Characterization of a purified bovine lung cGMP-binding cGMP phosphodiesterase.

A bovine lung cGMP-binding phosphodiesterase (cG-BPDE) was purified to homogeneity and exhibited specific cGMP hydrolytic (Km = 5.6 microM) and cGMP binding (half-maximum approximately 0.2 microM) activities which comigrated throughout the purification. A chimeric structure was suggested for cG-BPDE since DEAE chromatography of a partial alpha-chymotryptic digest of cG-BPDE separated cGMP-binding fragments from a cGMP hydrolytic fragment. Native cG-BPDE (178 kDa) appeared to be a homodimer comprised of two 93-kDa subunits. The order of potency of inhibitors of cG-BPDE hydrolysis of cGMP was as follows: zaprinast greater than dipyridamole greater than 3-isobutyl-1-methyl-8-methoxymethylxanthine greater than 3-isobutyl-1-methylxanthine greater than cilostamide greater than theophylline greater than rolipram. Minimum [3H]cGMP binding stoichiometry was 0.93 mol of cGMP bound/mol of monomer, but [3H]cGMP dissociation from cG-BPDE in the presence of excess unlabeled cGMP was curvilinear, suggesting multiple cGMP-binding sites. Two chymotryptic cGMP-binding fragments of 35 and 45 kDa were specifically photoaffinity labeled with [32P] cGMP, exhibited [3H]cGMP association and dissociation behavior indistinguishable from native cG-BPDE, and each had the amino-terminal sequence: Thr-Ser-Pro-Arg-Phe-Asp-Asn-Asp-Glu-Gly-. Cochromatography of the two cGMP-binding fragments suggested that both a dimerization domain and a cGMP-binding domain were located in a 35-kDa segment of cG-BPDE. Increased [3H]cGMP binding to or [32P]cGMP photoaffinity labeling of cG-BPDE binding sites in the presence of hydrolytic site-specific cyclic nucleotide analogs suggested communication between hydrolytic and binding sites. The principle of reciprocity thus predicts that cGMP binding to the binding sites may affect the hydrolytic site. In the presence of cGMP, the binding fragments or native cG-BPDE exhibited an electronegative shift on high performance liquid chromatography-DEAE, consistent with a cGMP-induced change in cG-BPDE conformation.     

Specificity of cGMP binding to a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue

The binding of [3H]cGMP (guanosine 3',5'-monophosphate) to purified bovine adrenal cGMP-stimulated phosphodiesterase was measured by Millipore filtration on cellulose ester filter. [3H]cGMP-binding activity was enhanced when the assay was terminated in buffer containing 70% of saturated ammonium sulfate to dilute the enzyme and wash the filters. The cGMP-binding activity was co-purified with the phosphodiesterase activity. The binding of [3H]cGMP to purified enzyme was measured in the presence or absence of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. 1-Methyl-3-isobutylxanthine showed linear competitive inhibition with respect to cGMP as substrate in the phosphodiesterase reaction but stimulated the [3H]cGMP-binding activity in the binding assay. The stimulatory effect appeared not to be the result of preservation from [3H]cGMP hydrolysis; no cGMP phosphodiesterase activity has been measured under the cGMP-binding assay conditions, in the absence or presence of the inhibitor. Half-maximal stimulation by 1-methyl-3-isobutylxanthine occurred in the 5-7 microM concentration range. The specificity of binding of [3H]cGMP was investigated by adding increasing concentration of unlabeled analogs of cAMP (adenosine 3',5'-monophosphate) and cGMP. The binding of [3H]cGMP (50 nM) was displaced by unlabeled cGMP and cAMP with the following potency: 50% displacement was reached at the 0.1 microM cGMP range and only at a fiftyfold higher cAMP concentration. Our data with comparative series of analogs (e.g. 5'-amino-5'-deoxyguanosine 3',5'-monophosphate and 3'-amino-3'-deoxyguanosine 3',5'-monophosphate) showed that the potencies of stimulation of cAMP phosphodiesterase activity parallels displacement curves or [3H]cGMP binding to purified enzyme with no correlation with phosphodiesterase inhibition sequences. Those experiments suggest that the cGMP-binding activity is directly related to the non-catalytic (allosteric) cGMP-binding site.     

