light-scattering changes (rod outer segment/retina/gtpase/transducin/light-activation)

Transcription

1 Proc. Natl Acad. Sci. USA Vol. 78, No. 11, pp , November 1981 Biophysics Interactions between photoexcited rhodopsin and GTP-binding protein: Kinetic and stoichiometric analyses from light-scattering changes (rod outer segment/retina/gtpase/transducin/light-activation) H. KUHNt, N. BENNETT, M. MICHEL-VILLAZ, AND M. CHABRE Laboratoire de Biologie Mol6culaire et Cellulaire, Equipe de Recherche, Centre National de la Recherche Scientifique, and D6partement de Recherche Fondamentale, Centre d'etudes Nucl~aires de Grenoble 85 X, Grenoble, France Communicated by John E. Dowling, July 27, 1981 ABSTRACT In rod outer segments, photoexcited rhodopsin (R*) activates a cyclic GMP phosphodiesterase through a sequence of reactions involving a GTP-binding protein. By measuring lightscattering changes above 700 nm, we have studied the kinetics and stoichiometry of the association of R* with this protein and of the dissociation of the complex upon GDP/GTP exchange. Two lightscattering signals were obtained upon photoexcitation of rhodopsin in bovine rod outer segment membranes as weil as in a reconstituted system consisting of purified GTP-binding protein and washed disc membranes; both signals depended specifically on the presence of GTP-binding protein. A "binding signal" that was observed in the absence of GTP as an increase in turbidity became saturated when a number of rhodopsin molecules equal to the number of GTP-binding protein molecules present (~=10% in rod outer segments) has been bleached, suggesting that the protein binds to R* in a 1:1 complex. A "dissociation signal" of opposite sign, observed in presence of GTP at.1,um, is half maximal at 0.04% bleaching and saturated at 0.5% bleaching; it is interpreted as reflecting the dissociation of GTP-binding protein-r* complexes after GDP/GTP exchange on the GTP-binding protein, one R* being able to interact sequentially with about 100 GTP-binding protein molecules. The early time course of the binding signal is faster than that of the dissociation signal, and both signals take place in the 100-msec range at 20rC. Photoexcitation of rhodopsin (to R*) in rod outer segments (ROS) activates a cyclic GMP phosphodiesterase (PDEase) (1, 2) leading to the hydrolysis of up to 4 x 10' molecules of cyclic GMP per molecule of bleached rhodopsin (3). This results from a cascade of steps involving GDP/GTP exchange on a GTPbinding-protein which has slow GTPase activity (1, 4-6). This protein consists ofthree polypeptides OfMr 37,000, 35,000, and =6000 (5). The name "transducin" has recently been proposed for this protein which, in its GTP-binding form, activates the PDEase (2). Both the GTP-binding protein and the PDEase are peripherally membrane associated at moderate ionic strength ( mm salts) but can be solubilized at low ionic strength ('10 mm) (7, 8). It has been shown by centrifugation studies (5) that light changes the mode of binding of the GTP-binding protein to the disc membrane; proteolytic studies strongly suggest that the light-induced binding site is located on the rhodopsin molecule (9). GTP specifically reverses this light-induced binding (5). However, these binding studies as well as the biochemical GDP/GTP exchange studies (2, 4, 6) do not provide information on the temporal sequence and kinetics of these reactions. In the present study, we demonstrate that monitoring light- The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact scattering changes ofros membrane suspensions in the far red is a highly sensitive technique with fast time resolution which provides insight into the sequence, kinetics, and stoichiometry of these interactions. A number of light-scattering studies on photoreceptor membranes have already been reported (10-13), leading to various interpretations, as for instance light-induced "rhodopsin cooperativity" (11). The involvement of proteins other than rhodopsin in these light-scattering signals was notconsidered except by; Bignetti et al (13). Using biochemically well-defined preparations including reconstituted systems with purified proteins, we show that two light-scattering signals evoked by photoexcitation of rhodopsin are strictly related to light-induced changes