Reciprocal effects of an inhibitory factor on catalytic activity and

Transcription

1 Proc. Natl cad. Sci. US Vol. 79, pp , June 1982 iochemistry Reciprocal effects of an inhibitory factor on catalytic activity and noncatalytic cgmp binding sites of rod phosphodiesterase (guanylyl nucleotide/heat-stable, inhibitor) KO YMZK, FRNCS RTUCC*, LEXNDER TNGt, ND MRK W. TENSKYt Life Sciences Division, University of California, Los lamos National Laboratory, Los lamos, New Mexico Communicated by Lewis Thomas, March 11, 1982 STRCT n illuminated rod outer segment membranes, GTP and guanosine 5'-[fyl-imido]triphosphate (p[nh]ppg) have reciprocal effects on cgmp phosphodiesterase (PDEase; 3':5'- cyclic-nucleotide 5'-nucleotidohydrolase, EC ) activity and cgmp binding to noncatalytic-sites on that enzyme. Two micromolar p[nh]ppg increased PDEase activity more than 2-fold while inhibiting cgmp binding more than 4%. Reduction of noncatalytic cgmp binding, which followed addition of p[nh]ppg, was not a result of PDEase activation. oth effects of p[nh]ppg were completely dependent on the presence of bleached rhodopsin. heat-stable factor has been found to inhibit PDEase activity and also to stimulate cgmp binding to noncatalytic cgmp binding sites. ddition of p[nh]ppg reversed the effects of this factor on both PDEase activity and cgmp binding. During purification of this material, the activity peaks for both PDEase inhibition and activation of noncatalytic cgmp binding comigrated on both lue Sepharose CL-6 column chromatography and sucrose density gradients centrifugation, suggesting that the same factor could be responsible for both inhibition of PDEase activity and enhancement of noncatalytic cgmp binding. Limited tryptic proteolysis of PDEase, which markedly reduced cgmp binding to the noncatalytic sites, and experiments using highly purified cmp (free of cgmp) as substrate for PDEase showed that the binding of cgmp to noncatalytic sites was not required for the heat-stable inhibitory factor to inhibit PDEase activity. We discuss possible relationships between the regulation of PDEase and the binding of cgmp to noncatalytic sites. Light and GTP are required to activate a peripheral protein cgmp phosphodiesterase (PDEase; 3':5'-cyclic-nucleotide 5'- nucleotidohydrolase, EC ) located on the disk membranes ofvertebrate rod outer segments (ROS) (1, 2). The lightmediated decrease in ROS cgmp concentrations may play a role in photoreceptor excitation or sensitivity control or both (1). PDEase activation depends on photon capture by rhodopsin (3) and binding ofgtp to a GTP-binding protein (4) can function as a soluble activator of PDEase (5, 6). Once formed in the presence of bleached. rhodopsin, this complex can transmit the influence of light to PDEase in the absence of rhodopsin (6). n addition, a number of studies have indicated the presence of heat-stable inhibitors of PDEase in ROS (7-12). The effect on PDEase of the activating complex appears to result from the release of an inhibitor or a change in the conformation of PDEase or the inhibitor or combinations thereof (1). Recently, we reported the presence of specific noncatalytic high-affinity cgmp binding sites on ROS PDEase (13). Similar cgmp-specific binding sites have also been found in association with the PDEase ofrat platelets (14) and rat lung (15). The physiological function of these sites is not yet understood. n this 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. 372 paper, we describe regulation of noncatalytic cgmp binding sites and catalytic activity of ROS PDEase by a heat-stable factor. The physiological activators of PDEase-i.e., light and GTP-are shown to reverse the effects ofthis heat-stable factor on both the enzymatic activity of PDEase and the binding of cgmp to its noncatalytic sites on PDEase. MTERLS ND METHODS Materials. [3H]cGMP and [3H]cMP were purchased from New England Nuclear. 