Noncatalytic cGMP-binding sites of amphibian rod cGMP phosphodiesterase control interaction with its inhibitory gamma-subunits. A putative regulatory mechanism of the rod photoresponse.

The cGMP phosphodiesterase (PDE) of retinal rods plays a central role in phototransduction. Illumination leads to its activation by a rod G-protein (Gt, transducin), thus causing a decrease in intracellular cGMP concentration, closure of plasma membrane cationic channels gated by cGMP, and development of the photoresponse. The PDE holoenzyme is an alpha beta gamma 2 tetramer. The alpha- and beta-subunits each contain one catalytic and one, or possibly two, noncatalytic cGMP-binding sites. Two identical gamma-subunits serve as protein inhibitors of the enzyme. Their inhibition is removed when they bind to Gt-GTP during PDE activation. Here we report that the noncatalytic cGMP-binding sites regulate the binding of PDE alpha beta with PDE gamma and as a result determine the mechanism of PDE activation by Gt. If the noncatalytic sites are empty, Gt-GTP physically removes PDE gamma from PDE alpha beta upon activation. Alternatively, if the noncatalytic sites are occupied by cGMP, Gt-GTP releases PDE gamma inhibitory action but remains bound in a complex with the PDE heterotetramer. The kinetic parameters of activated PDE in these two cases are indistinguishable. This mechanism appears to have two implications for the physiology of photoreceptor cells. First, the tight binding of PDE gamma with PDE alpha beta when the noncatalytic sites are occupied by cGMP may be responsible for the low level of basal PDE activity observed in dark-adapted cells. Second, occupancy of the noncatalytic sites ultimately controls the rate of PDE inactivation (cf. Arshavsky, V. Yu., and Bownds, M. D. (1992) Nature 357, 416-417), for the GTPase activity that terminates PDE activity is slower when these sites are occupied and Gt stays in a complex with PDE holoenzyme. In contrast GTPase acceleration is maximal when the noncatalytic sites are empty and Gt-PDE gamma dissociates from PDE alpha beta. Because cGMP levels are known to decrease upon illumination over a concentration range corresponding to the binding constants of the noncatalytic sites, the binding might be involved in determining the lifetime of activated PDE, after a single flash and/or during dark adaptation.     

Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

Retinal photoreceptor phosphodiesterase (PDE6), a key enzyme for phototransduction, consists of a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγs). Pαβ has two noncatalytic cGMP-binding sites. Here, using bovine PDE preparations, we show the role of these cGMP-binding sites in PDE regulation. Pαβγγ and its transducin-activated form, Pαβγ, contain two and one cGMP, respectively. Only Pαβγ shows [(3)H]cGMP binding with a K(d) ～ 50 nM and Pγ inhibits the [(3)H]cGMP binding. Binding of cGMP to Pαβγ is suppressed during its formation, implying that cGMP binding is not involved in Pαβγγ activation. Once bound to Pαβγ, [(3)H]cGMP is not dissociated even in the presence of a 1000-fold excess of unlabeled cGMP, binding of cGMP changes the apparent Stokes' radius of Pαβγ, and the amount of [(3)H]cGMP-bound Pαβγ trapped by a filter is spontaneously increased during its incubation. These results suggest that Pαβγ slowly changes its conformation after cGMP binding, i.e. after formation of Pαβγ containing two cGMPs. Binding of Pγ greatly shortens the time to detect the increase in the filter-trapped level of [(3)H]cGMP-bound Pαβγ, but alters neither the level nor its Stokes' radius. These results suggest that Pγ accelerates the conformational change, but does not add another change. These observations are consistent with the view that Pαβγ changes its conformation during its deactivation and that the binding of cGMP and Pγ is crucial for this change. These observations also imply that Pαβγγ changes its conformation during its activation and that release of Pγ and cGMP is essential for this change.     

Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of phosphodiesterase-5 by cyclic nucleotide-dependent protein kinase alters its catalytic and allosteric cGMP-binding activities.

In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem. 265, 14971-14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50-70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2-0.5 microM PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the Km value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 microM. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed.     

