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)

1. The kinetics of photoresponses to flashes and steps of light of rods, from the retina of the newt Triturus cristatus, were analysed by recording the membrane current with a suction electrode. 2. In dark-adapted conditions the relation between the normalized amplitude of the photoresponse at a fixed time 1 s after the onset of light and the light intensity could be fitted by an exponential or a polynomial relation. In the presence of a steady bright light the same relation could be fitted by a Michaelis-Menten relation. 3. The kinetics of photoresponses to light stimuli was reconstructed using a model in which: (i) three molecules of guanosine 3'.5'-cyclic monophosphate (cyclic GMP) open a light-sensitive channel; (ii) light activates the enzyme phosphodiesterase, which hydrolyses cyclic GMP, thus closing light-sensitive channels: (iii) Ca2+ ions permeate through light-sensitive channels: and (iv) intracellular Ca2+ inhibits, in a co-operative way, the enzyme cyclase, which synthesizes cyclic GMP. 4. The model reproduces the shortening of the time to peak of brief flash photoresponses from about 1080 ms to about 690 ms with brighter lights. The model also explains the shortening of the time to peak to 350 ms observed in the presence of a steady light and the lack of a further acceleration with brighter flashes of lights. 5. The presence in the model of an intracellular calcium buffer accounts for the partial reactivation of the photocurrent following a step of light, lasting several seconds. The time course of this reactivation is not accelerated by a steady bright light both experimentally and in the model. 6. After the extinction to a long step of light the photocurrent showed a rapid partial reactivation, which was followed by a slow component of the photoresponse which extinguished with a rate constant of about 0.05 s-1. The model explains the origin of this slow component by assuming that the inactivation of excited rhodopsin is partially reversible. 7. The model is also able to explain the particular changes of kinetics when different amounts of exogenous calcium buffers are incorporated into rods (Torre, Matthews & Lamb, 1986).
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PMID:Kinetics of phototransduction in retinal rods of the newt Triturus cristatus. 262 32

We have previously reported that the purified GDP-bound alpha-subunit of the GTP-binding protein transducin (TD), present in outer segments of retinal rod cells (ROS), serves as a high affinity substrate (Km = 1 microM) for protein kinase C (PKC) [Zick et al. (1986) Proc. natn. Acad. Sci., U.S.A. 83, 9294-9297]. In the present study we demonstrate that TD-alpha undergoes phosphorylation by PKC when present in its native form in intact ROS membranes. This phosphorylation is inhibited by GTP-gamma-S which activates TD, suggesting that it is only the inactive conformation of TD-alpha that serves as a substrate for PKC. Indeed, both vanadate and AlF4, that confer an active conformation on TD-alpha-GDP, inhibit PKC-mediated phosphorylation of purified TD-alpha-GDP. We demonstrate that the purified beta subunit of TD also serves as an in vitro substrate for PKC. Moreover, following their phosphorylation, both TD-alpha and beta form high affinity complexes with PKC. This is evident from the findings that PKC coprecipitates with both the alpha and beta subunits of TD when the latter are immunoprecipitated by their respective antibodies. PKC phosphorylates additional ROS proteins of 36, 48 and 92 kDa, tentatively identified as rhodopsin, arrestin and the cGMP-phosphodiesterase. Taken together our results strongly suggest that phosphorylation of TD is of physiological relevance and that through phosphorylation of endogenous ROS proteins, PKC could play a key role in regulating phototransduction.
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PMID:Protein kinase C-mediated phosphorylation of retinal rod outer segment membrane proteins. 264 84

Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4- acts as an analogue of the gamma-phosphate of GTP in the nucleotide site. We have investigated the action of AIF4- or of BeF3- on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4- or BeF3-, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha-GDP and T alpha-GDP alpha S are activable by fluorides, but not T alpha-GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino-fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be-F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal-fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP. 282 23

Recent progress in understanding phototransduction has come primarily from studies on cell-free systems. To investigate the transduction process under physiological conditions, a fully functional preparation of retinal rod outer segments without attached inner segments was developed that allows electrical recording of light-sensitive current during intracellular dialysis with defined solutions. No light-sensitive current is recorded from detached outer segments dialyzed with nucleotide-free solutions, whereas cells detached from the retina into Ringer's solution containing 3-isobutyl-1-methyl-xanthine (a phosphodiesterase inhibitor) develop a light-sensitive inward dark current. This indicates that there is a basal level of cGMP-specific phosphodiesterase activity in the dark. Detached outer segments dialyzed with greater than or equal to 20 microM cGMP rapidly develop a light-suppressible current. A current of similar magnitude is generated more slowly during dialysis with a 50-fold greater concentration of GTP. Apparently, cGMP can be synthesized from GTP by guanylate cyclase in the outer segment. Cells dialyzed with cGMP alone show a reduced light sensitivity that is restored to normal by addition of 20 microM GTP. This action of GTP is antagonized by guanosine 5'-[beta-thio]diphosphate. These findings are in good agreement with biochemical evidence indicating that a GTP-binding protein (transducin) plays a pivotal role in the generation of responses to light. The recovery of photocurrent following a brief flash is delayed or abolished by dialysis with solutions that lack ATP or contain guanosine 5'-[gamma-thio]triphosphate, a nonhydrolyzable GTP analog. These results support the view that both GTP hydrolysis by activated transducin and ATP-dependent phosphorylation of a rhodopsin photoproduct are necessary for termination of the transduction process.
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PMID:Intracellular biochemical manipulation of phototransduction in detached rod outer segments. 282 76