8-zidoinosine 3',5'-[32P]phosphate (8- N3-[32P]cMP) was prepared as described (16). cmp, purified by HPLC (contamination by cgmp was <.1%), was a gift from CN. Preparation of ROS. ROS membranes were prepared from (3-5 large) Rana catesbiana retinas by sucrose flotation (17) and suspended in 1 mm Tris HC, ph 7.5/1 mm dithiothreitov 5 mm MgSO4 (buffer ). ll manipulations were carried out on ice under R illumination. Preparation of Crude PDEase and Urea-Treated Disk Membranes. Crude PDEase was prepared as described (13). t was extracted with EDT, and the pellet was suspended in 3 vol of 1 mm Tris-HC, ph 7.5/1 mm dithiothreitol/1 mm EDT/5 M urea. The suspension was passed through a no. 21 G needle three times, kept on ice for 6 min, and then centrifuged (6 min) in a eckman SW 27.1 rotor (81, X g, 4 C). The resulting pellet was washed three times with 5-vol portions of 2 mm Tris HC, ph 7.5/2 mm MgSO1 mm dithiothreitol (buffer ). The pellet ("urea-treated membranes") was suspended in buffer (=2 mg of protein/ml), quick frozen in acetone/dry ice, and stored at -8 C. (This preparation was stable for >3 months.) Preparation of nhibitory Factor and PDEase-Membrane Complex. Lyophilized crude PDEase was combined with ureatreated membranes (prepared from the same number of R. catesbiana retinas as the crude PDEase) and centrifuged for 1 min (48,2 x g, 4 C). The resulting supernatant is referred to as "inhibitory factor." The pellet was washed twice with 3-4 vol of buffer and twice with buffer. The pellet ("PDEase-membrane complex") had 3-5% of the PDEase activity and 2-3% ofthe GTPase activity found in the starting material. nhibitory factor was incubated at 8 C for 3 min and centrifuged for 1 min (48, x g, 4 C). The resulting supernatant is referred to as "heated inhibitory factor." bbreviations: PDEase, cyclic nucleotide phosphodiesterase; p[nh]ppg, guanosine 5'-[p, y-imido]triphosphate; ROS, rod outer segment; 8-N3- fl2p]cmp, 8-azidoinosine 3',5' -132P]phosphate. * Present address: Yale University School of Medicine, Dept. of Pathology, New Haven, CT t Present address: The Lawrence Livermore Laboratory, Livermore, C t To whom reprint requests should be addressed

2 iochemistry: Yamazaki et al. Partial Purification of nhibitory Factor with lue Sepharose CL-6. nhibitory factor was dialyzed against buffer and then loaded onto a lue Sepharose CL-6 column (.9 X 15cm) that had been equilibrated with buffer. The column was washed with buffer and 1 mm Tris HCl, ph 7.5/1 mm dithiothreitol/2 mm EDT (buffer C) and then eluted with a KC gradient in buffer C. Miscellaneous. PDEase purification, assay of PDEase activity (with or without protamine), photoaffinity labeling with 8- N3-[32P]cMP, and sucrose density gradient centrifugation (5-2%) of inhibitory factor were carried out as described (13). Determination ofcgmp binding by Millipore filtration was also carried out as described (13). More than 9% of PDEase activity stayed on the filter after limited trypsin proteolysis of PDEasemembrane complex. Protein was measured by the procedure of radford (18). RESULTS Demonstration of cgmp inding in ROS Membrane Preparations. Photoincorporation of 8-N3-[32P]cMP (1 tm) was used to detect noncatalytic cgmp binding sites in ROS membranes (Fig. 1). ncorporation of 8-N3-[32P]cMP into a single band having the same electrophoretic mobility as that of purified PDEase (13) could be prevented by 1,uM unlabeled cgmp but not by 1 M cmp. These data confirm the finding (13) that cgmp binding in crude PDEase is confined to PDEase. Effects of Light