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)

The cationic conductances of purified bovine retinal rod membranes were studied by incorporation of vesicles into planar lipid bilayers. When the membranes were stripped of all peripheral proteins [guanine nucleotide-binding protein (G protein) and cGMP phosphodiesterase (3',5'-cyclic-GMP 5'-nucleotidohydrolase), EC 3.1.4.35], sodium and calcium fluxes were almost only observed in the presence of cGMP. Reconstitution experiments in which purified cGMP phosphodiesterase alone or with G protein were reassociated to the vesicles in proportions similar to those found in the native rod provide evidence for a direct interaction between the cGMP-dependent channel protein and the phosphodiesterase. (i) In its inhibited state, phosphodiesterase markedly stimulates the activity of the channels in the presence of cGMP (situation in the dark-adapted rod) but is not capable of activating the channels in the absence of cGMP. (ii) In the absence of cGMP, activation of the phosphodiesterase by G protein with GTP bound (equivalent to photoexcitation) induces the opening of cation channels that have the same conductance for sodium ions as cGMP-activated channels (20-22 pS, with two sublevels of about 7 pS and 13 pS).
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PMID:Direct activation of cGMP-dependent channels of retinal rods by the cGMP phosphodiesterase. 247 Nov 90

Cyclic AMP (cAMP) metabolism has been studied in rat aortic myocytes grown in primary culture to characterize this second messenger system in vascular smooth muscle cells that retain responses to vasoactive drugs. For comparison, cAMP metabolism was also studied in the aorta from donor rats. Adenylate cyclase activity from myocytes and from the aorta was stimulated to a similar degree by GTP, NaF, or forskolin, and the enzyme activation produced by isoproterenol or vasoactive intestinal polypeptide was observed only in the presence of GTP. A cAMP phosphodiesterase activity was found in homogenates from cultured myocytes and aorta as well, and it was similarly stimulated by calmodulin in both cases. The rates of cAMP production and degradation were about seven-fold higher in cultured myocytes than in aorta. Basal levels of cAMP were also higher in the cultured cells than in the aorta. Hormones and drugs acting on adenylate cyclase or cAMP-phosphodiesterase in cell-free preparations altered the cAMP content of undisrupted cultured myocytes and aorta in the expected manner. Differences between cultured myocytes and aorta resided in the courses of drug-induced cAMP increases and in the magnitude of the cAMP response to isoproterenol, which was markedly increased in cultured myocytes compared with aorta. It is concluded that, despite some quantitative differences, the cAMP system of rat aortic myocytes grown in primary culture has characteristics similar to those displayed in rat isolated aorta. These cells are therefore suitable for studying the effects of drugs involving cAMP as a second messenger.
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PMID:A comparison of cyclic AMP signaling system in rat aortic myocytes in primary culture and aorta. 247 90

1. The mechanism by which acetylcholine (ACh), by stimulation of muscarinic receptors, acts to inhibit activation of the hyperpolarization-activated 'pacemaker' current, if was investigated in isolated rabbit sino-atrial (SA) node myocytes. 2. Intracellular loading with GTP gamma S, a non-hydrolysable analogue of GTP, did not impair the ACh action on if, but made it irreversible. On the other hand, the ACh action on if disappeared after a few minutes of cell loading with GDP beta S, a GDP analogue known to bind to G-proteins and prevent their receptor-stimulated action. Furthermore, incubation of cells in a solution containing pertussis toxin (PTX) led to abolition of the if response to ACh. These results indicate that the inhibitory effect of ACh on if is mediated by G-proteins activated by muscarinic receptors. 3. Intracellular loading with phosphodiesterase (PDE) increased the rate of if current run-down, but did not abolish the inhibitory action of ACh on if. 4. Extracellular perfusion with isobutylmethylxanthine (IBMX), a PDE inhibitor, increased if activation by shifting the current activation range to more positive voltages, as inferred by a three-pulse protocol analysis; in the presence of IBMX, the inhibition of if by ACh was not abolished. 5. The ACh-induced if depression persisted also in cells loaded with cyclic GMP. In these cells, as in those loaded with PDE, the if run-down was fast. 6. Oxotremorine, a muscarinic agonist coupled to adenylate cyclase but not to phosphoinositide turnover in cardiac cells, simulated ACh in its inhibitory action on if. The above results rule against the ACh action being mediated by PDE or by phosphoinositide turnover. 7. To investigate the possible involvement of cyclic AMP as a second messenger in the ACh action on if, we loaded cells with cyclic AMP and IBMX; under these conditions the action of ACh disappeared within a few minutes of whole-cell recording. 8. In cells where the slow inward Ca2+ current (isi) was measured together with if, ACh was seen to depress both currents. 9. In cells superfused with forskolin, the if amplitude on stepping to the half-activation voltage range was enhanced as a consequence of a depolarizing shift of the activation curve; ACh was not effective on if following stimulation by forskolin, but strongly depressed in the same cell the if current stimulated to a similar degree by isoprenaline.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Muscarinic control of the hyperpolarization-activated current (if) in rabbit sino-atrial node myocytes. 247 9

