Mechanism of ATP Quench of Phosphodiesterase Activation in Rod Disc Membranes*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 2, Issue of January 25, pp , 1983 Prmted in U.S.A. Mechanism of ATP Quench of Phosphodiesterase Activation in Rod Disc Membranes* (Received for publication, May 24, 1982) Ari Sitaramayya and Paul A. Liebman From the Department of Anatomy, University of Pennsylvania, Philadelphia, Pennsylvania GTP-dependent light activation of cyclic GMP phosphodiesterase in bovine ro disc membranes was quenched by ATP. ATP reduced both initial velocity (V,) and turn off time (tor) of phosphodiesterase activated by a flash that bleached 1.5 X lo-' of the rhodopsin present. In the absence of rhodopsin kinase, ATP had no effect on either VO or toft of reconstituted preparations containing phosphodiesterase and GTP-binding protein. Addition of partially purified rhodopsin kinase to such reconstitutions again permitted ATP to quench both initial velocity and turn off time. It is thus likely that kinase-mediated phosphorylation of bleached rhodopsin reduces and arrests light-induced phosphodiesterase activation. Thermolysin cleavage of rhodopsin's COOH-terminal dodecapeptide eliminated ATP's effect on tor, but did not diminish its effect on Vo. Thus,the effects of ATPand kinaseon Vo maybe mediated by sites proximal to and effects on totr by sites distal to the thermolysin cleavage point at rhodopsin's COOH-terminal end. Bleached rhodopsin catalyzes the activation of cyclic GMP phosphodiesterase on rod disc membranes (1). An essential step in this activation is the conversion of inactive GTPbinding protein to the active form (2) which can combine with inactive phosphodiesterase to form the active enzyme that hydrolyzes cgmp. Return to the dark state is marked by cessation of cgmp hydrolysis. This is mediated by the simultaneous inactivation of R*' and hydrolysis of the bound GTP to again form inactive r-gdp. R* is also the substrate for rhodopsin kinase, an enzyme present in RDM (3, 4). The kinase prefers ATP over GTP in phosphorylating R* (5) and R* is multiply phosphorylated (4, 6). Sitaramayya et al. (7) showed that phosphorylated R* does not activate phosphodiesterase. Real time experiments of Liebman and Pugh (8, 9) showed that ATP rapidly quenches light activation of phosphodiesterase. The K, for ATP in the quench mechanism was about 4 PM, similar to that of the phosphorylation of R* by rhodopsin kinase (5). These studies suggested that phosphorylation of R* may be important in the quenching of phosphodiesterase activation. The present work adds weight to this hypothesis by finding that the ATP quench is not seen in reconstituted preparations * This work was supported by Grants EY00012 and EY01583 from the National Eye Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The abbreviations used are: R*, bleached rhodopsin; RDM, rod disc membranes; srdm, hypotonically washed RDM; r GTP-binding protein; AMP-P(NH)P, adenyl-5"yl(p,y-imido)diphosphate; Mops, 4- morpholinepropanesulfonic acid SDS, sodium dodecyl sulfate; TL+, thermolysin-treated; TL-, quenched thermolysin-treated consisting of rhodopsin-containing membranes, GTP-binding protein, and phosphodiesterase alone, but is restored by addition of rhodopsin kinase. It is further shown that ATP quenches phosphodiesterase activation by decreasing both the initial velocity (Vo) and the turn off time of the activation (teff). The teff effect is lost upon brief thermolysin treatment. EXPERIMENTAL PROCEDURES Materials-Cyclic GMP, GTP, and protease X (thermolysin) were purchased from Sigma. ATP and AMP-P(NH)P were products of Boehringer Mannheim. Frozen dark-adapted bovine retina were obtained from George A. Hormel Co., Austin, MN and stored at -30 "C until use. Preparation of