and Guanosine 5'-[8,y-imido~triphosphate (p[nh]ppg) on the Catalytic ctivity of PDEase and on Noncatalytic cgmp inding. The effects of various concentrations of p[nh]ppg on PDEase catalytic activity and on noncatalytic cgmp binding in illuminated ROS membranes are shown in Fig. 2. p[nh]ppg increased PDEase catalytic activity under these conditions by more than 2-fold while inhibiting noncatalytic cgmp-binding more than 4%. reciprocal pattern is observed when the data are plotted as percent effect (Fig. 2). These data support the hypothesis that both PDEase activation and inhibition of noncatalytic cgmp binding reflect a single p[nh]ppg-modulated process. Under identical conditions, addition of protamine caused a striking increase in PDEase activity (from.8 to 1.98,g of cgmp hydrolyzed per min per mg ofprotein) but had no effect on noncatalytic cgmp binding (52 pmol of cgmp bound/mg of protein). t has been shown that protamine can fully activate PDEase in ROS membranes (17). n the absence ofp[nh]ppg, noncatalytic cgmp binding was not significantly altered by protamine even though the maximal activation of PDEase by protamine significantly decreased the concentration of cgmp in the binding assay mixture. n addition, a 1:1 dilution of ROS membranes significantly reduced PDEase activity (data not shown) but did not affect the ability of p[nh]ppg to reduce noncatalytic cgmp binding. Thus, PDEase activation by p[nh]ppg and subsequent reduction in cgmp levels is not an adequate explanation for the effects of p[nh]ppg on noncatalytic cgmp binding. Nucleotide Requirements for ctivation of PDEase and nhibition of Noncatalytic cgmp inding. The capacity of a nucleotide to reduce noncatalytic binding of cgmp to PDEase correlates with its capacity to activate PDEase in illuminated ROS membranes (Table 1). The largest effects were seen with GTP and p[nh]ppg while GDP, GMP, and TP had little effect on either PDEase activity or binding of cgmp to noncatalytic sites. These data indicate that the GTP-GTP-binding complex (5, 6) can regulate both the catalytic activity of PDEase and noncatalytic cgmp binding associated with PDEase. Effects of nhibitory Factor on PDEase ctivity and Noncatalytic cgmp inding. We previously found (13) that the con- Proc. Natl cad. Sci. US 79 (1982) FG. 1. utoradiogram of photoactivated incorporation of 8-N3- [32P]cNP into PDEase. Photoaffinity labeling with 8-N3-[32P]cMP was carried out in the absence (lane 1) or presence of 1 um cgmp (lane 2) or cmp (lane 3). Each lane received 44 lg of ROS protein. rrow, 32P-labeled band having RF (.19) identical to that of extensively purified PDEase. centration ofcgmp (.9 um) necessary for half-maximal binding in crude PDEase preparations is significantly less than the concentration of cgmp (.75 M) necessary for half-maximal binding to highly purified PDEase. This observation suggested that there may be a factor in ROS preparations that can influence noncatalytic cgmp binding to PDEase. We have now found that there is a reciprocal relationship between the effects of inhibitory factor on PDEase activity and on noncatalytic cgmp binding (Fig. 3). These data suggest that the same protein may regulate both PDEase activity and noncatalytic cgmp binding. n support of this hypothesis, 8-N3-[32P]cMP was incorporated into the same gel band irrespective ofwhether ROS membranes (Fig. 1), crude PDEase (13), or extensively purified PDEase (13) were studied. These data indicate that the inhibitory factor enhances the binding of cgmp to the noncatalytic cgmp sites previously studied (13). The inhibitory factor was quite thermostable; 86%, 87%, and