1. Analogues of GTP and GDP were introduced into isolated rod photoreceptors using the whole-cell patch clamp technique, while simultaneously recording the photocurrent with a suction pipette. After several minutes of whole-cell recording the patch pipette was disengaged, thus trapping the analogue inside the cell. 2. During the introduction of the hydrolysis-resistant GTP analogues guanosine-5'-O-(3-thio-triphosphate) (GTP-gamma-S) and guanylyl-imidodiphosphate (GMP-PNP) the dark current progressively declined, and the duration of responses to flashes of light which had previously been just-saturating increased slightly. The form of the rising phases of the responses to dim or bright flashes was little affected. 3. Following the incorporation of these GTP analogues the response to an intense flash was prolonged by a factor of up to 300, and the circulating current remained suppressed for up to 1 h. Ultimately the circulating current recovered and the duration of the flash response returned to near its control value. 4. Superfusion of the outer segment with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) during the extended period of saturation resulted in a rapid increase in the circulating current, suggesting that the analogues had their major effect on the duration of phosphodiesterase activation by light. 5. Introduction of the phosphorylation-resistant GDP analogue guanosine-5'-O-(2-thio-diphosphate) (GDP-beta-S) resulted in a decrease in light sensitivity and a reduction in the slope of the rising phase of the flash response. 6. The response to an intense flash was also prolonged in cells containing GDP-beta-S, recovery becoming progressively slower on successive presentations of the flash following the withdrawal of the patch pipette. This observation suggests that GDP-beta-S may be slowly converted within the cell to form a hydrolysis-resistant product. 7. These results indicate that the presence of a hydrolysis-resistant analogue of GTP within the cell causes light activation of the transduction mechanism for an extended period. Our interpretation of this finding is that hydrolysis of the bound guanosine nucleotide is necessary for the quenching of activated GTP-binding protein.
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PMID:Incorporation of analogues of GTP and GDP into rod photoreceptors isolated from the tiger salamander. 247 54

Of 120 laboratory-maintained strains of Listeria monocytogenes and two of L. ivanovii examined for haemolytic and lipolytic activity, 62 exhibited haemolytic activity alone, 20 of these showed haemolytic and lipolytic activity and 40 had neither activity. The L. ivanovii strains showed both activities. The results indicated a relationship between haemolysin production and lipolytic activity which was not explained by the serotype of the organism. In addition, the following hydrolytic activities were detected in the cell-free growth media of strains L. monocytogenes Boldy and L. ivanovii (formerly L. monocytogenes) Type 5 (substrates acted upon are given in parentheses): acid phosphate (4-nitrophenylphosphate, naphthyl phosphate, glycerophosphate, phosphorylcholine and GTP); neutral phosphatase (4-nitrophenylphosphate, naphthyl phosphate, phosphorylcholine, NADP and UDPG); phosphodiesterase (bis-4-nitrophenylphosphate, ATP and NADP); NADase (NAD); phospholipase C (4-nitrophenylphosphoryl-choline, phosphatidyl choline and ethanolamine, and sphingomyelin); and lipase and esterase (triacetin, tributyrin, triolein, naphthyl-laurate,-myristate,-caprylate,-palmitate and -oleate, 4-nitrophenyl-acetate-laurate and Tween 80). The preparations also showed weak catalase activity. No evidence was found for the presence of RNAase, DNAase, peptidase/amidase, phosphoamidase, alpha-amylase, glucosidase, galactosidase, pyranosidase or glucose aminidase.
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PMID:Haemolysins and extracellular enzymes of Listeria monocytogenes and L. ivanovii. 250 86