RDM-All operations were done at 0-4 "C in darkness with the aid of infrared viewing devices. Frozen retinas were thawed and vortexed in 1 ml/retina of 45% sucrose (w/v) in Mops buffer (20 mm 4-morpholinepropanesulfonic acid, 100 mm KCl, 2 mm MgC12, 1 mm dithiothreitol, 0.1 mm EDTA, ph 8.0). The suspension was overlaid with buffer and spun at 27,000 X g for 30 min. Crude RDM were collected from the sucrose-buffer interface and diluted in buffer. The suspension was centrifuged at 27,000 X g for 15 min and the resulting pellet was taken up in buffered sucrose. The flotation and pelleting were repeated and the final pellet was suspended in Mops buffer. Preparation of srdm and Peripheral Proteins-RDM were washed in hypotonic buffer (10 mm Tris-HC1, 1 nm dithiothreitol, ph 7.5) to extract peripheral proteins, including phosphodiesterase, r, rhodopsin kinase, etc. Usually, the RDM from 10 retinas were extracted in 50 ml of buffer at 48,000 X g for 40 min. The extractiqn was repeated once and the two extracts were pooled. The pellet was washed again in the same buffer and suspended in Mops buffer (srdm). The extracted peripheral proteins were concentrated by Millipore immersible-cx ultrafilters. Reconstitution of Peripheral Proteins with srdm-increasing amounts of concentrated peripheral proteins were added to a 4 ~ L M rhodopsin-containing srdm suspension until the phosphodiesterase activity in the reconstitution matched that of the original RDM when light-activated by a photoflash that bleached 22% of the contained rhodopsin. This usually required a 2- to 3-fold excess of peripheral protein over that originally present in RDM. Preparation of TL+ and TL- Membranes-Hargrave and Fong (10) described the controlled digestion of peripheral protein-depleted disc membranes with thermolysin to remove a 12-amino acid peptide from the COOH-terminal end of rhodopsin. We employed the procedure of Kiihn and HargravF (11). srdm were incubated with 0.21 mg/d of thermolysin in 5 mm Ca2+ containing medium at 22 "c for 5 min at a concentrationpf 2.6 mg of rhodopsin/ml. The reaction was terminated by addition of EDTA and the membranes were extensively washed with hypotonfc buffer and suspended in Mops buffer to give TL+ membranes. &DM were incubated under similar conditions with EDTA-inaqtivated thermolysin and washed to give TL- (control) membranes. Fig. 1 shows the electrophoretic mobility of rhodop- sin from TL+ and TL- membranes in SDS-polyacrylamide gels. The molecular weights given for untreated rhodopsin and TL+ rhodopsin by Hargrave and Fong (10) were 35,000 and 30,500, close to the 35,500 and 32,000 values obtained by us. Purification of Phosphodiesterase and r-the protocol for purification was that of Baehr et al. (12). Bleached RDM were washed 5 times in isotonic buffer. The washed membranes retained phosphodiesterase and r. Extraction with hypotonic buffer released phospho-

2 in RDM ATP Quench of Phosphodiesterase Activation 1206 diesterase which was further purified on a DE52/G-100 column. I was then eluted in hypotonic buffer containing 50 p~ GTP and needed no further enrichment. Phosphodiesterase andr were dialyzed overnight against Mopsbuffer containing 50%glycerol in which they were finally stored a t -20 C. The purity of the proteins is evident from the SDS-polyacrylamide gels shown in Fig. 1. Preparation of Kinase-free srdm-srdm obtained after dark hypotonic washes were extracted 3 times, with 50 ml of 1 M NH,CI a t 48,000 X g for 30 min (4). The extracted pellet was treated for 30 min in 4% alum (13) followed by extensive washing in hypotonic buffer. The final pellet was suspended in Mops buffer. Preparation of Partially Purified Rhodopsin Kinase-A crude preparation of rhodopsin kinase was obtained according to the fmt two steps in the