3 374 iochemistry: Yamazaki et al. Proc. Natl. cad. Sci. US 79 (1982) to 15 4fi to 5~ ~ ~ zl 5 - X asw al: as ' c. ~~~~~~~~ , U 15 ~~~~~~ Heated inhibitory factor/pdease-membrane complex FG. 3. Effect of heated inhibitory factor on PDEase activity and cgmp binding. Various amounts of heated inhibitory factor were added to 135 ng of PDEase-membrane complex for PDEase assay (a) and to 5.4 jig of PDEase-membrane complex for cgmp binding assay (). Maximum (without heated inhibitory factor) and minimum activities (with 11 ng of heated inhibitory factor) were 95.3 and 9.91 jimol of (cgmp hydrolyzed/min)/mg of protein, respectively. Maximal (with 4.1 jig of heated inhibitory factor) and minimal (without heated inhibitory factor) cgmp binding values were 13 and 27.2 pmol of cgmp/mg of protein, respectively. Results are expressed as functions of the ratios of the amounts of heated inhibitory factor to those of complex (ng/mg for PDEase activity and jg/,jg for cgmp binding)..x p.4 5 ol Z, _ p[nh]ppg, pum FG. 2. Effect of light and p[nh]ppg on PDEase activity and cgmp binding in ROS suspensions. ROS membranes, 3.3 lug or 33 p.g, were used for assay of PDEase activity (e) and cgmp binding (), respectively. PDEase activity and cgmp binding were assayed in illuminated membranes () and under ER illumination (C). () Data shown in were plotted as percent, where 1% represents the difference in PDEase activity between and 2. jim p[nh]ppg and also the difference in cgmp binding observed between and 2.,uM p[nh]ppg. 88% of PDEase inhibitory activity were preserved when the inhibitory factor was heated (8C, 3 min) at ph values of 2, 7.5, and 1, respectively. lso, 8% ofthe capacity of inhibitory fac- Table 1. Effect of purine nucleotides on PDEase activity and cgmp binding to high-affinity sites in illuminated ROS membrane preparations PDEase activity, Concen- (j.mol of cgmp cgmp binding, tration, hydrolyzed/min)/ pmol/mg ddition im mg of protein of protein None GTP p[nhppg GDP GMP TP p[nh~pp p[nh]pp, adenosine 5'-[P3,y-imido]triphosphate, a nonhydrolyzable analogue of TP. tor to enhance noncatalytic cgmp binding was preserved after heating (8C, 3 min) at ph 7.5. When this heated inhibitory factor was treated with bovine pancreatic trypsin at 375 pug/ ml for 5 min at 37C, both the PDEase inhibitory activity and cgmp binding effects were decreased by =75%. Reversal of oth Effects of nhibitory Factor by p[nh]ppg. The PDEase-membrane complex and the inhibitory factor were used to examine the effects ofp[nh]ppg on both PDEase activity and noncatalytic cgmp binding (Table 2). These data illustrate the following points. (i) ntrinsic PDEase activity found in the PDEase-membrane complex (without added inhibitory factor) was not further activated by addition of p[nh]ppg. n this same preparation, cgmp binding to noncatalytic sites on PDEase was not affected by p[nh]ppg. (ii) When inhibitory factor was added to the PDEase-membrane complex, PDEase activity was markedly diminished and non- Table 2. Reversal of the effect of inhibitory factor on PDEase activity and cgmp binding by p[nh]ppg PDEase activity, (gmol of cgmp hydrolyzed/min)/ cgmp binding, mg of protein pmol/mg of protein nhibitory factor nhibitory factor bsent Present bsent Present PDEase-membrane complex alone PDEase-membrane complex/2 pm p[nh]ppg PDEase activity and cgmp binding were assayed under ambient light. Results are corrected for residual PDEase activity and cgmp binding in the inhibitory factor preparation. PDEase activity was not detected (in this preparation) in the absence of p[nh]ppg and was.82 (pnol of cgmp hydrolyzed/min)/mg of protein in the presence of 2 pt p[nh]ppg. n the inhibitory factor preparation, cgmpbinding was 18.1 pmol/mg of protein in the absence of p[nh]ppg and 11.8 pmol/ mg of protein in the presence of 2,uM p[nh]ppg.