We report the purification of a CaATPase of high specific activity from Paramecium tetraurelia. The enzyme is preferentially released into solution upon deciliation of cells by a Ca2+ shock procedure. Purification by ion exchange and gel filtration chromatography yields major peptides of 68 and 53 kDa and a minor peptide of 58 kDa, as determined by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. These three peptides yield similar proteolytic peptide maps. Rabbit antisera to the purified enzyme inhibit enzyme activity and specifically label 68- and 53-kDa bands on nitrocellulose blots of the deciliation supernatant from which the enzyme is isolated. Concanavalin A-Sepharose precipitates about 60% of ATPase activity; only the 53-kDa band binds concanavalin A on nitrocellulose blots. The purified enzyme has a specific activity of 620 +/- 70 mumol/min/mg with ATP as substrate in the presence of Ca2+, which is required for enzyme activity. As substrates, ATP and GTP are strongly preferred to UTP and CTP. The Km for ATP in the presence of 3 mM Ca2+ is approximately 20 microM. Enzyme activity is strongly inhibited by the calmodulin antagonists trifluoperazine, fluphenazine, W7, and calmidazolium. However, calmodulin is not associated with the purified enzyme, based on the enzyme's inability to bind anti-calmodulin antibodies or to stimulate brain phosphodiesterase. The intracellular origin of this ATPase, its possible function, and its relationship to several other ATPases of Paramecium are discussed.
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PMID:Purification and characterization of a calcium-dependent ATPase from Paramecium tetraurelia. 252 45

Aluminum ion perturbs the activity of a number of physiologically important enzymes, including members of a family of guanine nucleotide-binding proteins (G-proteins). G-proteins couple cellular receptor proteins to a variety of effector enzymes (including adenylate cyclase, phospholipase C, and the rod photoreceptor phosphodiesterase). We show herein that subnanomolar concentrations of free aluminum ion, produced in a carefully defined and kinetically stable manner through the buffering of total aluminum at 0.1-1.0 mM with calculated ratios of chelating agents, inhibit both the receptor-mediated activation and the self-inactivating GTPase activity of the rod photoreceptor G-protein, Gv. In the presence of 4 X 10(-10) M free aluminum ion, GTPase activity is inhibited from about 25-60% as the magnesium ion concentration is reduced from 10(-3) to about 5 X 10(-5) M. The principal effect of aluminum ion upon Gv is to inhibit receptor catalyzed nucleotide exchange. Binding of the GTP analog 5'-guanylyl imidodiphosphate can be reduced by as much as 90% by aluminum ion following subsaturating rhodopsin stimulation. Aluminum ion can produce either competitive or mixed noncompetitive inhibition of rhodopsin-catalyzed Gv activation and GTPase activity, as a function of whether Gv undergoes single (competitive), or multiple (mixed noncompetitive) nucleotide exchanges. The rod photoreceptor phosphodiesterase is only slightly inhibited by similar aluminum ion activities. Light- and Gv-coupled phosphodiesterase activation exhibits both a lower maximum rate of cyclic guanosine monophosphate hydrolysis and a slower inactivation in the presence of aluminum ion activities from about 10(-12) - 10(-10) M. These data suggest that intracellular free aluminum ion concentrations in the subnanomolar range could markedly affect the ability of cells to transduce extracellular signals. Interestingly, the combination of Al3+ and F- to produce the fluoro-aluminate species (AlFx) also inhibits the GTPase of G-proteins, although the mechanism of inhibition (e.g. binding to the G-protein.Mg2+.GDP complex) is totally distinct from that observed for free Al3+ and the overall effect on signal transduction (e.g. enhanced signal amplification) is in complete opposition to that observed for free Al3+.
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PMID:Inhibition of transducin activation and guanosine triphosphatase activity by aluminum ion. 253 40