proceduredescribed by Shichi and Somers(5). RDM were extracted 3 times with 1 M NH&I and the extract was subjected to ammonium sulfate fractionation. The 20-45%saturation pellet was dissolved inand dialyzed againstmops buffer and used without further purification. The kinase activity was very unstable (5) and the amount of partially purified preparation employed in the reconstitutions was about 3 times greater than the amount in theoriginal RDM preparations. Reconstitution with Purified Components-When srdm or kinase-free RDM were reconstituted with purified r and phosphodiesterase, the proportion was 4 p~ rhodopsin to 0.8 p~ r (M, = 80,000) to 0.08 phosphodiesterase ( M, = 180,000). The reconstituted mixture could not be light-activated without added GTP suggesting that the GTP used to elute r from RDM was either completely dialyzed out or was hydrolyzed to GDP. Rhodopsin Assay-The concentration of rhodopsin in RDM or srdm suspensions was determined from the absorbance difference spectrum of a suitably diluted suspension before and after bleaching in 50 mm NHzOH using the molar extinctioncoefficient for rhodopsin of 40,000 cm2 mmol a t 500 nm. Phosphodiesterase Assay-Phosphodiesterase activity was measured at 38 C, ph8, according to the phrecording method of Liebman and Evanczuk (14). The reaction mixtures contained RDM or reconstituted systems a t 4 PM rhodopsin, 5 m cgmp, 250 p~ GTP, and 500 p~ ATP or AMP-P(NH)P where indicated. The reaction was initiated by a green light flash of I-ms duration appropriately attenuated by neutral filters and calibrated to bleach a desired fraction of rhodopsin in the assay mixture. Except where otherwise indicated, the flashbleached 1.5 X of thecontained rhodopsin. Assay of Rhodopsin Phosphorylation-To measure the rate of phosphorylation in RDM or srdm, membranes were suspended in Mops buffer a t 4 p~ rhodopsin. Partially purified kinase was present where indicated. Fifty p~ ATP containing about4 pci of [y-. P]ATP was added to the mixtures15 s before a light flash that bleached 22% of the rhodopsin. Atvarioustimesafterthe flash, aliquots were rapidly expelled into electrophoresis sample buffer containing SDS. The samples were electrophoresed according to Baehr et al.(15) and the gels were stained for protein with Coomassie brilliant blue. After destaining, the rhodopsin band was cut out and incorporated P,was counted in Triton/toluene scintillation fluid. Reaction mixtureswhich were not bleached were sampled under similar conditions to serve as dark controls. Electrophoresis-SDS-polyacrylamide gel electrophoresis on 6-20% continuousacrylamidegradients was done according to O Farrell (16). T o resolve the higher molecular weight subunits of phosphodiesterase and r and to distinguish between the bands of rhodopsin and r, electrophoresis was done according to Baehr etal. (15). RESULTS Effect of ATP on Phosphodiesterase Activation in RDMFig. 2 shows RDM phosphodiesterase activation after a 1.5 X bleach in 250 PM G T P (curve a ). The initialvelocity, Vo,was about 4.9 PM cgmp hydrolyzed/s and the l / e time to turnoff (tom)was about36 s. Five hundred PM ATP reduced Vo to 2.4 PM/S and the reaction turned off much faster with tom about 11 s (curve b). Vo and torrvaried from preparation to preparation but 500 ELM ATP, whenincluded,consistently reduced both. This effect has previously been shownnot tobe due to ATP competition with the GTP activator mechanism of RDM phosphodiesterase(9). Influence of Thermolysin Treatment of Rhodopsin on ATP Effects-Brief thermolysin treatmentcleaves a 12-amino acid fragment from the COOH-terminal end of rhodopsin. This fragment contains2 serines and 2 threonines that are among those residues phosphorylated by kinase and ATP (10, 17). MWx a b c d e f g h i j FIG. 