4 iochemistry: Yamazaki et al. catalytic cgmp binding was markedly enhanced. These data also show the reciprocal relationship shown in Fig. 3. (iii) n the presence of inhibitory factor, addition of p[nh]ppg caused maximal activation of PDEase and maximal reduction of noncatalytic cgmp binding. These data indicate the virtual absence of inhibitory factor from the PDEase-membrane complex. n addition, the concentration ofcgmp necessary for half-maximal binding to illuminated ROS membranes was increased from.1,um to.8,um by the addition of 2,uM p[nh]ppg. This effect was not found with unilluminated ROS membranes. These data show that preparations of PDEase that are free of inhibitory factor are fully active and that inhibitory factor probably functions, in the intact rod, as part of an endogenous PDEase regulation system. n the absence of light and GTP, inhibitory factor maintains PDEase in an inactive mode. nhibitory factor may be released or conformationally altered by the GTP-GTP-binding protein complex in a manner that both permits expression of PDEase activity and diminishes noncatalytic cgmp binding to PDEase. dditional Evidence that nhibitory Factor ffects both PDEase ctivity and Noncatalytic cgmp inding. Using two different separation procedures (lue Sepharose CL-6 column chromatography and sucrose density gradient centrifugation), we found that those fractions containing the peak of PDEase inhibitory factor activity also maximally enhanced noncatalytic cgmp binding to PDEase (Fig. 4). nhibition of PDEase by nhibitory Factor Does Not Depend on Enhanced Noncatalytic inding ofcgmp. We assayed PDEase activity in the presence ofinhibitory factor using 1 JM cmp as substrate (purified by HPLC). The data (Fig. 5) show rq :; ~ ba d 21 o Proc. Natl. cad. Sci. US 79 (1982) 375 C av. -a) 6 4 li 2, Ei N C.) o E "a) U a)._~ - Q 2' as ottom 4' * _,. " * " --O Fraction Top FG. 4. Partial purification of inhibitory factor by lue Sepharose CL-6 column chromatography and sucrose density gradient centrifugation. Fractions of the different preparations were added to PDEase-membrane complex and PDEase activity (W), cgmp binding (o), and KCl concentrations () were measured. () nhibitory factor (containing 9,ug of protein) was applied to a lue Sepharose CL-6 column, washed, and eluted as described. () nhibitory factor (containing 16 Mg of protein) was layered on top of a continuous sucrose gradient (5-2%) and then centrifuged at 96,6 x g for 37 hr at 4 C _ o bd. a ~ C.) 5 Time of trypsin proteolysis, min FG. 5. Evidence that inhibition of the PDEase activity is inde- 1. pendent of noncatalytic cgmp binding. PDEase-membrane complex > (42 ug of protein/1,ul) was incubated for 2 min at 3 and mixed with a suspension of 1.1,g of trypsin in 1,ul of buffer. Trypsin S proteolysis was carried out for the times shown and stopped by addition.5 of 4.4,tg of soybean trypsin inhibitor in 1,ul of buffer. nhibitory factor was extensively dialyzed against buffer C, heated, and centrifuged. The supernatant was used as inhibitory factor. PDEase activity was assayed with 1,uM cmp () and with 1,M cgmp ().-, PDEase without inhibitory factor; ---, PDEase activity in the presence of heated inhibitory factor at 1 jg per tube. cgmp binding (C) was assayed in the presence of inhibitory factor at 2 jig per tube. rrow, cgmp binding to PDEase-membrane complex without inhibitory factor. that PDEase activity was fully sensitive to inhibitory factor with cmp as substrate. We have consistently found that cmp cannot bind to or displace cgmp from the noncatalytic sites on PDEase (13). n a complimentary experiment, the PDEasemembrane complex was incubated with trypsin for various periods of time. Limited trypsin proteolysis caused a striking decrease in noncatalytic cgmp binding. t those incubation times when noncatalytic cgmp binding sites had been reduced by >75%, the inhibitory factor remained fully effective as an inhibitor of PDEase activity. These data suggest that binding of cgmp to noncatalytic sites on PDEase is not required for inhibition of PDEase catalytic function by inhibitory factor. DSCUSSON We found that a heat-stable factor in ROS can inhibit PDEase activity and also stimulate the binding of cgmp to noncatalytic