Allosteric sites of phosphodiesterase-5 (PDE5). A potential role in negative feedback regulation of cGMP signaling in corpus cavernosum.

To date, relative cellular levels of cGMP and cGMP-binding proteins have not been considered important in the regulation of smooth muscle or any other tissue. In rabbit penile corpus cavernosum, intracellular cGMP was determined to be 18 +/- 4 nM, whereas the cGMP-binding sites of types Ialpha and Ibeta cGMP-dependent protein kinase (PKG) and cGMP-binding cGMP-specific phosphodiesterase (PDE5) were 58 +/- 14 nM and 188 +/- 6 nM, respectively, as estimated by two different methods for each protein. Thus, total cGMP-binding sites (246 nM) greatly exceed total cGMP. Given this excess of cGMP-binding sites and the high affinities of PKG and PDE5 for cGMP, it is likely that a large portion of intracellular cGMP is associated with these proteins, which could provide a dynamic reservoir for cGMP. Phosphorylation of PDE5 by PKG is known to increase the affinity of PDE5 allosteric sites for cGMP, suggesting the potential for regulation of a reservoir of cGMP bound to this protein. Enhanced binding of cGMP by phosphorylated PDE5 could reduce the amount of cGMP available for activation of PKG, contributing to feedback inhibition of smooth muscle relaxation or other processes. This introduces a new concept for cyclic nucleotide signaling.     

cGMP binding to noncatalytic sites on mammalian rod photoreceptor phosphodiesterase is regulated by binding of its gamma and delta subunits.

The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, alpha beta gamma2) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, alpha beta gamma2 delta) can release and bind cGMP at both noncatalytic sites; the delta subunit is likely responsible for this difference in cGMP exchange rates. Removal of the delta and/or gamma subunits yields a catalytic alphabeta dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (KD >1 microM); cGMP binding affinity at the second site (KD approximately 60 nM) is reduced 3-4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of gamma subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the gamma subunit with the alphabeta dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells.     

Direct interaction of the inhibitory gamma-subunit of Rod cGMP phosphodiesterase (PDE6) with the PDE6 GAFa domains

Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.     

Structural and functional features in human PDE5A1 regulatory domain that provide for allosteric cGMP binding, dimerization, and regulation

The cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for dimerization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human PDE5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-dimerization occurs between GAF a modules (K(D) < 30 nM) and between GAF b modules (K(D) = 1-20 pM), and the sequence between the GAFs (Thr322-Asp403) contributes to dimer stability; 2) 176 amino acids (Val156-Gln331) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K(D) < 40 nM) than does the isolated R domain (K(D) = 110 nM) or holoenzyme (K(D) = 200 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met1-Glu321) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser102 phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into PDE5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to dimerization.     

Adenine nucleotide binding sites on beef heart F1-ATPase. Asymmetry and subunit location

Previously we have shown that beef heart mitochondrial F1 contains a total of six adenine nucleotide binding sites. Three "catalytic" sites exchange bound ligand rapidly during hydrolysis of MgATP, whereas three "noncatalytic" sites do not. The noncatalytic sites behave asymmetrically in that a single site releases bound ligand upon precipitation of F1 with ammonium sulfate. In the present study, we find this same site to be the only noncatalytic site that undergoes rapid exchange of bound ligand when F1 is incubated in the presence of EDTA at pH 8.0. Following 1000 catalytic turnovers/F1, the site retains the unique capacity for EDTA-induced exchange, indicating that the asymmetric determinants are permanent and that the three noncatalytic sites on soluble F1 do not pass through equivalent states during catalysis. Measurements of the rate of ligand binding at the unique noncatalytic site show that uncomplexed nucleotide binds preferentially. At pH 7.5, in the presence of Mg2+, the rate constant for ADP binding is 9 X 10(3) M-1 s-1 and for dissociation is 4 X 10(-4) s-1 to give a Kd = 50 nM. The rate of dissociation is 10 times faster in the presence of EDTA or during MgATP hydrolysis, and it increases rapidly at pH below 7. EDTA-induced exchange is inhibited by Mg2+, Mn2+, Co2+, and Zn2+ but not by Ca2+ and is unaffected by dicyclohexylcarbodiimide modification. The unique noncatalytic site binds 2-azido-ADP. Photolysis results in the labeling of the beta subunit. Photolabeling of a single high-affinity catalytic site under conditions for uni-site catalysis also results in the labeling of beta, but a different pattern of labeled peptides is obtained in proteolytic digests. The results demonstrate the presence of two different nucleotide binding domains on the beta subunit of mitochondrial F1.     

Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase.

The structure of cyclic GMP (cGMP)-binding (cGB), cGMP specific phosphodiesterase (PDE5) comprises several domains. We have used RT-PCR methods to clone the noncatalytic cGB domains of PDE5 from human colon cancer cell RNA and constructed glutathione-S-transferase (GST) fusion proteins to express and study the domains. One fragment showed 94% identity to bovine PDE5 and coded for the high affinity cGB domain of PDE5 (Val(156)-Asp(394), cGB-I). Another cloned fragment showed 92% identity to bovine PDE5 and coded for the phosphorylation site plus both high and low affinity cGB domains of PDE5 (Val(36)-Glu(529), cGB-II). Both fragments expressed as GST-cGB fusion proteins bound cGMP specifically, as determined by competitive [3H]-cGMP ligand binding. We found that cGB-I showed high affinity cGMP binding with K(d)=0.33 microM. cGB-II showed two cGMP binding sites with similar affinities and specificity to the native enzyme. cGB-II was phosphorylated by cGMP-dependent protein kinase (PKG) as reported for bovine PDE5. These data show that recombinant regulatory regions of PDE5 form cGB sites similar to native enzyme sites and confirm proposed domain functions. These results establish that recombinant fusion proteins of PDE5 domains may be used to further characterize the structure of PDE5.     

Purification and subunit composition of guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung.

The guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung was purified to apparent homogeneity by affinity chromography using 8-2-aminoethylthio-cGMP coupled to Sepharose 4B. The kinase activity was purified approximately 6000-fold with an overall recovery of approximately 20%. The product isolated by affinity chromatography contained both cGMP-binding and cGMP-dependent histone kinase activity, indicating that the enzyme was not dissociated into regulatory and catalytic components by the immobilized cGMP derivative. The enzyme had a molecular weight of approximately 165,000 and a sedimentation coefficient of 7.8 S. The purified kinase displayed several characteristics similar to that of the partially purified enzyme including specificity for cGMP and stimulation by high concentrations of magnesium. On sodium dodecyl sulfate gels, only one major polypeptide chain was present having a molecular weight of approximately 81,000. This subunit bound 1 mol of cGMP and exhibited cGMP-dependent protein kinase activity. It is proposed that the native enzyme consists of two identical subunits (Mr=81,000), each of which binds cGMP and catalyzes protein phosphorylation.     

Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 mumol min-1 (mg of protein)-1 when 1 microM cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 microM, respectively. Addition of calcium and calmodulin reduces the apparent Km for cGMP to 2-3 microM and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki's close to their relative Km values. Highly purified preparations of the enzyme contain a major protein band of Mr 74 000 that best correlates with enzyme activity. Proteins of Mr 59 000 and Mr 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150 000 by gel filtration suggests that the enzyme exists as a dimer of Mr 74 000 subunits. Phosphorylation of the enzyme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated gGMP phosphodiesterase and the Mr 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.     

cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1

cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.     

PDE5 is converted to an activated state upon cGMP binding to the GAF A domain

cGMP-specific, cGMP-binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N-terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time- and concentration-dependent reversible activation in response to cGMP, with the highest activation (9- to 11-fold) observed at low substrate concentrations (0.1 micro M cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non-activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildenafil than non-activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain-containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.     