1. SDS-polyacrylamide gel electrophoresis of preparations used in this study. a-h, electrophoresis in 6-20% gradient polyacrylamide gel according to O Farrell (16); i-k, 15%acrylamide gel according to Baehr etal. (12). a, molecular weight markers; b, RDM (15 pg); c, srdm (15 pg); d, peripheral proteins (corresponding to 30 pg of RDM); e, TL+ srdm (15 pg); f, TL- srdm (15pg); g,purified phosphodiesterase (8pg); h, purified I (8 Fg); i, purified I (8 pg);j,purified phosphodiesterase (8 pg); k, RDM (50 pg). In lanes i-k, proteins of molecular welght below 30,000 are not shown. k

3 x_ ATP Quench of Phosphodiesterase Activation in RDM /"I O L Y ' I FIG. 2. Effect of ATP on phosphodiesterase activation in RDM. RDM phosphodiesterase was activated in 250 p~ GTP (curve a) or 250 ~ LGTP M and ATP ~ ~ (curve b) with a flash (A bleaching 1.5 x fraction of the 4 p~ rhodopsin in the reaction mixture. TABLE I Light activation ofphosphodiesterase in preparations reconstituted with TL+ and TL- membranes Reconstitutions consisted of srdm treated with thermolysin (TL+) or quenched thermolysin (TL-) plus the peripheral proteins previously extracted from the same membranes and concentrated. GTP was 250 p~ and cgmp was 5 mm. Reactions were initiated with a light flash calibrated to bleach the specified fraction of rhodospin. TL- activity was the same as that of identical but untreated reconstitutions. Samples contained 4 p rhodopsin. 200 I A Initial velocity of phosphodiesterase TL- TL+ PM cgmp hydrolyzed/s/pm Counts/min R 1.5 X bleach bleach reconstituted with peripheral proteins in the presence of GTP or GTP and ATP. For TL- membranes with GTP (curue c), VO was 2.9 ~ M/S and teff 42 s with GTP. ATP reduced VO to 0.9 ~M/S and toff to 21 s (curue d). TL+ membranes in GTP alone (curue a) gave Vo of 5.8 ~ M/S and t,h of 37 s. Addition of ATP reduced Vo to 2.2 ~M/S but toe was unaltered at about 41 s (curue b). The normalized inset curves emphasize the unique separability of these differences (Fig. 3). Reconstitution with Purified Proteins-In order to test more directly the hypothesis that kinase-mediated reactions are involved in the ATP effects on VO and torr, it was necessary to design a reconstitution system lacking kinase and to show that ATP had no effect in such a system. Thus, srdm were reconstituted with purified phosphodiesterase and r instead of with peripheral protein extract. However, we were surprised to find that Vo and t,ff were still reduced by ATP in these reconstitutions to the same extent as in TL- membranes reconstituted with peripheral proteins (data not shown). Reasoning that such a result might be due to kinase contamination of the srdm themselves, attempts were made to check the constituents for kinase activity. Table I1 and Fig. 4 show that srdm were contaminated. Addition of purified r and phosphodiesterase to srdm did not increase the phosphorylation TABLE I1 Phosphorylation level of various membrane fractions Normal RDM, srdm, and srdm supplemented with purified r and phosphodiesterase were mixed with [y-32p]atp. One portion of each was left in the dark and the other portion was activated with a 22% bleach. The reactions were stopped after 2 min, samples were electrophoresed in SDS-polyacrylamide gel, and the rhodopsin band was cut out and counted in scintillation fluid. Counts/min shown are light minus dark values. Fraction RDM, 4 p~ rhodopsin 11,255 srdm, 4 p~ rhodopsin 1,871 SRDM PM r 1,179 srdm 0.08 p~ phosphodiesterase 1,318 srdm p~ r p~ phosphodiesterase 1,101 srdm washed with 1 M NH4C1 and alum I F, I d, 20 I I 40 I I I I I 0 Q 80 FIG. 3. Effect of ATP on themolysin-treated (TL+) and control (TL-) srdm reconstituted with peripheral protein extract. TL+ membranes activated in250 p~ GTP (curve a) or 250 PM GTP and 500 p~ ATP (curue