ofinteraction between GTP-binding protein and R*. The first signal, termed "binding signal," is observed in the absence of GTP; it becomes saturated at ca. 10% bleaching and appears to reflect the binding of GTP-binding protein to R* in a 1:1 complex. The second signal, termed "dissociation signal," occurs in the presence of GTP; it becomes saturated at about 0.5% bleaching, and it most probably reflects the dissociation of the GTP-binding protein-r* complex after GDP/GTP exchange. Another signal, termed "rhodopsin signal," is observed after saturation of the GTP-binding protein-related signals and also in disc membranes washed free of GTP-binding protein; it does not become saturated until all of the rhodopsin is bleached. All three signals occur in the 100-msec range which is within the time of the electrical response of the rod cell. MATERIALS AND METHODS All experiments were performed in darkness or dim red light. The "standard buffer" used for all light-scattering experiments was 100 mm KCV1 mm MgClJ10 mm Tris HCl, ph 7.4. All buffers used contained 1 mm dithiothreitol. ROS Membrane Preparation. ROS were purified from fresh bovine retinas as described (14) and were stored frozen as pellets at - 70 C under argon. Thawed pellets were homogenized (Teflon/glass) with buffer followed by passage through a syringe needle. With standard buffer, this treatment yielded suspensions of fragmented ROS consisting of irregular-sized stacks of discs termed "ROS membranes." Further fragmentation by sonication decreased the absolute turbidity but did not strongly influence the relative amplitudes of the light-induced lightscattering signals. Rhodopsin concentrations were.determined in detergent-solubilized aliquots by measurement ofaas0o (14). Abbreviations: ROS, rod outer segments; PDEase, cyclic GMP phosphodiesterase; R*, photoexcited rhodopsin; p[nh]ppg, guanosine 5'- [,y-iimido]triphosphate; p[s]pg, guanosine 5'-[(3thio]diphosphate. t Present address: Institut fir Neurobiologie der Kernforschungsanlage Julich, Postfach 1913, D-5170 Julich, Fed. Rep. Germany.

2 U60074 Biophysics: Kfihn et al. Washed Discs and Total Extract. ROS (4 mg of rhodopsin per ml) were homogenized in 10 mm Tris HCl (ph 7.4) and centrifuged (30 min at 80,000 x g). The clear supernatant is termed "total extract." The pellet was resuspended and centrifuged twice again in 10 mm Tris-HCl (0.5 mg of rhodopsin per ml). The resulting "washed discs" were homogenized in standard buffer. Other Preparations of Extractable ROS Proteins. Purified GTP-binding protein (see upper gel in Fig. 1B Inset) and purified PDEase were prepared as described (5, 8). GTPase activity was assayed as described (5, 8). The concentration ofgtpbinding protein in extracts was determined by densitometry of its polypeptide bands on Coomassie blue-stained gels (15); rabbit muscle aldolase (Sigma) was used as a calibration standard (SD, ±5%). Light-Scattering Measurements. Flash-induced changes of light-scattering were recorded as optical transmission changes at 708 nm (1.2 nm band width) in a Durrum D 117 spectrometer equipped with a Data Lab DL 905 transient recorder of L-msec time constant. The sample, suspended in standard buffer, was contained in a thermostated (20TC) 1 x 1 cm cuvette. The photomultiplier window was 7.5 cm from the sample and subtended an angle of 120. Exposing the sample to the measuring beam FIG. 1. Light-induced signals in ROS membranes and in a reconstituted system containing purified GTP-binding protein (G). Lightscattering changes were observed as transmittance changes at scattering angle (Measurements at 90 scattering angle yielded signals of opposite sign.) An amplitude change of 20 mv corresponds to a relative change in transmittance, ATIT, of 2 x Rhodopsin concentrations were 5-7 pm. (A) Binding signals in absence of GTP. Tracings a-d, ROS membrane suspension subjected to four consecutive flashes, each bleaching 4% of the rhodopsin. Tracings e and f, rapid recordings after bleaching 10% of the rhodopsin in ROS membranes (e) and in a reconstituted system of washed discs and purified GTPbinding protein (f). Tracing g, washed discs alone (8% bleached). The sharp peak at the start of the tracings is the flash artifact. (B) Dissociation signals in presence of 17 pm GTP, inducedby a flash