The hypothesis that cyclic GMP is the internal transmitter of retinal rod phototransduction, when combined with the observations that 8-bromo-cyclic GMP opens the cyclic GMP-dependent outer segment conductance and that rods into which 8-bromo-cyclic GMP has been injected still respond to light, predicts that the light-activated phosphodiesterase (EC 3.1.4.17) must catalyze the hydrolysis of 8-bromo-cyclic GMP. This hypothesis was tested by measuring light-activated toad rod disk membrane phosphodiesterase with a pH assay technique. Phosphodiesterase-catalyzed hydrolysis of 8-bromo-cyclic GMP was confirmed: at pH 8.0, total proton production after flash activation was identical to total amount of 8-bromo-cyclic GMP added as substrate. Photoactivated phosphodiesterase was remarkably less efficient in catalyzing the hydrolysis of 8-bromo-cyclic GMP than of cyclic GMP: Vmax for 8-bromo-cyclic GMP was 0.063 M/M rhodopsin/s, whereas that for cyclic GMP was 11 M/M rhodopsin/s--170 times greater. The Km for 8-bromo-cyclic GMP was 160 microM, and for cyclic GMP, 590 microM. 8-bromo-cyclic GMP competitively inhibited phosphodiesterase-catalyzed hydrolysis of cyclic GMP with a Ki of 1.2 mM. Complete reaction progress curves were analyzed for obedience to Michaelis-Menten kinetics: cyclic GMP hydrolysis, 8-bromo-cyclic GMP hydrolysis, and cyclic GMP hydrolysis in the presence of 8-bromo-cyclic GMP as competitive inhibitor were found to follow the integrated form of the Michaelis-Menten equation over the time course of the reactions, assuming phosphodiesterase was activated as a step. The kinetic parameters extracted from reaction progress curves were consistent with those derived from analysis of the initial velocity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetics of the hydrolysis of 8-bromo-cyclic GMP by the light-activated phosphodiesterase of toad rods. 282 48

The bifunctional reagents para-phenyldimaleimide and maleimidobenzoyl-N-hydroxysuccinimide ester were used to chemically cross-link the subunits of the transducin and cGMP phosphodiesterase (PDE) complexes of bovine rod photoreceptor cells. The cross-linked products were identified by Western immunoblotting using antisera against purified subunits of transducin (T alpha and T beta gamma) and PDE. Oligomeric cross-linked products of transducin subunits as large as (T alpha beta gamma)3 were observed in the latent form of transducin with bound GDP. In addition to the expected T alpha beta and T beta gamma cross-linked products, a (T alpha gamma)2 structure was detected. The close proximity of T alpha and T gamma suggests that T gamma may play a role in conferring the specificity of the interaction between T alpha and rhodopsin. Most of the oligomeric cross-linked structures between T alpha and T beta gamma were diminished in the activated form of transducin, with guanosine 5'-(beta, gamma-imidotriphosphate) (Gpp(NH)p) bound. However, cross-linking between T beta and T gamma was not altered. These results suggest that transducin exists as an oligomer in solution which dissociates upon the binding of Gpp(NH)p. To identify the possible interacting domains between the T alpha, T beta, and T gamma subunits, the cross-linked products were subjected to limited tryptic proteolysis. Several cross-linked tryptic peptides of transducin subunits were found and include the cross-linked products of the N terminus 15-kDa fragment of T beta and the C terminus 5-kDa fragment of T alpha, T gamma and the 12-kDa fragment of T alpha, T gamma and the 15-kDa as well as the 23-kDa fragments of T beta, and an intra-T alpha cross-linked product of the 2- and 21-kDa fragments. These results have allowed the construction of a topographical model for the transducin subunits. The organization of the subunits of PDE (P alpha, P beta, and P gamma) was also studied. The formation of the high molecular size cross-linked products of PDE resulted in the concurrent loss of the P beta and P gamma subunits, suggesting that they are in close proximity. Finally, the interaction between transducin and PDE was examined by chemical cross-linking of transducin-Gpp(NH)p and PDE. Two additional cross-linked products of 180 and 210 kDa were obtained which could be due to the cross-linking of T alpha or T beta with P alpha beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Chemical cross-linking of bovine retinal transducin and cGMP phosphodiesterase. 283 96

