Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Heterotrimeric G-proteins mediate between receptors and effectors, acting as molecular clocks. G-protein interactions with activated receptors catalyze the replacement of GDP bound to the alpha-subunit with GTP. alpha-Subunits then modulate the activity of downstream effectors until the bound GTP is hydrolyzed. In several signal transduction pathways, including the cGMP cascade of photoreceptor cells, the relatively slow GTPase activity of heterotrimeric G-proteins can be significantly accelerated when they are complexed with corresponding effectors. In the phototransduction cascade the GTPase activity of photoreceptor G-protein, transducin, is substantially accelerated in a complex with its effector, cGMP phosphodiesterase. Here we characterize the stimulation of transducin GTPase by a set of 23 mutant phosphodiesterase gamma-subunits (PDE gamma) containing single alanine substitutions within a stretch of the 25 C-terminal amino acid residues known to be primarily responsible for the GTPase regulation. The substitution of tryptophan at position 70 completely abolished the acceleration of GTP hydrolysis by transducin in a complex with this mutant. This mutation also resulted in a reduction of PDE gamma affinity for transducin, but did not affect PDE gamma interactions with the phosphodiesterase catalytic subunits. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDE gamma to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of PDE gamma in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules.
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PMID:An effector site that stimulates G-protein GTPase in photoreceptors. 778 90

We seek to define the influence of retinal cGMP phosphodiesterase (PDE) on the GTPase activity of transducin (T). A novel stopped-flow/fast filtration apparatus [Antonny, B., et al. (1993) Biochemistry 32, 8646-8653] is used to deliver T alpha GTP free of rod outer segment (ROS) membranes to a suspension of phospholipid vesicles bearing holoPDE. As measured by a pH electrode, the decay of cGMP hydrolysis from these samples, which contain no other proteins but T alpha and holoPDE, requires GTP hydrolysis and occurs in 40 s. The addition of T beta gamma to the vesicles does not accelerate this deactivation. When ROS membranes are urea-stripped, reconstituted with transducin + holoPDE, and illuminated, the injection of an amount of GTP that is substoichiometric to holoPDE gives a cGMP hydrolysis pulse that lasts for 30 s. However, the same reconstitution performed with ROS stripped by extensive dilution in isotonic buffer results in a deactivation time of only 8 s, which resembles the 7 s observed with native ROSs. With these isotonically stripped ROSs, when GTP injection comes after a first injection with GTP gamma S, the cGMP hydrolysis pulse is lengthened and lasts for 17 s; with urea-washed ROS, no such lengthening is observed. These results clearly demonstrate that holoPDE by itself cannot enhance the GTPase activity of transducin, even when the two proteins are localized on a membrane surface. Instead, they point to the existence of a membrane-bound, urea-sensitive protein factor that activates the GTPase of T alpha in the transducin-holoPDE complex.
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PMID:Modulation of the GTPase activity of transducin. Kinetic studies of reconstituted systems. 780 83

The photoreceptor G-protein, transducin, belongs to the class of heterotrimeric GTP-binding proteins that transfer information from activated seven-span membrane receptors to effector enzymes or ion channels. Like other G-proteins, transducin acts as a molecular clock. It is activated by photoexcited rhodopsin which catalyzes the exchange of transducin-bound GDP for GTP and then stays active until bound GTP is hydrolyzed by an intrinsic GTPase activity. Our previous study on the components of the amphibian phototransduction cascade (Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417) has shown that transducin GTPase can be significantly accelerated by the target enzyme, cGMP phosphodiesterase (PDE), and more specifically its gamma-subunit (PDE gamma). Here we report that an analogous mechanism is present in bovine photoreceptors. Addition of recombinant PDE gamma to the test photoreceptor membranes which retain transducin but are depleted of endogenous PDE causes a significant acceleration of transducin GTPase activity. A similar effect was observed with the PDE holoenzyme, but not with the complex of PDE alpha- and beta-subunits prepared by a limited proteolysis of PDE with trypsin. The activating effect of PDE gamma is increased as test membrane concentration increases, exceeding 20-fold at rhodopsin concentrations over 80 microM and approaching the rate of the photoresponse turnoff. This suggests either that photoreceptor membranes contain a further factor which is essential for PDE-dependent regulation of transducin-bound GTP hydrolysis or that components of the phototransduction cascade interact in a cooperative manner. We also report that the GTPase-activating epitope is located within the C-terminal third of PDE gamma: the peptide corresponding to the 25 C-terminal amino acid residues of PDE gamma can accelerate transducin GTPase almost as well as the full-length PDE gamma. A part of the GTPase activating epitope is located within the 3 C-terminal amino acid residues: the truncation PDE gamma mutant lacking these residues accelerates transducin GTPase considerably less than the whole length PDE gamma.
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PMID:Regulation of transducin GTPase activity in bovine rod outer segments. 805 Oct 70