bleaching of 0.8% of the rhodopsin in ROS membranes (tracing h) or in a mixture of washed discs and purified GTP-binding protein (tracing i). A second flash (tracing h) evoked no further dissociation signal. Tracing k, washed discs in presence of GTP but absence of GTP-binding protein (0.8% bleach). (Inset) NaDodSO4polyacrylamide gels (15) showing washed discs [bottom gel; 7,gg of rhodopsin (R)I, total extract (middle gel; amount corresponding to 40 tg of rhodopsin in unextracted ROS), and purified GTP-binding protein (top gel; 7 ug of protein). Numbers on gels indicate Mr 1;03. Proc. Natd Acad. Sci. USA 78 (1981) for up to 20 min did not influence the light-evoked signals. Photoexcitation was performed with an electronic flash (Sunpak Autozoom 3400; msec), at 900 from the measuring beam, through a Balzers K3 interference filter (Am., 500 nm) supplemented with 1 cm of 15% CuS04 in 0.25 M H2SO4 and neutral density filters when necessary. The photomultiplier was protected from the flash by an interference filter (Am., 708 nm). Calibration of bleaching extents was obtained from spectrophotometric measurements on ROS suspensions. RESULTS AND DISCUSSION Binding Signal in ROS Membranes. Flash illumination of a suspension of ROS membranes in the absence of GTP led to a rapid decrease in the transmitted light intensity, termed "binding signal" in this study (Fig. 1A). Its total amplitude was roughly proportional to the amount of rhodopsin bleached by the flash, ifless than about 10% ofthe rhodopsin was bleached. A series of consecutive flashes delivered on the same ROS membrane suspension, each flash bleaching 4% of the rhodopsin, evoked no more than three consecutive signals, the response becoming saturated with the third flash. Further flashes produced small rapid signals (Fig. LA, tracing d), termed ''rhodopsin signal," which differed from the binding signal by their opposite sign, much smaller amplitude, and. the fact that they did not become saturated until all of the rhodopsin was bleached. The binding signal resembles in many respects the so-called P-signal, and the rhodopsin signal resembles the N- signal described by Hofmann et al (10). The amount of rhodopsin bleached at saturation of the binding signal (ca. 10%) is approximately equimolar to the amount ofgtp-binding protein present in ROS (8); this suggests that the binding signal may reflect a 1:1 stoichiometric association of GTP-binding protein with R*, reminiscent of the binding observed in centrifugation studies (5). The time course of the binding signal was complex, consisting of rapid ( msec) and slow (several seconds) components. The half-time of the rapid phase (Fig. 1A, tracing e) depended on the flash intensity. At 20'C, it ranged from ca. 25 msec (10% bleach) to 60 msec (1.3% bleach). The time course was slowed by freezing/thawing or by transient hypoosmotic shock of the ROS suspensions, as well as by strong sonication. The time course ofthe rapid phase ofthe binding signal always was slower than that of metarhodopsin II formation. Dissociation Signal in ROS Membranes. Flash illumination of ROS membranes in the presence of GTP led to an increase in the transmitted light intensity, the dissociation signal (Fig. 1B, tracing h). It increased with a sigmoidal shape, and slow phases like those seen in the binding signal normally were absent; only ROS preparations that had been treated by repeated freezing or by hypoosmotic shock often showed a slow decrease in transmittance after the dissociation signal. Previous binding studies (8) performed under ionic. conditions similar to those used here have shown that GTP does not merely reverse the light-induced binding of the GTP-binding protein to R* but it leads to a new state of solubility ofthe GTPbinding protein, different from the dark-adapted state before the flash. The GTP-binding protein becomes partially dissociated from the membrane upon illumination in presence of GTP. This fits with the observation (Fig. 1) that GTP does not merely suppress the binding signal but leads to a new signal of opposite sign which we have therefore termed "dissociation signal." Signals in Reconstituted System. Hypoosmotic washing of ROS kept in the dark removed a component required for both the binding signal and the dissociation signal. The washed disc membranes exhibited only the rhodopsin signal (Fig. 1B, trac-