An antibody (AS/7) prepared against the carboxyl-terminal decapeptide of the alpha subunit of transducin (alpha T) has been used in various reconstitution studies aimed at characterizing the role of the carboxyl-terminal domain in the different functional activities of transducin. The peptide-specific antibody is a potent inhibitor of the rhodopsin-stimulated GTPase activity in phospholipid vesicle systems containing pure rhodopsin and pure holo-transducin, or rhodopsin and the purified alpha T and beta/gamma (beta gamma T) subunit components, with the highest levels of inhibition (80-95%) occurring under conditions where the molar ratio of holo-transducin (or alpha T) to AS/7 approximately equal to 1. The inhibition of the receptor-stimulated GTPase does not represent an interference in the interactions between the alpha T subunit and the beta gamma T complex, since essentially identical levels of inhibition are observed when AS/7 is preincubated with either free alpha T, holo-transducin, or alpha T in the presence of excess beta gamma T, prior to assay. The AS/7-induced inhibition also does not appear to reflect an alteration in the ability of alpha T to bind or hydrolyze GTP and, in fact, the incubation of alpha T with AS/7 results in a stimulation of the intrinsic GTPase activity for alpha T alone (i.e. in the absence of rhodopsin). Thus, we conclude that the inhibition of the rhodopsin-stimulated GTPase activity by AS/7 is due to the direct blocking (by the antibody) of rhodopsin-alpha T interactions. While AS/7 is capable of uncoupling rhodopsin-transducin interactions, it appears to promote the stimulation of the cyclic GMP phosphodiesterase (PDE) by an activated alpha T subunit. Specifically, when the pure alpha T-guanosine 5-O-(3-thiotriphosphate) (alpha TGTP gamma S) species is preincubated with AS/7 prior to its addition to an assay solution containing PDE, there is at least a 4-fold increase in the resultant cyclic GMP hydrolysis relative to the activities measured with alpha TGTP gamma S, alone, or with alpha TGTP gamma S preincubated with nonimmune (control) rabbit IgG. The AS/7-induced promotion is specific for the active form of alpha T; the inactive alpha TGDP species does not stimulate PDE activity either in the presence or absence of the antibody. The different effects by AS/7 on the various activities of the alpha T subunit highlight the existence of distinct functional domains on alpha T.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:An antibody directed against the carboxyl-terminal decapeptide of the alpha subunit of the retinal GTP-binding protein, transducin. Effects on transducin function. 283 85

The successful reconstitution of rhodopsin, the rod outer segment (ROS) G protein, and the ROS phosphodiesterase (PDE) into partially polymerized bilayer membranes is described. Purified bovine rhodopsin (Rh) was inserted into performed partially polymerized lipid vesicles. Sonicated vesicles composed of approximately equal moles of dioleoylphosphatidylcholine (DOPC) (or 1-palmitoyl-2-oleoyl-phosphatidylcholine) and 1,2-bis(octadeca-2,4-dienoyl)phosphatidylcholine (DENPC) were photolyzed with 254-nm light to polymerize the DENPC and form domains of DOPC and polyDENPC in the vesicle wall. Rh-octyl glucoside (OG) micelles were slowly added to the vesicle suspension to give 15 mM OG (below the OG critical micelle concentration). The suspension was incubated and then dialyzed and purified on a sucrose gradient. Ultracentrifugation revealed a major Rh-lipid band which was harvested and found to contain a 100 +/- 10 phosphatidylcholine to rhodopsin ratio (Rh-polyDENPC/DOPC). The orientation of Rh in the membrane was determined by limited proteolytic digestion of Rh and by competitive inhibition of monoclonal antibody binding to solubilized disk membranes. Results were compared with control membranes of Rh-DOPC (1:43) prepared by insertion and Rh-phospholipid membranes prepared by detergent dialysis. Visual inspection of thermolysin proteolytic patterns of Rh indicates one major population cleaved at the carboxy terminus, as is found in disk membranes with an asymmetric arrangement of Rh. In contrast, proteolysis of a Rh-egg PC/PE (1:50/50) membrane (detergent dialysis) produced two Rh populations, which indicates a symmetric arrangement of Rh. The Rh-polyDENPC/DOPC (1:100) membranes were allowed to compete with solubilized, immobilized disk membranes for the monoclonal antibody R2-15 (specific for the amino-terminal region of Rh). They were intermediate between the asymmetric ROS disk membranes and the symmetric dialysis membranes in their ability to bind the R2-15 monoclonal antibody. The data indicate approximately 80% of the Rh's in Rh-polyDENPC/DOPC are in the normal orientation found in disks. These Rh-containing polymerized bilayer membranes demonstrated functionality as determined by chemical regeneration, kinetic spectrophotometry, and cGMP cascade reconstitution experiments. In the latter experiments the peripheral proteins, ROS G protein and PDE, bound with comparable efficiency to both the polymerized PC bilayers and egg PC bilayers. Thus the biocompatibility of the phosphatidylcholine membrane surface was maintained after polymerization of DENPC.
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PMID:Reconstitution of rhodopsin and the cGMP cascade in polymerized bilayer membranes. 284 Sep 46

