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)

Agents such as prostaglandins E1 and I2 which elevate cAMP levels in platelets also increase cAMP phosphodiesterase activity. Since much of the cAMP phosphodiesterase activity in human platelets is due to the cGMP-inhibited isozyme (Macphee, C. H., Harrison, S. A., and Beavo, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6600-6663), we examined the regulation of this isozyme by prostaglandins E1 and I2 in intact platelets. Because this isozyme is a minor component of platelet protein, normally requiring several thousand-fold purification to achieve homogeneity, a specific monoclonal antibody (CGI-5) was utilized to identify and isolate the cGMP-inhibited phosphodiesterase activity. Treatment of intact platelets with the prostaglandins promoted an increase in the phosphorylation state of the cGMP-inhibited phosphodiesterase and a corresponding increase in phosphodiesterase activity. The effect on activity and phosphorylation of the cGMP-inhibited phosphodiesterase was observed within 2 min after intact platelets were exposed to the prostaglandins. The half-maximal effective dose for prostaglandin I2 (10 nM) was approximately 10-fold lower than that for prostaglandin E1. The phosphorylated, cGMP-inhibited isozyme migrated as a 110-kDa peptide following sodium dodecyl sulfate gel electrophoresis. Direct in vitro phosphorylation of the platelet cGMP-inhibited phosphodiesterase by the catalytic subunit of cAMP-dependent protein kinase caused a similar increase in phosphodiesterase activity. Treatment with PKI peptide, a specific inhibitor of cAMP-dependent protein kinase, blocked the phosphorylation and the effect on activity. Taken together, the data strongly suggest that the effects of prostaglandins E1 and I2 on platelet phosphodiesterase activity are mediated by a direct cAMP-dependent protein kinase-catalyzed phosphorylation of the cGMP-inhibited phosphodiesterase isozyme.
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PMID:Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets. 283 85

The purified catalytic subunit (C) of cAMP-dependent protein kinase produced a 2-fold activation of the low Km phosphodiesterase in crude microsomes (P-2 pellet) of rat adipocytes. This activation was C subunit concentration-dependent, ATP-dependent, blocked by a specific peptide inhibitor, and lost if the C subunit was first heat denatured. The concentration of ATP necessary for half-maximal activation of the low Km phosphodiesterase was 4.50 +/- 1.1 microM, which was nearly the same as the known Km of C subunit for ATP (3.1 microM) using other substrates. The concentration of C subunit producing half-maximal activation of phosphodiesterase was 0.22 +/- 0.04 microM, slightly less than the measured concentration of total C subunit in adipocytes (0.45 microM). The activation of the low Km phosphodiesterase by C subunit was specific, since on an equimolar basis, myosin light chain kinase, cGMP-dependent protein kinase, or Ca2+/calmodulin-dependent protein kinase II did not activate the enzyme. The percent stimulation of phosphodiesterase by C subunit was about the same as that produced by incubation of adipocytes with a cAMP analog, and the enzyme first activated in vivo with the analog was not activated to the same extent (on a percentage basis) by in vitro treatment with C subunit. Treatment of the crude microsomes with trypsin resulted in transfer of phosphodiesterase catalytic activity from the particulate to the supernatant fraction, but the enzyme in the supernatant was minimally activated by C subunit, suggesting either loss or dislocation of the regulatory component. The C subunit-mediated activation of phosphodiesterase was preserved after either transfer of phosphodiesterase activity to the supernatant fraction by nonionic detergents or partial purification of the transferred enzyme. The present findings are consistent with the suggestion that protein kinase regulates the concentration of cAMP through phosphodiesterase activation and provide direct evidence that the mechanism of activation involves phosphorylation.
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PMID:Activation of the particulate low Km phosphodiesterase of adipocytes by addition of cAMP-dependent protein kinase. 283 86