3 Biophysics: Kuhn et al ings g and k). These discs were nearly devoid of GTP-binding protein and GTPase activity (5). Addition ofpurified GTP-binding protein to the washed discs restored both the binding signal in the absence of GTP and the dissociation signal in the presence of GTP (Fig. IA, tracings f and i). The signals obtained in the reconstituted system closely resembled the corresponding signals in ROS with respect to their saturation behavior. The reconstituted binding signal had a similarly complex time course, consisting of rapid and slow phases, as the binding signal in ROS membranes. Under the ionic conditions used (standard buffer), both the GTP-binding protein and the PDEase become membrane-reassociated when their solutions are added to disc membrane suspensions (9). When total extract was used rather than purified protein, the binding signal often was slower and the dissociation signal was followed by the slow decrease in transmittance described above. These complications may be due to irregularities in the reconstitution process. The following preparations did not give binding signals or dissociation signals when added to washed discs: 10 mm Tris HCl extract from illuminated ROS from which GTP-binding protein was absent due to its light-induced binding to the membranes but PDEase was present (5); extract containing the proteins that are soluble in standard buffer but not GTP-binding protein or PDEase; purified PDEase. This clearly demonstrates that both the binding signal and the dissociation signal specifically reflect light-induced interactions between GTP-binding protein and the disc membrane. We further argue that the binding signal reflects the association of GTP-binding protein with R* and that the dissociation signal reflects the GTP-induced dissociation of the complex. Saturation of Binding Signal in Reconstituted System. Saturation is defined as the level of R*, reached by a succession offlashes, beyond which a further flash evokes no further binding signal. Extracted GTP-binding protein was mixed with washed disc membranes in various ratios in order to determine the stoichiometry of interaction between R* and GTP-binding protein with more accuracy than is possible in ROS membranes. No more than 1 min separated subsequent flashes in order to avoid recovery from saturation. Whatever the amount of GTPbinding protein added, from 0.25 to 4 times the native GTPbinding protein/rhodopsin ratio, saturation always was reached when the molar amount ofbleached rhodopsin was equal to that of GTP-binding protein present (Fig. 2). The total amplitude, including slow phases, accumulated for the sum of the signals evoked until saturation was proportional to the amount of GTP-binding protein present in the mixture (Fig. 2, Lower Inset). This indicates that the total amplitude of the binding signal is a quantitative measure of binding. The saturation is independent of the total amount of rhodopsin but depends only on the amount ofgtp-binding protein over a wide range of mixing ratios. Addition of more GTP-binding protein (Fig. 2, Upper Inset) to a mixture already flash-bleached until close to saturation, led to the appearance offurther binding signals in response to further flashes. This again demonstrates the additive behavior of the interaction between GTP-binding protein and R*. Saturation of the Dissociation Signal in Presence of GTP. The saturation of the dissociation signal is in sharp contrast to the 1:1 stoichiometric relationship of the binding signal. The amplitude- of the dissociation signal reached its maximum at flash intensities that bleached less than 0.8% of the rhodopsin in ROS membranes (Figs. 3 and 4A). Furthermore, a second flash delivered after a-first flash bleaching 0.8% or more did not evoke a subsequent signal except the small rhodopsin signal. This shows that, in the presence ofgtp, the system is saturated Proc. Nad Acad. Sci. USA 78 (1981) 6875 Z 200 Q p %/cbleach.30 Wl C) x~~~~~~~~~~~~~~~~ 200P s0io 1.5% aded to contan nmol amutgfwse G PROTEIN icmmrns(. o FIG. 2. Dependence of saturation of the binding signal upon the amount of GTP-binding protein present in the reconstituted system. Various amounts of total extract containing GTP-binding