The vast majority of extracellular signals alters cell function by activating cell surface receptors. The transmembranous signalling process initiated by an activated receptor leads to the generation of an intracellular signal and eventually to a cellular response. In contrast to receptors that are permanently coupled to an enzyme or an ion channel representing the effector, a large number of surface receptors for hormones, neurotransmitters and receptors for exogenous chemical or physical stimuli reversibly interacts with membranous signal transduction components which, in turn, regulate intracellular messenger-generating effectors. The transducer molecules isolated so far form a family of guanine nucleotide-binding proteins (G- or N-proteins). All isolated G-proteins are composed of three different subunits (alpha, beta, gamma). The alpha-subunit, which is specific for the individual G-protein, binds and hydrolyzes GTP and is target of ADP-ribosylating bacterial toxins. Hormone-induced activation of a receptor causes interaction with the alpha-subunit of a G-protein and the exchange of bound GDP with GTP. The GTP-bound form of the alpha-subunit represents the active form of the G-protein, which is capable of stimulating or inhibiting the respective effector. The active state of the alpha-subunit is terminated by its inherent GTPase activity causing hydrolysis of bound GTP. The beta gamma-complexes of G-proteins are structurally very similar and functionally interchangeable; they appear to dissociate from the alpha-subunits during receptor activation of the G-protein. Possible functions of the beta gamma-complex are to anchor the non-activated G-protein in the membrane, to facilitate G-protein-receptor interaction, and to promote the inactive state of the alpha-subunit. G-protein-regulated effectors include enzymes, ion channels and probably transporters. The best studied G-protein-regulated enzyme is the retinal cyclic GMP-phosphodiesterase which is activated by bleached rhodopsin via the tissue-specific G-protein, termed transducin. The ubiquitously occurring membrane-bound adenylate cyclase is under dual control by families of stimulatory and inhibitory receptors, acting via G-proteins called Gs and Gi, respectively. Moreover, the receptor control of phospholipases A2 and C and probably of phospholipase D most likely involves G-proteins which have not yet been identified. Finally, the activity of NADPH oxidase of neutrophils and that of cyclic AMP phosphodiesterases in liver and fat cells may be regulated via G-proteins. Modulations of non-enzymatic effectors are reviewed elsewhere.
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PMID:[Guanidine nucleotide binding proteins as membrane signal transduction components and regulators of enzymatic effectors]. 284 11

Light activation of GTP binding to G-protein and its eventual hydrolysis are hypothesized to lead to activation and inactivation of cGMP phosphodiesterase (PDE) in vertebrate rod disk membranes (RDM). However, the reported GTPase rate of 3 per minute is too slow to account for the observed rapid inactivation of PDE. Our investigations on GTPase activity showed that RDM isolated in the dark have considerable dark GTPase activity, which is enhanced by light. In dark and light, the enzyme exhibits biphasic substrate dependence with two Km's for GTP of 2-3 and 40-80 microM at 22 degrees C and less than 1 and 10-25 microM at 37 degrees C. The Km's were not influenced by light. On the basis of G-protein content of the RDM, the Vmax's for the two activities at 37 degrees C in light are 4-5 and 20-30 GTPs hydrolyzed per minute per G-protein. RDM washed free of soluble and peripheral proteins do not have measurable GTPase activity in the dark or light. Purified G-protein alone also did not turn over GTP, apparently because bleached rhodopsin is required for it to bind GTP. Reconstitution of washed membranes with purified G-protein restores both the low- and high-Km GTPase activities. Inactivation of G-protein as measured by PDE turnoff and dissociation signal recovery is found to be faster at higher than lower [GTP], consistent with the observation that the higher GTPase activity associated with the higher Km alos resides in the G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contribution of the guanosinetriphosphatase activity of G-protein to termination of light-activated guanosine cyclic 3',5'-phosphate hydrolysis in retinal rod outer segments. 284 43


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