Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A number of 2-substituted cyclic nucleotide derivatives were synthesized and investigated as activators of cAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterase. Ring closure of 5-amino-1-beta-D-ribofuranosylimidazol-4-carboxamide cyclic 3',5'-phosphate (1) with various aldehydes according to a new procedure (Meyer, R. B., Jr., Shuman, D.A., and Robins, R. K. (1974), J. Am. Chem. Soc. 96, 4962) gave new derivatives of adenosine cyclic 3',5'-phosphate with the following 2-substituents: n-propyl, n-hexl, n-octyl, n-decyl, styryl, o-methoxyphenyl, and 2-thienyl. Alkylation of 2-mercaptoadenosine cyclic 3',5'-phosphate (20, Meyer et al., 1974) gave new cAMP derivatives with the following 2-substituent: ethylthio, n-propylthio, isopropylthio, allylthio, n-decylthio, and benzylthio. Deamination of 2-methyl-,2-n-butyl-, and 2-ethylthioadenosine cyclic 3',5'-phosphate. Using multiple regression analysis, a striking relationship was found between the relative potency of the compounds as activators of bovine brain cAMP-dependent protein kinase and parameters describing the hydrophobic, steric, and electronic character of the substituents on these compounds. All compounds were substrates for a cyclic nucleotide phosphodiesterase preparation from rabbit kidney. Additionally, the compounds were as a group, good inhibitors of the hydrolysis of cAMP by phosphodiesterase preparations from rabbit lung, beef heart, and dog heart.
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PMID:2-substituted derivatives of adenosine and inosine cyclic 3',5'-phosphate. Synthesis, enzymic activity, and analysis of the structural requirements of the binding locale of the 2-substituent on bovine brain protein kinase. 16 24

The recently discovered heat-stable inhibitor protein of the Ca2+-activated cyclic nucleotide phosphodiesterase (Sharma, R. K., Wirch, E. & Warg, J. H. (1978) J. Biol. Chem., in press) has been purified 238 214-fold from bovine brain extract using an affinity column of the modulator protein--Sepharose 4B conjugate. The purified sample appears to be homogeneous as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The protein band has a mobility corresponding to that of a polypeptide of molecular weight 68 000. Since the heat-stable inhibitor protein has a molecular weight of 70 000 under nondenaturing conditions, it suggests that it is a monomeric protein. The protein has no inhibitory activity toward the cAMP-dependent protein kinase or protein phosphatase. The purified sample has been tested for various enzyme activities which include ATPase, GTPase, cAMP phosphodiesterase, cGMP phosphodiesterase, 5'-nucleotidase, and protein kinase. None of these activities are exhibited by the purified sample.
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PMID:Purification of the heat-stable inhibitor protein of the Ca2+-activated cyclic nucleotide phosphodiesterase by affinity chromatography. 20 31

Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.
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PMID:Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain. 132 38