protein were added to constant amounts of washed disc membranes (4.8 nmol of rhodopsin per sample). The suspensions were illuminated by a succession of flashes, each bleaching 1.9% of the rhodopsin, until a subsequent flash no longer evoked a measurable binding signal. The total amount of rhodopsing bleached when this saturation was reached (determined within 10% accuracy) is plotted against the amount of GTPbinding protein in the mixture. The linearity of this plot demonstrates a 1:1 stoichiometry of GTP-binding protein to R* at saturation. The circled data point corresponds to a mixing ratio of 1 GTP-binding protein molecule for 10.7 rhodopsins, close to the natural stoichiometry, and saturation was reached with this sample for 11% bleaching (0.53 of 4.8 nmol), as in ROS. (Lower Inset) Dependence of the total amplitude of the binding signals, summed up until saturation, upon the amount of GTP-binding protein in mixtures with various amounts of rhodopsin. For the lowest data point, saturation was reached with two flashes; for the highest, one had to sum the amplitudes of 31 signals. T, 4.8 nmol rhodopsin, same data as in main figure; v,2.4 mol; v, 9.5 nmol; a,19 nmoi. This plot demonstrates that the total amplitude of the binding signal at'saturation is proportional only to the amount of GTP-binding protein and is independent of the amount of total rhodopsin over a wide range of rhodopsin/gtp-binding protein ratios around the natural stoichiometry. (Upper Inset) Effect of addition of GTP-binding protein after saturation. Two identical mixtures of washed discs and -an extract containing GTP-binding protein were flashedi(bleaching, 4% per flash) until close to saturation. One sample (e)was further flashed to control that saturation was reached. To the other (n), another aliquot of GTP-binding protein was added before further flashing, giving rise to another series of binding signals. Ordinate is total accumulated amplitude. at bleaching levels below 0.8%. It is important to note that the second flash evoked neither a second dissociation signal nor a binding signal although the level of bleached rhodopsin (0.8%) was far below the level of saturation of the binding signal (ca. 10%). This indicates that, after the first small flash, all of the GTP-binding protein molecules are already transformed into a form that is unresponsive to further flashes, and it leads to the following interpretation ofthe dissociation signal. GTP-binding

4 6876 Biophysics: 'Uhn et al. FIG. 3. Saturation of dissociation signal. Each tracing represents a separate sample of ROS membranes (6.6 pm rhodopsin; 17 /M GTP) subjected to a series of flashes (indicated by the arrows). The flash intensity was constant within each series. The fraction of rhodopsin bleached per single flash is indicated at each tracing. protein which normally contains tightly bound GDP (4, 6) associates first with R*; this is followed, in the presence of GTP, by an exchange of GTP for the bound GDP (4, 6). The GTPbinding form of the protein must have a low affinity to R*. This leads to its dissociation from R*, revealed as the dissociation signal, and to its unresponsiveness to further flashes yielding neither dissociation nor binding signals. Because bleaching only 0.8% ofthe rhodopsin obviously transforms all ofthe GTP-binding protein into this unresponsive form, one has to conclude that one R* can turn over many GTP-binding protein molecules. Bleaching less than 0.1% of the rhodopsin per flash resulted in responses of submaximal amplitude, followed by further small responses upon further flashes, until the final saturating level of total amplitude was reached (Fig. 3). For example, a flash bleaching of 0.08% led to a dissociation signal of 80% of the maximal amplitude, bleaching of 0.04% led to 50% of max- FIG. 4. Dependence of dissociation signal in ROS membranes on flash intensity and GTP concentration. Each tracing represents one sample. (A) Constant GTP concentration (17 jum) but varied bleaching extents (% R*) as indicated at each tracing. Rhodopsin concentration was 6.6AM. (B) Constant flash intensity (bleaching4% in each sample) but varied GTP concentration as indicated above each tracing. Rhodopsin concentration was 3.9 pm. Proc. Natl. Acad. Sci. USA 78 (1981) imum, and bleaching