The cGMP phosphodiesterase (PDE) of retinal rod outer segments (ROS) is activated by the GTP-bound form of the G protein, transducin (Gt alpha). This activation can be reversed by the inhibitory gamma subunit of PDE through two distinct mechanisms: acceleration of GTP hydrolysis and direct inactivation independent of GTP hydrolysis. We have found that acceleration of Gt alpha GTPase by PDE gamma does not occur upon formation of a Gt alpha PDE gamma complex but rather reflects enhanced activity toward this complex of a membrane-bound GTPase accelerating protein. GTPase rate constants for Gt alpha in the presence of 3.3 microM PDE gamma were as high as 0.7 s-1 with hypotonically washed ROS membranes at 40 microM rhodopsin but were more than 10-fold lower when protein-free vesicles containing ROS lipids were substituted for ROS membranes. Acceleration of Gt alpha GTPase by PDE gamma was also barely detectable at low ROS concentrations (e.g. 4 microM rhodopsin) or if ROS treated with trypsin or urea were used. GTPase-independent inactivation by PDE gamma occurred efficiently at much lower membrane concentrations. Inhibition of Gt alpha-activated PDE was much slower than inhibition of PDE alpha beta by PDE gamma. Effects of PDE gamma upon successive additions of GTP suggested formation of a complex of PDE gamma and Gt alpha-GDP that is refractory to reactivation.
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PMID:Enhancement of rod outer segment GTPase accelerating protein activity by the inhibitory subunit of cGMP phosphodiesterase. 820 35

Hydrolysis of GTP by the photoreceptor G protein transducin (Gt alpha) was found to occur with kinetics identical to the inactivation of its effector cGMP phosphodiesterase (PDE), but was too slow (tens of seconds) in dilute rod outer segment (ROS) suspensions to account for subsecond recovery of the light response. Raising the concentration of ROS membranes increased the rates of GTP hydrolysis and PDE inactivation in parallel as much as 6-fold. Holo-PDE and its gamma subunit had weak effects on GTPase kinetics (< 1.6-fold and < 1.3-fold, respectively). ROS membranes stripped of PDE retained approximately 90% of a GTPase accelerating activity that was protease sensitive, indicating that they contain a GTPase-accelerating factor distinct from PDE.
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PMID:A GTPase-accelerating factor for transducin, distinct from its effector cGMP phosphodiesterase, in rod outer segment membranes. 824 Aug 15

In retinal rods photoexcited rhodopsin (R*) catalyses the activation of transducin (T) by GTP, which in turn activates the cGMP phosphodiesterase (PDE). The ensuing decrease in cGMP concentration reduces the cell membrane's channel conductance. To account for the kinetics of the response to light, all underlying biochemical reactions must reach maximum speed and be turned off within a second. Kinetic analysis of transducin activation suggests that because of the fast lateral diffusion of T, the rate-limiting step is not the collision between R* and T but the entry of GTP after the release fo GDP from the R*-bound T alpha. T alpha-GTP dissociates from both R* and T beta gamma and diffuses through the cytoplasm to activate PDE. In suspensions of bovine rod outer segments, time-resolved microcalorimetry yields rates of approximately 1-2 s-1 for the GTPase of T alpha and the correlated deactivation of PDE. But for isolated T alpha-GTP the single turnover GTPase rate measured by a stopped-flow technique is only 0.05 s-1. To activate PDE, T alpha-GTP binds tightly to the PDE gamma subunit. In vitro the soluble T alpha-GTP.PDE gamma complex dissociates from activated PDE alpha beta. Thus PDE gamma might be the GTPase activator of T alpha, but no GTPase acceleration was observed in isolated T alpha-GTP.PDE gamma. The GTPase activation must depend on the interaction of T alpha-GTP.PDE gamma with membrane-bound PDE alpha beta.
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PMID:The G protein cascade of visual transduction: kinetics and regulation. 829 14