b). TL- membranes activated in 250 p~ GTP (curve c) or 250 p~ GTP and 500 p~ ATP (curve d). Inset a plots curve a together with curve b normalized by the ratio of initial velocities, a/b. The two curves superimpose. Inset b plots curve c (lower) together with curue d (upper) normalized by the ratio of initial velocities, c/d. Rhodopsin concentration in both assays was 4 p ~ 1.5. x bleach in each case at F. Table I summarizes results of reconstitution of treated and control srdm with peripheral proteins. Treated srdm (TL+) activated phosphodiesterase marginally better than TL- at 22% bleach, but were almost twice as good as TL- at weak bleaches where only 1.5 X was bleached. Fig. 3 shows light activation of TL+ and TL- membranes 40w 0 I I I I I FIG. 4. Phosphorylation of rhodopsin in various preparations used in this study. RDM (O), srdm (A), srdm washed with NKC1 and alum (A), and washed RDM supplemented with partially purified rhodopsin kinase (0) were incubated in Mops buffer with 50 p~ ATP containing 4 pci of [y3'p]atp. Rhodopsin concentration was 4 p~ in all cases and, when added (O), the kinase corresponded to that obtained from 12 p~ rhodopsin-containing RDM. After a light flash bleaching 22% of rhodopsin, aliquots were taken out at different times, dissolved in SDS-buffer, and electrophoresed. "P, incorporated into the rhodopsin band was determined. Dark controls were sampled under similar conditions and counts/min were subtracted from paired experiments. Picomoles of Pi incorporated into rhodopsin were then calculated using the calibration data.

4 1208 ATP Quench of Phosphodiesterase Activation in RDM FIG. 5. Effect of ATP on phosphodiesterase activation in reconstitutions of kinase-free srdm with purified phosphodiesterase and I'. Four p~ kinase-free rhodopsin in srdm was reconstituted with 0.8 p~ r and 0.08 PM phosphodiesteraseandlightactivated in either 250 p~ GTP (lower curve) or 250 ~ LGTP M and 500 p~ ATP (upper curve). Light flash (F) bleached 1.5 X fraction of rhodopsin. "I +/ I 1 I O " FIG. 6. Effect of ATP on phosphodiesterase activation when kinase-free reconstitutions were supplemented with partially purified rhodopsin kinase. Kinase-free srdm (4 PM rhodopsin), 0.8 PM r, and 0.08 p~ phosphodiesterase were supplemented with partialiy purified kinase in the same amounts used in Fig. 4 and lightactivated in 250 p~ GTP (a) or 250 p~ GTP and 500 p~ ATP (b), or 250 p~ GTP and 500 p~ AMP-P(NH)P (c). Light flash (F) bleached 1.5 X fraction of rhodopsin. of srdm (Table 11) showing that these proteins were free of kinase activity. When r and phosphodiesterase were reconstituted with kinase-free membranes, ATP was no longer effective in reducing VO or t,ff (Fig. 5). As shown, ATP marginally increased the phosphodiesterase activity of kinase-free srdm instead of diminishing it as for normal RDM. Effect of Partially Purified Kinase on Kinase-free Reconstitutions-When a partially purified kinase preparation was added to reconstitutions of kinase-free srdm with phosphodiesterase and r, ATP reduced VO from 1.0 p~ s-' to 0.5 p~ s-l and torr from 66 s to 29 s (Fig. 6). AMP-P(NH)P was unable to mimic the effects of ATP on Vo or t,ff, activation remaining the same as that with GTP alone. Fig. 4 shows the phosphorylation of rhodopsin in normal RDM, srdm, kinase-free srdm, and kinase-free srdm supplemented with partially purified rhodopsin kinase in amounts that elicited the effects of ATP ashown in Fig. 6. The relative phosphorylation activity of each of these preps clearly parallels the effect of kinase on Va and ton of the real time records of light-activated phosphodiesterase activity (Figs. 2-6). DISCUSSION Light rapidly converts rhodopsin to R*, which is simultaneously the enzyme that activates phosphodiesterase and the substrate