of 0.008% led to 10% of maximum. Assuming that the amplitude measured at the first flash is approximately proportional to the number ofgtp-binding protein molecules turned over after the flash and using the ratio of molecule of GTP-binding protein present per 10 rhodopsins, we estimate that, in each ofthe three examples given, photolysis ofrhodopsin leads to the turnover of molecules ofgtpbinding protein and therefore to GDP/GTP exchanges. Several experiments confirm that, once photoexcited, R* is able to react in the dark with many GTP-binding protein molecules for some time. (i) Bleaching 0.8% rhodopsin in ROS membranes in the absence ofgtp and then adding GTP shortly before a second flash yielded neither a dissociation signal nor a binding signal upon the second flash, indicating that all of the GTP-binding protein had been turned over after the addition ofgtp before the second flash. (ii) A mixture ofdisc membranes and extract containing GTP-binding protein, similar to that shown in Fig. 2 Upper Inset, was flashed (0.8% R*) in presence of GTP, leading to a dissociation signal. A second aliquot ofboth GTP binding protein and GTP was then added; however, a subsequent flash did not evoke any further signal, indicating that all of the freshly added protein had been- turned over into the GTP-binding form before the second flash. Kinetics of the Dissociation Signal. The time course strongly depended on both the flash intensity (Fig. 4A) and the GTP concentration (Fig. 4B). The half-time of rise at 17,uM GTP (20TC) ranged from 80 msec at 10% bleaching to =1 sec at 0.08% bleaching and even slower at lower bleaching extents (Fig. 3). Diffusion of proteins within the disc membrane may be rate limiting. The signal rose faster at higher GTP concentration. At very low GTP ('2 MM), the response started with a binding signal and was followed by a delayed dissociation signal, the amplitude and delay of which depended on the GTP concentration. This separation of the two signals may be due to one (or both) ofthe following reasons. (i) At low GTP concentrations, the associated complex between R* and GTP-binding protein has a sufficiently long life-time, and therefore may accumulate to a sufficient extent, that it is revealed as a transient binding signal, before it reacts with GTP. (ii) On the other hand, because the concentration of GTP ( ,u M) is not much higher than that of GTP-binding protein (0.4 A.LM), it is also possible that the two signals reflect two different portions of the GTP-binding protein, one which undergoes GDP/GTP exchange and another one which only binds to R* and never reacts with GTP. Whichever of the two possibilities predominates in this special case, we assume that the association between GTP-binding protein and R* always precedes the GDP/GTP exchange. The fact that at higher GTP concentrations no transient binding signal is seen indicates that the life-time ofthe associated complex must be very short and its steady-state concentration therefore too low to be noticeable as a light-scattering transient. The onset of rise of the dissociation signal was never observed to be faster than that of the binding signal at the same flash intensity. It should be stressed that only the very early time course of the binding signal is of interest in this context because only a very small proportion of R* is needed to catalyze the GDP/GTP exchange. Nucleotide Specificity. The dissociation signal appeared to be specific for GTP and its y-blocked analogue, guanosine 5'- [(3, y-imido]triphosphate (p[nh]ppg). This analogue yielded slightly modified signals, resembling dissociation signals obtained at lower GTP concentrations; this appears to reflect a lower affinity of p[nh]ppg. Neither cyclic GMP nor ATP (up to 200,tM) yielded dissociation signals. GDP (>50,uM) yielded signals resembling dissociation signals obtained at very low GTP concentrations, probably due to trace amounts of GTP present