Mechanism of transducin activation of frog rod photoreceptor phosphodiesterase. Allosteric interactiona between the inhibitory gamma subunit and the noncatalytic cGMP-binding sites

The rod photoreceptor phosphodiesterase (PDE) is unique among all known vertebrate PDE families for several reasons. It is a catalytic heterodimer (alphabeta); it is directly activated by a G-protein, transducin; and its active sites are regulated by inhibitory gamma subunits. Rod PDE binds cGMP at two noncatalytic sites on the alphabeta dimer, but their function is unclear. We show that transducin activation of frog rod PDE introduces functional heterogeneity to both the noncatalytic and catalytic sites. Upon PDE activation, one noncatalytic site is converted from a high affinity to low affinity state, whereas the second binding site undergoes modest decreases in binding. Addition of gamma to transducin-activated PDE can restore high affinity binding as well as reducing cGMP exchange kinetics at both sites. A strong correlation exists between cGMP binding and gamma binding to activated PDE; dissociation of bound cGMP accompanies gamma dissociation from PDE, whereas addition of either cGMP or gamma to alphabeta dimers can restore high affinity binding of the other molecule. At the active site, transducin can activate PDE to about one-half the turnover number for catalytic alphabeta dimers completely lacking bound gamma subunit. These results suggest a mechanism in which transducin interacts primarily with one PDE catalytic subunit, releasing its full catalytic activity as well as inducing rapid cGMP dissociation from one noncatalytic site. The state of occupancy of the noncatalytic sites on PDE determines whether gamma remains bound to activated PDE or dissociates from the holoenzyme, and may be relevant to light adaptation in photoreceptor cells.     

Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium

Recently, we recognized two genes, gbpA and gbpB, encoding putative cGMP-binding proteins with a Zn(2+)-hydrolase domain and two cyclic nucleotide binding domains. The Zn(2+)-hydrolase domains belong to the superfamily of beta-lactamases, also harboring a small family of class II phosphodiesterases from bacteria and lower eukaryotes. Gene inactivation and overexpression studies demonstrate that gbpA encodes the cGMP-stimulated cGMP-phosphodiesterase that was characterized biochemically previously and was shown to be involved in chemotaxis. cAMP neither activates nor is a substrate of GbpA. The gbpB gene is expressed mainly in the multicellular stage and seems to encode a dual specificity phosphodiesterase with preference for cAMP. The enzyme hydrolyses cAMP approximately 9-fold faster than cGMP and is activated by cAMP and cGMP with a K(A) value of approximately 0.7 and 2.3 microM, respectively. Cells with a deletion of the gbpB gene have increased basal and receptor stimulated cAMP levels and are sporogeneous. We propose that GbpA and GbpB hydrolyze the substrate in the Zn(2+)-hydrolase domain, whereas the cyclic nucleotide binding domains mediate activation. The human cGMP-stimulated cAMP/cGMP phosphodiesterase has similar biochemical properties, but a completely different topology: hydrolysis takes place by a class I catalytic domain and GAF domains mediate cGMP activation.     

Conformational conversion of PDE5 by incubation with sildenafil or metal ion is accompanied by stimulation of allosteric cGMP binding

Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1 h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30 °C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn⁺⁺ or Mg⁺⁺ caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.     

Allosteric activation of cGMP-specific,cGMP-binding phosphodiesterase (PDE5) by cGMP

The effects of cGMP binding on the catalytic activity of cGMP-specific, cGMP-binding phosphodiesterase (PDE5) are unclear because cGMP interacts with both allosteric and catalytic sites specifically. We studied the effects of cGMP on the hydrolysis of a fluorescent substrate analogue, 2'-O-anthraniloyl cGMP, by PDE5 partially purified from rat cerebella. The preparation contained PDE5 as the major cGMP-PDE activity and was not contaminated with cAMP- or cGMP-dependent protein kinases. The Hill coefficients for hydrolysis of the analogue substrate were around 1.0 in the presence of cGMP at concentrations <0.3 microM, while they increased to 1.5 at cGMP concentrations >1 microM, suggesting allosteric activation by cGMP at concentrations close to the bulk binding constant of the enzyme. Consistent with an allosteric activation, increasing concentrations of cGMP enhanced the hydrolysis rate of fixed concentrations of 2'-O-anthraniloyl cGMP, which overcame competition between the two substrates. Such activation was not observed with cAMP, cyclic inosine 3',5'-monophosphate, or 2'-O-monobutyl cGMP, indicating specificity of cGMP. These results demonstrate that cGMP is a specific and allosteric activator of PDE5, and suggest that in cells containing PDE5, such as cerebellar Purkinje cells, intracellular cGMP concentrations may be regulated autonomously through effects of cGMP on PDE5.