for rhodopsin kinase. Phosphodiesterase activity cannot terminate until R* is itself inactivated. The reports of Sitaramayya et al. (7) that phosphorylated (bleached) rhodopsin does not activate phosphodiesterase and that of Liebman and Pugh (8,9) that ATP rapidly quenches phosphodiesterase activation suggest that the function of rhodopsin kinase is to mediate termination of phosphodiesterase activation by incapacitating R* through rapid phosphorylation. We have tested this hypothesis in two ways. First, by determining whether the removal of kinase removes the ATPdependent quenching of activation and whether kinase replacement leads to recovery and second, by determining whether proteolytic removal of a segment of rhodopsin normally phosphorylated in the presence of kinase alters the ATP-dependent quenching. The results of these tests add further weight and refinement to the hypothesis. Normal RDM preparations showed both ATP-dependent quenching of phosphodiesterase activation (Fig. 2) and y-"p incorporation into rhodopsin (Fig. 4,O) while kinase-free preparations showed neither ATP-dependent quenching (Fig. 5) nor y3'p incorporation into rhodopsin (Fig. 4, A). When repleted with partially purified rhodopsin kinase, the later preparations showed parallel recovery of quenching (Fig. 6) and of y-"'p incorporation into rhodopsin (Fig. 4,O). Both kinase and ATP were required for quenching. AMP-P(NH)P could not substitute for ATP. Neither I' nor phosphodiesterase was phospho- rylated, nor did they increase 32P incorporation into rhodopsin in the presence of kinase. Instead, these proteins caused "P incorporation to diminish as might be expected from the mutually exclusive nature of the transient binding interactions between such large peripheral proteins and rhodopsin. Together, we take these observations as confirmatory evidence that phosphorylation of bleached rhodopsin and quenching of phosphodiesterase activation are causally linked. It may be argued that the time course of phosphorylation shown in Fig. 4 is not fast enough to be causal to the faster phosphodiesterase quenching times shown. It should be pointed out, however, that such a comparison is only appropriate where both phosphodiesterase activation and phosphorylation rates are measured at comparably weak bleaches that are in the linear response range. Its great amplification allows phosphodiesterase activity to be readily measured in the linear range using a 1.5 x W 5 bleach (18). However, its much smaller stoichiometry does not permit phosphorylation to be readily measured with such weak bleaches. The rhodopsin phosphorylation measurements of Fig. 4 were thus made using a bleach sufficiently strong to permit accurate assays, but that also generated a saturating quantity of substrate for the small amounts of kinase present. This figure therefore provides information only about kinase-(enzyme)-limited maximum rates while the relaxation, toff, seen in the phosphodiesterase activity figures occurs in R*-(substrate)-limited conditions (weak bleach). It is of interest that the phosphodiesterase quenching speed (l/toff) of various preparations increases in parallel with the increase in the kinase-limited rate of phosphorylation shown in Fig. 4. It is expected from these data that phosphorylation and quenching speed would become still faster at a higher kinase concentration. This has in fact been demonstrated in a very recent paper (6). Our preparations are made in darkness where the smallest amount of kinase is membrane-bound and perhaps most of the kinase is lost and unavailable to the bleached rhodopsins of isolated RDM. In vivo conditions may provide a much larger local concentration of kinase that would yield far higher phosphorylation and quench rates approaching the physiologic time scale of vision. Phosphorylation of rhodopsin is known to be multiple, occurring at 7-9 sites near the cytoplasmic surface, COOHterminal end (6). Brief exposure to thermolysin removes the