The single-turn GTP hydrolysis by isolated and soluble transducin has been time-resolved using a rapid flow filtration technique which takes advantage of the GTP-requiring detachment of transducin alpha-subunits (T alpha) from photoactivated rhodopsin (R*). Illuminated rod outer segment (ROS) fragments to which holo-transducin is tightly bound are retained on a syringe filter that is washed continuously with a buffer containing no GTP. When the flow is switched to a buffer with GTP, T alpha GTP is specifically eluted and injected into a cuvette where GTP hydrolysis is monitored via the associated change in the T alpha intrinsic tryptophan fluorescence. Low concentrations of GTP elute the complete pool of T alpha from the filter-retained ROS fragments in less than 1 s. This directly demonstrates that, upon GTP loading, T alpha becomes instantly soluble in physiological buffers (120 mM KC1 and 2 mM MgCl2). When all alone, T alpha hydrolyzes its bound GTP in 21 +/- 1 s (1/e time at 25 degrees C). Replacing chloride by other anions increases the GTPase rate by 2-fold. The K50 for chloride inhibition of GTPase is approximately 2 mM. Slower GTP hydrolysis is observed for cholera-toxin-modified transducin or when GTP alpha S (Sp) replaces GTP in the eluting buffer. No signal is observed when GTP gamma S is used. The GTPase rate is unaffected when T alpha GTP binds to the inhibitory subunit (PDE gamma) of the cGMP phosphodiesterase (PDE), although this binding is fast and of high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:GTP hydrolysis by purified alpha-subunit of transducin and its complex with the cyclic GMP phosphodiesterase inhibitor. 839 13

The activation and recovery phases of the murine rod photo-response were determined from corneal electroretinograms (ERGs) obtained in response to pairs of full-field flashes producing 50-10(5) photoisomerized rhodopsins (R*) per rod. The a-wave component of the ERG in response to the initial flash provided a well established measure of the activation phase of the rod response. The amplitude of the a-wave response to an intense second flash (45,000 R*) delivered 0.2-5 seconds (s) after the first flash was used to reconstruct the recovery phase of the response. For 160-3000 R* rod-1, recovery curves were isomorphic, translating on the time axis such that each e-fold increase in R* produced an incremental recovery delay of tau c = 210 +/- 50 ms (mean +/- SD). For initial flashes producing > 3000 R*, recovery curves lost their initial isomorphism and half-times had intensity dependence exceeding 1 s per e-fold increase in R*. We conclude that for flashes producing < 3000 R*, the effective lifetime of these R* is not > 210 ms. Two extant and non-mutually exclusive hypotheses are discussed that can account for the sharp increase in recovery times from flashes producing > 3000 R*. They are as follows: (1) approximately 0.03% of R* have a lifetime exceeding 1 s; and (2) the gamma subunit of phosphodiesterase (PDE gamma) serves as a GTPase-activating factor, and 3000 R* produce sufficient activated G-protein (G*) to exceed the total quantity of PDE gamma subunits such that excess G* must wait for unoccupied PDE gamma to inactivate via GTP hydrolysis.
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PMID:Recovery phase of the murine rod photoresponse reconstructed from electroretinographic recordings. 855 40