5 376 iochemistry: Yamazaki et al sites on PDEase. The effects of inhibitory factor, both on PDEase activity and noncatalytic cgmp binding, were almost completely reversed by the PDEase activators light and GTP. Recent studies (1, 5, 6) have found that GTP-GTP-binding protein complex, which is formed in the presence of bleached rhodopsin, can serve as a PDEase activator in the absence of rhodopsin. These data suggest that physiological regulation of PDEase depends on the inhibitory factor and on the reversal of its effect by the GTP-GTP-binding protein complex (1). number of reports (8-12) have focused on the rod PDEase inhibitory factor since the study of Dumler and Etingoff (7). Molecular size estimates include 38, daltons (7), 8, daltons (9), and 54, daltons (8). recent report has also suggested the possibility that the inhibitor participates in the light regulation of PDEase (1). n this study, we have found that both the enhancement of noncatalytic cgmp binding and the inhibition of PDEase activity reside in a heat-stable trypsin-labile factor that showed coincident migration in two different separation procedures. More data are needed to ascertain whether in fact a single molecule is responsible for both effects. t is noteworthy that methylisobutylxanthine, a PDEase inhibitor, was effective in stimulating noncatalytic cgmp binding to photoreceptor PDEase (13) and to PDEases in rat platelets and lung (14, 15). Francis et al (15) have suggested that the order ofpotency for the stimulation ofnoncatalytic cgmp binding by PDEase inhibitors is the same as that for the inhibition of cgmp hydrolysis by PDEase. Experiments in which trypsin decreased noncatalytic cgmp binding and in which extensively purified cmp was used as substrate for ROS PDEase showed that noncatalytic cgmp binding was not necessary for PDEase inhibition by inhibitory factor. While the consequences of noncatalytic cgmp binding on rod function are not yet understood, our data imply a twoconformational model in which the catalytic function and the noncatalytic cgmp binding sites may be allosterically coupled; i.e., the active form (in the absence of inhibitory factor) of PDEase has poor affinity for cgmp (at the noncatalytic sites) while the inhibited form of PDEase has enhanced affinity for cgmp (at the noncatalytic sites). Surprisingly, protamine activated PDEase without changing noncatalytic cgmp binding and thus induced an active conformation that was independent of the noncatalytic cgmp binding sites. fter we had completed this paper, Hurley et al. (19) and Stryer et al (2), working with bovine rods, suggested that a 13,-dalton heat-stable protein inhibits trypsin-activated Proc. Nad cad. Sci. US 79 (1982) PDEase. They also suggested that activation of PDEase may occur when the GTP-GTP-binding protein complex alters the interaction of the inhibitory protein with PDEase, as previously suggested (1) and in agreement with our results. We thank Drs. Mark M. Rasenick, George L. Wheeler, Kenji Yamamoto, and Walter Goad for critical reading of this manuscript. This work was supported by National nstitutes of Health Grant ROL M itensky, M. W., Wheeler, G. L., Yamazaki,., Rasenick, M. & Stein, P. J. (1981) in Current Topics in Membranes and Transport, eds. ronner, F. & Kleinzeller,. (cademic, New York), Vol. 15, pp Wheeler, G. L. & itensky, M. W. (1977) Proc. Natl. cad. Sci. US 75, Wheeler, G. L., Matuto, Y. & itensky, M. W. (1977) Nature (London) 269, Shinozawa, T., Uchida, S., Martin, E., Cafiso, D., Hubbell, W. & itensky, M. W. (198) Proc. Natl. cad. Sci. US 77, Fung,. K. K., Hurley, J.. & Stryer, L. (1981) Proc. Natl. cad. Sci. US 78, Uchida, S., Wheeler, G. L., Yamazaki,. & itensky, M. W. (1981) J. Cyclic Nucleotide Res. 7, Dumler,. L. & Etingoff, R. N. (1976) iochim. iophys. cta 429, Liu, Y. P. & Wong, V. G. (1979) iochim. iophys. cta 583, aehr, W., Devlin, M. J. & pplebury, M. L. (1979) J. iol Chem. 254, Hurley, J.. (198) iochem. iophys. Res. Commun. 92, Robinson, P. R., Kawamura, S., bramson,. & ownds, M. D. (198) J. Gen. Physiol. 75, Hurley, J.. & Ebrey, T. G. (1979) iophys. J. 25, 314a. 13. Yamazaki,., Sen,., itensky, M. W., Casnellie, J. E. & Greengard, P. (198)J. iol. Chem. 255, Hamet, P. & Coquil, J. F. (1978) J. Cyclic Nucleotide Res. 4, Francis, S. H., Lincoln, T. M. & Cornin, J. D. (198) J. iol Chem. 255, Casnellie, J. E., ves, M. E., Jamieson, J. D. & Greengard, P. (198) J. iol Chem. 255, Mild, N., araban, J. M., Keirns, J. J., oyce, J. J. & itensky, M. W. (1975) J. iol Chem. 25, radford, M. (1976) nal iochem. 72, Hurley, J.., arry,. & Ebrey, T. C. (1981) iochim. iophys. cta 675, Stryer, L., Hurley, J.. & Fung,. K.-K. (1981) Trends iochem. Sri. 6,