower light sensitivity in cones than in rods.