Experiments have been performed to characterize guinea-pig peritoneal eosinophil cyclic nucleotide phosphodiesterase (PDE) activity and establish whether it is involved in regulating superoxide (.O2-) generation. Eosinophils were found to contain a predominantly membrane-bound cAMP PDE(s) (92.5 +/- 2.4% of total activity) which was resistant to solubilization with Triton X-100 (1%). This particulate PDE exhibited complex kinetics (Km = 1.3 and 31.4 microM) and was unaffected by cGMP (IC50 greater than 100 microM) or CaCl2 (2 mM) + calmodulin (10 units/mL). Little cGMP PDE activity was detected in either the soluble or particulate fractions. Inhibitors of the Ro-20-1724-inhibited (Type IV) cAMP PDE, namely Ro-20-1724 (IC50 = 0.92 +/- 0.43 microM), rolipram (IC50 = 0.20 +/- 0.04 microM) and denbufylline (IC50 = 0.20 +/- 0.01 microM), potently inhibited the particulate cAMP PDE, as did the non-selective inhibitors trequinsin (IC50 = 0.11 +/- 0.02 microM) and AH-21-132 (IC50 = 2.57 +/- 0.02 microM). Eosinophil cAMP PDE was resistant to SK&F 94120 (IC50 greater than 1000 microM), the cGMP-inhibited (Type III) cAMP PDE inhibitor, and the cGMP PDE (Type I) inhibitor, zaprinast, was only weakly active (IC50 = 35.33 +/- 10.74 microM). .O2- release from resting cells was potently inhibited by rolipram (IC50 = 0.05 +/- 0.03 microM) and denbufylline (IC50 = 0.06 +/- 0.04 microM) but surprisingly, in view of its potent cAMP PDE inhibitory activity, was only weakly decreased by trequinsin (IC50 = 8.0 +/- 2.7 microM). AH-21-132 (IC50 greater than 10 microM), SK&F 94120 (IC50 greater than 10 microM) and zaprinast (IC50 greater than 10 microM) were without effect. Rolipram and denbufylline alone exerted little effect on cAMP in intact cells but, in the presence of 10 microM isoprenaline, potently increased intracellular accumulation (EC50 = 0.45 +/- 0.16 and 0.28 +/- 0.08 microM, respectively). Trequinsin and AH-21-132 only weakly enhanced isoprenaline-stimulated cAMP accumulation. Although it induced a marked rise in cAMP only in the presence of isoprenaline, rolipram (50 microM) alone was able to increase the activity ratio of cAMP-dependent protein kinase from 0.24 to 0.84. The results suggest that Ro-20-1724-inhibited cAMP PDE plays a role in regulating eosinophil .O2- generation. The poor correlation between the PDE inhibitory actions of certain compounds and their effectiveness in elevating cAMP and inhibiting .O2- suggests the existence of a barrier impeding access to the enzyme.
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PMID:Characterization of guinea-pig eosinophil phosphodiesterase activity. Assessment of its involvement in regulating superoxide generation. 165 Oct 83

Activation of cAMP-dependent protein kinase II by static and dynamic steady-state cAMP levels was studied by reconstituting an in vitro model system composed of hormone-sensitive adenylate cyclase, cyclic nucleotide phosphodiesterase, and cAMP-dependent protein kinase II. The rates of cAMP synthesis were regulated by incubating isolated membranes from AtT20 cells with various concentrations of forskolin. In the presence of 3-methylisobutylxanthine, the rate of protein kinase activation was proportional to the rate at which cAMP was synthesized, and there was a direct relationship between the degree of activation and the level of cAMP produced. The activation profiles of protein kinase generated in the presence of exogenous cAMP or cAMP produced by activation of adenylate cyclase in the absence of cAMP degradation were indistinguishable. Dynamic steady-state levels of cAMP were achieved by incubating the membranes with forskolin in the presence of purified cyclic nucleotide phosphodiesterase. Under these conditions, the apparent activation constant of protein kinase II for cAMP was reduced by 65-75%. This increased sensitivity to activation by cAMP was seen when phosphotransferase activity was measured directly in reaction mixtures containing membranes, protein kinase, and histone H2B or when regulatory and catalytic subunits were first separated by immunoprecipitation of holoenzyme and regulatory subunits with specific anti-serum. Our results are consistent with the hypothesis that rapid cAMP turnover may function as a mechanism for amplifying hormonal signals which use the cAMP-dependent protein kinase system.
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PMID:Enhanced activation of cAMP-dependent protein kinase by rapid synthesis and degradation of cAMP. 243 Sep 60