5 Biophysics: Kuhn et al in the GDP or formed during incubation. The GDP analogue guanosine 5'-[,f3thio]diphosphate (p[s]pg), which is not metabolized (16) and therefore free of GTP, yielded normal-appearing binding signals (even at 800 ILM p[s]pg). The rise time of the dissociation signal was significantly slowed when excess GDP or p[s]pg was added together with the GTP, suggesting competition among the nucleotides. Requirement of Previously Bound GDP for the Dissociation Signal (but not for the Binding Signal). Extensive washing of ROS with buffered 100 mm KCV3 mm MgCl2 removes the bound nucleotides and the soluble proteins, whereas most of the GTP-binding protein and PDEase remain membrane-associated (8). Such membranes yielded normal binding signals in the absence of added nucleotides, indicating that R* associates with the GTP-binding protein regardless of whether or not it contains bound GDP. In the presence ofp[nh]ppg, however, these membranes yielded neither a dissociation signal nor a binding signal. This lack ofresponse suggests that, before the flash, the p[nh]ppg binds to the vacant binding sites on the GTP-binding protein such that the system is "saturated" before the flash. On the other hand, when low concentrations of p[s]pg or GDP were added before the addition of p[nh]ppg, a flash evoked a dissociation signal. This demonstrates that the light-triggered exchange of p[nh]ppg for bound GDP is an obligatory step for the appearance of the dissociation signal. Recovery from Saturation. When sufficient time (tens of minutes) was allowed to elapse after a saturating flash, a subsequent flash again evoked a response. This spontaneous "recovery from saturation" was observed for both the binding signal and the dissociation signal. Recovery of the dissociation signal was highly accelerated by the presence ofatp and kinase-containing extract. CONCLUSION By using reconstituted systems, we have demonstrated that two pronounced light-scattering signals evoked by photoexcitation of rhodopsin in ROS membranes are related to interactions of the peripheral GTP-binding protein with R*. The results are summarized in the scheme shown in Fig. 5. R* has a high affinity for the dark-adapted, GDP-binding (4, 6) form of GTP-binding protein. This leads to the formation, revealed by the binding signal, of a 1:1 stoichiometric complex GTP-binding protein-r* GDP which is stable for tens of minutes in the absence of GTP. In the presence of GTP, however, rapid exchange of bound GDP for GTP takes place, followed by rapid dissociation of the putative complex GTP-binding protein-r* GTP into R* and GTP-protein which is revealed by the dissociation signal. After dissociation R* is recycled up to about 100 times, binding further GDP-protein molecules and catalyzing GDP/GTP exchange on them. This amplification number, 100, compares well with published biochemical GDP/ p[nh]ppg exchange data [ranging from 71 to 500 exchanges per R* (2, 6)], as well as with PDEase activation data suggesting that 500 PDEase molecules can be activated through 1 R* (3). After dissociation from R*, GTP-protein activates the PDEase (2) until the GTP is hydrolyzed later on. The energy required for the multiple interactions ofone R* with many GDP-protein Proc. NatL Acad. Sci. USA 78 (1981) 6877 VJN d \ p. RGDP GG F'IG. 5. Scheme of reaction cycle of R* leading to amplified GDP/ GTP exchange. The light-scattering signals that accompany the different reaction steps are: 1, rhodopsin signal; 2, binding signal; 3, dissociation signal. For details see text. PDE, PDEase; GGDP, GTP-binding protein in the GDP-binding form; R. R*. molecules, leading to amplified GDP/GTP exchange and therefore to amplified PDEase activation, seems to be provided by a high-energy state of GDP-protein due to prior hydrolysis of GTP rather than by the photon energy absorbed by rhodopsin. We thank 0. Mommertz and C. Roche for technical assistance. This work was supported by grants from Centre National de la Recherche Scientifique (contrat ATP Internationale 1979), Fondation de la Recherche Medicale (to N.B.), and Dedegation Gen~rale a la Recherche Scientifique et Technique (Comite Membranes Biologiques). 1. Wheeler, G. L. & Bitensky, M. W. (1977) Proc.Natj Acad. Sci. USA 74, Fung, B. K. K., Hurley, J. B. & Stryer, L. (1981) Proc. NatL Aced. Sci. USA 78, Yee, R. & Liebman, P. A. (1978) J. BwLt Chem. 253, Godchaux, W., III & Zimmerman, W. F. (1979) J. BwLt Chemn. 254, Kuhn, H. (1980) Nature (London) 283, Fung, B. K.The& Stryer, L. (1980) Proc. Nat Aced. Sci. USA 77, Bignetti, E., Gavaggioni, A. & Sorbi, R. T. (1978) J. 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