Regulation of cardiac beta-adrenergic receptors during hypoxia and ischemia is an area of active investigation, with some investigators reporting an increase in sarcolemmal beta-receptor number after ischemia. Previous studies have been limited by the necessity of examining beta-adrenergic receptor properties in membrane preparations from hypoxic or ischemic cardiac tissue and drawing conclusions about receptor localization in intact tissue from the behavior of a fraction of total receptors in membrane populations. As an approach to examining beta-receptor properties under well-defined pathophysiological conditions in intact heart cells, we studied cell-surface beta-receptors and adenylate cyclase activity in intact cultured chick embryo ventricular cells under conditions of controlled hypoxia and reoxygenation. During 2 h of hypoxia (PO2 less than 1.5 Torr) there was a progressive decline in cell surface beta-receptors from 26 +/- 2 to 10 +/- 6 fmol/mg (P less than 0.003) with no change in antagonist or agonist affinity. Receptor number recovered fully during 2 h of reoxygenation. Basal adenosine 3',5'-cyclic monophosphate (cAMP) production was unchanged, but response to isoproterenol in the absence or presence of a phosphodiesterase inhibitor decreased to about half of the response for normoxic cells but fully recovered during reoxygenation in a pattern similar to that for receptor number. Although [ATP] declined significantly during hypoxia (from 35 to 25 nmol/mg), the decline in [GTP] was marginal (4.3 to 3.9 nmol/mg), making it unlikely that substrate for guanine nucleotide regulatory protein was limiting for beta-adrenergic signal transduction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Beta-adrenergic receptor regulation during hypoxia in intact cultured heart cells. 253 45

The functional interactions of the retinal G protein, transducin, with the cyclic GMP phosphodiesterase (PDE) have been examined using the different purified subunit components of transducin and the native and trypsin-treated forms of the effector enzyme. The limited trypsin treatment of the PDE removes the low molecular weight gamma subunit (Mr approximately 14,000) of the enzyme, yielding a catalytic moiety comprised of the two larger molecular subunits (alpha, Mr approximately 85,000-90,000; beta, Mr approximately 85,000-90,000), which is insensitive to the addition of either the pure alpha T.GTP gamma S species or the pure beta gamma T subunit complex. However, the addition of the pure alpha T.GDP species to the trypsin-treated PDE (tPDE) results in a significant (90-100%) inhibition of the enzyme activity. This inhibition can be reversed by excess beta gamma T, suggesting that the holotransducin molecule does not (functionally) interact with the tPDE. However, the inhibition by alpha T.GDP is not reversed by the alpha T.GTP gamma S complex, over a range of [alpha T.GTP gamma S] which elicits a marked stimulation of the native enzyme activity, suggesting that the activated alpha T species does not effectively bind to the tPDE. The alpha T.GDP complex also is capable of inhibiting the alpha T.GTP gamma S-stimulated cyclic GMP hydrolysis by the native PDE. This inhibition can be reversed by excess alpha T.GTP gamma S, as well as by beta gamma T, indicating that the binding site for the activated alpha T species is in close proximity and/or overlaps the binding site for the alpha T.GDP complex on the enzyme. Overall, these results are consistent with a scheme where (a) both the small and larger molecular weight subunits of PDE participate in alpha T-PDE interactions, (b) the activation of PDE by the alpha T.GTP gamma S (or alpha T.GTP) species does not result in the complete dissociation of the gamma subunit from the enzyme, and (c) the deactivation of this signal transduction system results from a direct interaction between the alpha T.GDP species and the catalytic moiety of the effector enzyme.
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PMID:The regulation of the cyclic GMP phosphodiesterase by the GDP-bound form of the alpha subunit of transducin. 253 46

Cross-linking of surface Ig receptors (sIg) by mitogenic forms of anti-Ig antibodies (e.g. F(ab')2 fragments of rabbit anti-Ig) causes the rapid, and prolonged breakdown of phosphatidylinositol 4,5-bisphosphate. This response involves an unidentified guanine nucleotide regulatory protein (termed Gp), which couples sIg to the polyphosphoinositide-specific phosphodiesterase. Intact (IgG) rabbit anti-Ig antibodies, which co-cross-link sIg and Fc gamma receptors on B cells, only induce short-lived inositol phospholipid breakdown and abortive B cell activation. We show here that in permeabilized B cells intact anti-Ig inhibits the reconstituted breakdown of inositol phospholipids given by a combination of F(ab')2 anti-Ig and the non-hydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), but not the basal stimulation of Gp induced by GTP gamma S alone. These results therefore indicate that co-cross-linkage of sIg and Fc gamma receptors on B cells uncouples the antigen receptors from the associated G protein, but does not affect coupling between Gp and the phosphodiesterase. These observations therefore provide further insight into the mechanisms whereby engaging Fc receptors on B cells, by antigen-antibody complexes for example, could modulate antigen-induced B cell activation.
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PMID:Co-cross-linking of surface immunoglobulin Fc gamma receptors on B lymphocytes uncouples the antigen receptors from their associated G protein. 254 10


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