Activation of glycolysis by insulin in cultured adult rat hepatocytes is accompanied by an activation of phosphofructokinase 2 (PFK 2). PFK 2 activation might be caused by insulin-dependent changes of (a) metabolite levels, (b) basal and (c) Br8cAMP-stimulated cAMP-dependent protein kinase activity; this problem was investigated. 1. Cells cultured with 0.1 nM insulin for 48 h exhibited a low glycolytic rate and low fructose 2,6-bisphosphate [Fru(2,6)P2] levels. Addition of insulin increased Fru(2,6)P2 and Fru(1,6)P2 levels sequentially which points to PFK 2 as first target enzyme of insulin action. 2. Concentrations of Glc6P, Fru6P, phosphoenolpyruvate, glycerol 3-phosphate and citrate, which modulate PFK 2/fructose 2,6-bisphosphatase 2 activity, were not altered by insulin. 3. Activation of PFK 2 by insulin occurred without changes in the levels of total and protein-bound cAMP. Bound cAMP amounted to about 14% of total cAMP. 4. Insulin neither decreased the basal dissociation state of the cAMP-dependent protein kinase nor lowered the sensitivity of the kinase towards cAMP in cell extracts. 5. Addition of the phosphodiesterase-resistant Br8cAMP to the cultures increased cAMP levels 3-4-fold, elevated the protein kinase activity ratio from 0.14 to 0.6 and decreased the Fru(2,6)P2 level and the rate of glycolysis. When Br8cAMP and insulin were given together, insulin was capable of counteracting Br8cAMP in that it activated glycolysis and PFK 2 and elevated the Fru(2,6)P2 level; however, it did not decrease the elevated protein kinase activity ratio. It is concluded that insulin presumably does not activate PFK 2 through changes in cAMP and effector levels or through inhibition of cAMP-dependent protein kinase dissociation. The data support the hypothesis that insulin may act via activation of PFK 2 phosphatase.
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PMID:Activation of phosphofructokinase 2 by insulin in cultured hepatocytes without accompanying changes of effector levels or cAMP-stimulated protein kinase activity ratios. 284 74

The mechanical and biochemical responses of the canine trachealis to SK&F 94836 [2-cyano-1-methyl-3-[4-(4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazine-3-yl)phenyl]guanidine], a selective inhibitor (ki = 1-3 microM) of the low km cyclic AMP (cAMP) phosphodiesterase, were assessed. Time course studies indicated that SK&F 94836-induced relaxation of trachealis strips contracted with 0.1 microM methacholine was accompanied by an activation of cAMP-dependent protein kinase (cAMP-PK). In subsequent experiments, trachealis strips were contracted with three concentrations of methacholine (0.1, 1.0 or 3.0 microM) or two concentrations of histamine (10 or 300 microM) before being relaxed by the cumulative addition of SK&F 94836. The relaxant response to SK&F 94836 (EC50 = 1-10 microM) decreased progressively as tissues were contracted with higher concentrations of methacholine. In parallel with its inhibitory effect on SK&F 94836-induced relaxation, methacholine suppressed the ability of SK&F 94836 to activate cAMP-PK. Interestingly, the inhibition of cAMP-PK activity was not accompanied by a significant inhibition of SK&F 94836-stimulated cAMP accumulation. Unlike the results with methacholine, the concentration of histamine used to contract tissues had no effect on SK&F 94836-induced relaxation or cAMP-PK activation. To determine the effect of SK&F 94836 on the mechanical and biochemical responses to the beta adrenoceptor agonist isoproterenol, tissues were first contracted with 3.0 microM methacholine and then incubated with 0, 0.3, 3.0 or 30 microM SK&F 94836 before being relaxed by the cumulative addition of isoproterenol. In these experiments, SK&F 94836 potentiated isoproterenol-induced relaxation, cAMP accumulation and cAMP-PK activation in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of the low km cyclic AMP phosphodiesterase in intact canine trachealis by SK&F 94836: mechanical and biochemical responses. 284 31

Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.
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PMID:Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain. 284 74

Methylxanthines are phosphodiesterase inhibitors and are therefore capable of increasing cyclic AMP levels, thereby stimulating cyclic nucleotide-dependent protein kinases. The direct action of several xanthine derivatives on enzyme-dependent phosphorylations involving red blood cell membrane proteins was studied in vitro. Pentoxifylline and caffeine exhibited no effect on the activity of the membrane cAMP-dependent protein kinase. Conversely, methylxanthines proved capable on inhibiting cyclic nucleotide-independent protein kinases present in the membrane and cytosol. This inhibition involves competition with ATP. Comparison of the inhibitory effect of two xanthine derivatives, ie propentofylline and pentoxifylline, demonstrated significant differences. Xanthine derivatives showed no activity on red blood cell tyrosine kinase. Furthermore, three xanthines, ie caffeine, pentoxifylline and propentofylline, inhibited phosphatidylinositol kinase.
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PMID:[Methylxanthines and phosphorylation of the constituents of the membrane of the human red blood cell]. 285 63