Dissociation and reassociation of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases I and II were studied in intact AtT20 cells. Cells were stimulated with 50 microM forskolin to raise intracellular cAMP levels and induce complete dissociation of R and C subunits. After the removal of forskolin from the incubation medium cAMP levels rapidly declined to basal levels. Reassociation of R and C subunits was monitored by immunoprecipitation of cAMP-dependent protein kinase activity using anti-R immunoglobulins. The time course for reassociation of R and C subunits paralleled the loss of cellular cAMP. Total cAMP-dependent protein kinase activity and the ratio of protein kinase I to protein kinase II seen 30 min after the removal of forskolin was the same as in control cells. Similar results were seen using crude AtT20 cell extracts treated with exogenous cAMP and Mg2+. Our data showed that after removal of a stimulus from AtT20 cells inactivation of both cAMP-dependent protein kinase isoenzymes occurred by the rapid reassociation of R and C subunits to form holoenzyme. Our studies also showed that half of the type I regulatory subunit (RI) present in control cells contained bound cAMP. This represented approximately 30% of the cellular cAMP in nonstimulated cells. The cAMP bound to RI was resistant to hydrolysis by cyclic nucleotide phosphodiesterase but was dissociated from RI in the presence of excess purified bovine heart C. The RI subunits devoid of C may function to sequester cAMP and, thereby, prevent the activation of cAMP-dependent protein kinase activity in nonstimulated AtT20 cells.
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PMID:In situ reassociation of the regulatory and catalytic subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase isoenzymes in AtT20 cells. 284 55

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

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

We have isolated and sequenced cDNA clones representing portions of the polyadenylylated transcripts of the dunce+ gene. These define an open reading frame of 1086 bases and some of the 5'- and 3'-untranslated regions of the transcripts. The deduced amino acid sequence is strikingly homologous to the amino acid sequence of a Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase isolated from bovine brain and more weakly related to the predicted amino acid sequence of a yeast cAMP phosphodiesterase. These homologies, together with prior genetic and biochemical studies, provide unambiguous evidence that dunce+ codes for a phosphodiesterase. In addition, the dunce+ gene product shares a seven-amino acid sequence with a regulatory subunit of cAMP-dependent protein kinase that is predicted to be part of the cAMP binding site. We also identify a weak homology between a region of the dunce+ gene product and the egg-laying hormone precursor of Aplysia californica. The open reading frame is divided in the genome by four introns.
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PMID:Molecular analysis of cDNA clones and the corresponding genomic coding sequences of the Drosophila dunce+ gene, the structural gene for cAMP phosphodiesterase. 302 34

High-affinity antibodies against calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase and protein phosphatase (calcineurin) were purified and characterized. Rabbit anti-phosphodiesterase antibody did not react with other phosphodiesterases or with the regulatory subunits of cAMP-dependent protein kinase. Affinity-purified goat anti-calcineurin antibody recognized both the 61-kDa catalytic subunit and the 18-kDa Ca2+-binding subunit of the phosphatase. Neither antibody reacted with CaM, several CaM-binding proteins (calmodulin-dependent protein kinase, myosin light chain kinase, fodrin), or other cytosolic proteins from brain. The antibodies were used to compare the cellular localization of these two CaM-dependent enzymes in rat brain. Both calcineurin and phosphodiesterase were found predominantly in nerve cells; however, phosphodiesterase was restricted to very specific neuronal populations. Phosphodiesterase was prominent in the somatic cytoplasm and dendrites of regional output neurons--e.g., cerebellar Purkinje cells and hippocampal and cortical pyramidal cells. The extensive and uniform staining in the dendrites was consistent with postsynaptic localization and suggested an important function for this enzyme in neurons that integrate multiple convergent inputs. Calcineurin was present in virtually all classes of neurons, with immunoreactivity confined primarily to cell bodies. Both diffuse cytoplasmic staining and characteristic punctate staining of cell bodies were observed; the latter suggested compartmentalization of calcineurin at or near the plasma membrane. The results of this study demonstrate that calcineurin and phosphodiesterase are differentially localized in the central nervous system. Thus, the expression and compartmentalization of CaM-binding proteins may be highly regulated and specific for particular differentiated nerve cell types.
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PMID:Differential localization of calmodulin-dependent enzymes in rat brain: evidence for selective expression of cyclic nucleotide phosphodiesterase in specific neurons. 302 62


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