A rich variety of mechanisms govern the inactivation of the rod phototransduction cascade. These include rhodopsin phosphorylation and subsequent binding of arrestin; modulation of rhodopsin kinase by S-modulin (recoverin); regulation of G-protein and phosphodiesterase inactivation by GTPase-activating factors; and modulation of guanylyl cyclase by a high-affinity Ca(2+)-binding protein. The dependence of several of the inactivation mechanisms on Ca2+i makes it difficult to assess the contributions of these mechanisms to the recovery kinetics in situ, where Ca2+i is dynamically modulated during the photoresponse. We recorded the circulating currents of salamander rods, the inner segments of which are held in suction electrodes in Ringer's solution. We characterized the response kinetics to flashes under two conditions: when the outer segments are in Ringer's solution, and when they are in low-Ca2+ choline solutions, which we show clamp Ca2+i very near its resting level. At T = 20-22 degrees C, the recovery phases of responses to saturating flashes producing 10(2.5)-10(4.5) photoisomerizations under both conditions are characterized by a dominant time constant, tau c = 2.4 +/- 0.4 s, the value of which is not dependent on the solution bathing the outer segment and therefore not dependent on Ca2+i. We extended a successful model of activation by incorporating into it a first-order inactivation of R*, and a first-order, simultaneous inactivation of G-protein (G*) and phosphodiesterase (PDE*). We demonstrated that the inactivation kinetics of families of responses obtained with Ca2+i clamped to rest are well characterized by this model, having one of the two inactivation time constants (tau r* or tau PDE*) equal to tau c, and the other time constant equal to 0.4 +/- 0.06 s.
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PMID:The kinetics of inactivation of the rod phototransduction cascade with constant Ca2+i. 874 28

The naturally occurring phospholipid, lysophosphatidylcholine (lyso-PC), regulates a broad range of cell processes, including gene transcription, mitogenesis, monocyte chemotaxis, smooth muscle relaxation, and platelet activation. Despite the growing list of cellular effects attributable to lyso-PC, the mechanism(s) by which it alters cell function have not been elucidated. In this report, we have examined the effects of exogenous lyso-PC on signal transduction processes within a variety of lyso-PC-responsive cells, including human platelets, monocyte-like THP-1 cells, and the megakaryoblastic cell line, MEG-01. Pretreatment of each of these cells with increasing concentrations of lyso-PC (25-150 microg/ml) was associated with a progressive increase in the cytosolic concentration of cAMP. The accumulation of cAMP in platelets correlated closely with the ability of lyso-PC to inhibit multiple platelet processes, including platelet aggregation, agonist-induced protein kinase C activation, thromboxane A2 generation, and the tyrosine phosphorylation of platelet proteins. In each of the cell types examined, the ability of lyso-PC to increase the cellular levels of cAMP was synergistically enhanced by pretreating the cells with the cAMP phosphodiesterase inhibitor, theophylline (5 mM), and was specifically inhibited by the P-site inhibitor of adenylyl cyclase, 2,5-dideoxyadenosine. A role for the stimulatory G-protein, Gs, in the lyso-PC-induced activation of adenylyl cyclase was suggested by the ability of the GTPase inhibitor, guanylyl 5'-thiophosphate (0.2 mM), to inhibit the lyso-PC-stimulated increase in cAMP, and also by the ability of cholera toxin to inhibit increases in membrane GTPase activity in response to lyso-PC. The functional significance of lyso-PC-induced activation of adenylyl cyclase was investigated in MEG-01 cells. Treatment of these cells with either lyso-PC or dibutyryl cAMP for 36-40 h resulted in a 3-5-fold increase in the surface expression of the natural anticoagulant protein, thrombomodulin (TM). The ability of lyso-PC to increase TM expression was abolished by pretreating these cells with the adenylyl cyclase inhibitor, 2,5-dideoxyadenosine, whereas the dibutyryl cAMP-induced increase in TM remained insensitive to adenylyl cyclase inhibition. These studies define an important role for the adenylyl cyclase signaling system in mediating cellular effects induced by lyso-PC.
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PMID:The bioactive phospholipid, lysophosphatidylcholine, induces cellular effects via G-protein-dependent activation of adenylyl cyclase. 890 Feb


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