A study has been made of the action of 5-hydroxytryptamine (5-HT) on the radio-sodium efflux from single barnacle muscle fibres. (i) Stimulation of the Na efflux by external application of 5-HT is seen in both unpoisoned and ouabain-poisoned fibres. (ii) Concentrations of 5-HT as low as 10(-9)M are effective. (iii) Characteristically, the response to 5-HT is prompt in onset, reaches a peak within 20 min and then decays rather rapidly. Fibres from certain barnacle specimens are sometimes unresponsive to 5-HT. Such fibres, however, can be rendered responsive by preinjecting into them the non-hydrolysable GTP analogue, Gpp(NH)p. The response of the ouabain-insensitive Na efflux to 5-HT depends on external Ca2+ and, to a certain extent, on external Na+. (i) The response to 5-HT is unaffected by prior external application of Ca2+ antagonists, viz. verapamil, Cd2+ and WB-4101. (ii) The calmodulin antagonist, trifluoperazine (10(-5)M), completely abolishes the response to 5-HT, even in fibres preinjected with Gpp(NH)p. (iii) Diphenylhydantoin is less effective than trifluoperazine (TFP). Whereas the receptor antagonist methysergide is ineffective, cyproheptadine is very effective. (i) Prior application of the phosphodiesterase inhibitor 1-propyl-3-methyl-7-(5-hydroxyhexyl)-xanthine (PMX) or the inhibitor 1-isoamyl-3-isobutyl-xanthine (IAX) augments the size of the response to 5-HT, but fails to stop the response from decaying. (ii) Augmentation of the response to 5-HT by IAX is seen despite the presence of 10(-5) M-TFP. Prior injection of Mg2+ or protein kinase inhibitor (PKI) leads to abolition or reduction of the response to 5-HT. These results demonstrate that barnacle fibres are a useful preparation for investigation the natriferic action of 5-HT. They also support the view that the response to 5-HT involves a receptor-adenylate cyclase complex and is the result of activation by newly formed cAMP of cAMP-dependent protein kinase.
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PMID:The modulatory action of 5-hydroxytryptamine on sodium efflux: the barnacle muscle fibre as a model system. 286 Oct 30

The interaction of T-killers with target cells was studied to reveal the biochemical changes in the latter. On specific binding of target cells with T-killers the activity in target cells of cAMP phosphodiesterase increased 2.1-fold, the level of cAMP decreased 1.5-fold, the adenylate cyclase activity decreased 2.0-fold, the phosphorylation of intracellular proteins decreased 1.8-fold, the cAMP-dependent protein kinase activity decreased 1.7-fold. No change in the activity of lysosomal enzymes was observed. At the "independent target cells lysis" stage the level of cAMP increased 1.8-fold, the phosphodiesterase activity decreased 1.7-fold, the cAMP-dependent protein kinase activity increased 1.8-fold, the released activity of acid phosphatase increased up to 40% compared with the control cells. In the presence of 1 mM dibutyryl cAMP the released activity of the acid phosphatase in target cells was inhibited by 29%, the target cells lysis was decreased by 23,5%. The data obtained allowed to suppose that the activation of the host lysosomal enzymes causes target cells autolysis and that cAMP takes part in the regulation of these processes.
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PMID:[Activation of adenylate cyclase system enzymes and lysosomal acid phosphatase in target cells interacting with T-killer cells]. 298 45

Exposure of 3T3-L1 adipocytes to 1 nM insulin for 10 min results in activation of particulate cAMP phosphodiesterase and suppression of lipolysis stimulated by 10 nM isoproterenol. When lipolysis was increased by cilostamide, a selective inhibitor of the particulate phosphodiesterase, the antilipolytic effect of insulin was not observed. Insulin did suppress lipolysis stimulated by Ro 20-1724, an inhibitor of soluble cAMP phosphodiesterase activity. Cilostamide did not interfere with insulin stimulation of glucose uptake, nor did it have any direct effect on cAMP-dependent protein kinase. Thus, inhibition of particulate but not soluble cAMP phosphodiesterase blocked the antilipolytic effect of insulin. Our findings support the idea that insulin inhibits lipolysis, perhaps in large part by activating particulate "low Km" cAMP phosphodiesterase, which seems to be functionally closely coupled with the hormone-sensitive lipase-regulatory system influencing primarily a pool of cAMP utilized by the relevant protein kinase.
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PMID:Antilipolytic action of insulin: role of cAMP phosphodiesterase activation. 298 73

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.
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PMID:Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain. 298 83


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