5 ATP Quench of Phosphodiesterase Activation in RDM 1209 COOH-terminal dodecapeptide of rhodopsin containing 4 of these sites (10, 17). If phosphorylation of rhodopsin arrests phosphodiesterase activation as hypothesized, partial prevention of phosphorylation as would be caused by thermolysin, for' example, might be expected to prolong activation. We found brief thermolysin treatment to have two effects. First, it increased the Vo obtainable with both strong and weak bleaches suggesting that part or all of this peptide may sterically hinder access of rhodopsin to components of the phosphodiesterase activation mechanism with which it interacts and confirming the importance of the rhodopsin phosphorylation region to the phosphodiesterase activation mechanism in the unphosphorylated state. Peptide removal may thus increase rhodopsin's efficiency of interaction with other membrane proteins. Second, brief thermolysin treatment significantly increases the ATP/kinase-dependent teff while leaving the V, suppression unaffected. Thermolysin treatment therefore seems to have separated the ATP quench mechanism into two distinct components. Removal of rhodopsin's COOHterminal dodecapeptide together with its 4 phosphorylation sites makes it impossible for ATP and kinase to phosphorylate these sites and to reduce ton to give a rapid quench of phosphodiesterase activation. Nevertheless, ATP and kinase-dependent Vo suppression remains. Together with the inability of either kinase or ATP alone to suppress Vo in the absence of the other, it is not unreasonable to infer that suppression of V, may require phosphorylation of one or more of the remaining serines and threonines proximal to the thermolysin cleavage point. The fact that Vo suppression must occur earlier than teff suggests that a causal phosphorylation mechanism would have to phosphorylate the region proximal to C'12 before that distal to C'12 in reconstitution. Litman' also found * B. J. Litman, personal communication. enhanced phosphodiesterase activity after limited rhodopsin proteolysis. Acknowledgments-We wish to thank Drs. Wolfgang Baehr and Meredithe Applebury for help and advice with the purification of r and phosphodiesterase, Dr. S. Walter Englander for the use of his scintillation counter, and Dr. Vivian T. Nachmias for the use of her ultracentrifuge and accessories. REFERENCES 1. Yee, R., and Liebman, P. A. (1978) J. Biol. Chem. 253, Fung, B. K.-K., Hurley, J. B., and Stryer, L. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, Kuhn, H., and Dreyer, W. J. (1972) FEBS Lett. 20, Bownds, D., Dawes, J., Miller, J., and Stahlman, M. (1972) Nature New Biol Shichi, H., and Somers, R. L. (1978) J. Biol. Chem. 253, Wilden, U., and Kuhn, H. (1982) Biochemistry 21, Sitaramayya, A., Virmaux, N., and Mandel, P. (1977) Neurochem. Res Liebman, P. A,, and Pugh, E. N., Jr. (1979) Vision Res. 19, Liebman, P. A,, and Pugh, E. N., Jr. (1980) Nature 287, Hargrave, P. A., and Fong, S.-L. (1977) J. Supramol. Struct. 6, Kuhn, H., and Hargrave, P. A. (1981) Biochemistry Baehr, W., Morita, E. A., Swanson, R. J., and Applebury, M. L. (1982) J. Biol. Chem. 257, McDowell, J. H., and Kuhn, H. (1977) Biochemistry 16, Liebman, P. A,, and Evanczuk, A. T. (1982) Methods Enzymol. 81, Baehr, W., Devlin, M. J., and Applebury, M. L. (1979) J. Biol. Chem. 254, OFarrell, P. H. (1975) J. Biol. Chem. 250, Hargrave, P. A,, Fong, S.-L., McDowell, J. H., Mas, M. T., Curtis, D. R., Wang, J. K., Juszczak, E., and Smith, D. P. (1980) Neurochem. Znt. 1, Liebman, P. A,, and Pugh, E. N., Jr. (1981) Curr. Topics Membr. Trans. 15,