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)

A procedure for the purification of phosphodiesterase from Crotalus venom on DEAE-cellulose at alkaline pH is described. The enzyme gives a single band in polyacrylamide gels and is free of contaminating nucleolytic enzymes. The molecular weight is about 115000. Concentration in an Amicon ultrafiltrator gave a highly concentrated active enzyme. Phosphodiesterase is relatively stable and can be stored at 4 degrees C in the presence of Mg2 and serum albumin for years. For the detection of contaminating endonuclease, an assay was used in which tRNA was the substrate and possible internal breaks were detected in polyacrylamide gel after denaturation. With bis(p-nitrophenyl) phosphate as substrate, 15mM Mg2 was necessary for optimal activity. The reaction remained linear for at least 15 min at 22 degrees C. At 45 degrees C, the liberation of p-nitrophenol was highest within 25 min of incubation. At 75 degrees C, inactivation of the enzyme occurred after 4 min.
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PMID:Purification and characterization of phosphodiesterase from Crotalus venom. 0 Feb 95

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca2+-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca2+-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in Km for cyclic GMP and a decrease in V. In the presence of calcium ions and purified activator protein, the Ca2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was minimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca2+-independent activation of Ca2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phoshodiesterase activity.
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PMID:Calcium-dependent cyclic nucleotide phosphodiesterase. Inhibition of basal activity by heat-stable factors from rat cerebrum. 3 21

Graafian follicles were isolated by microdissection from the ovaries of PMS-injected immature rats killed at specific times on the day before ovulation. They were incubated in Krebs bicarbonate buffer containing 5 mM glucose and 1% bovine serum albumin. The oocytes were recovered after incubation and examined by Nomarski interference contrast microscopy. The amount of lactate and glucose in the incubation medium was analysed enzymatically. Oocytes recovered from follicles extirpated in the morning, i.e. before the endogenous LH surge, and incubated for 2-10 h were in the dictyate stage, while addition of LH or FSH to the medium resulted in oocyte maturation as revealed by germinal vesicle breakdown (GVB) and polar body formation. The time-course of GVB in vitro was similar to that seen in vivo after exogenous LH. Both gonadotrophins markedly enhanced lactate accumulation in the medium and, as studied for LH, glucose uptake by the follicles. The effects of LH but not those of FSH, on GVB and lactate formation were prevented by the presence of an antiserum against the beta-subunit of LH. The gonadotrophic effects on GVB could be mimicked by the addition of prostaglandin E2 to the medium. When the follicles were extirpated in the late afternoon, i.e. after the LH surge, and incubated in hormone-free medium for 4 h the oocytes showed GVB in a progressively increasing proportion depending on the time of follicle extirpation. Lactate formation by this group of follicles was markedly enhanced compared to that of "morning" follicles, but it could be even more stimulated by in vitro exposure to LH. A preliminary series of experiments on the effect of phosphodiesterase inhibitors showed that theophylline and isobutylmethylxanthine at certain concentrations completely blocked the LH effect on GVB, whereas a newly developed compound, ICI 63.197, in itself induced GVB.
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PMID:Oocyte maturation and glycolysis in isolated pre-ovulatory follicles of PMS-injected immature rats. 18 38

An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...
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PMID:Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics. 18 86

A new Ca2+/calmodulin-dependent serine kinase was isolated from rat parotid gland acinar cells following chronic treatment with the beta-agonist isoproterenol. A single-step purification was performed on a calmodulin-agarose affinity column, following solubilization with Triton X-100. Among various substrates tested, bovine galactosyltransferase was the preferred substrate of the kinase, followed by glycogen synthetase greater than histone greater than phosphodiesterase greater than phenylalanine hydroxylase greater than phosphorylase b greater than bovine serum albumin. In comparison, a spleen preparation of Ca2+/calmodulin-dependent kinase did not show galactosyltransferase to be the preferred substrate. Thus, the enzyme would appear to be similar to the human galactosyltransferase-associated kinase. The kinase activity was saturable with 100 microM Ca2+ and 2 microM calmodulin. The molecular mass determined by nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoreses was 75 kDa with a pI of 4.3. The Vmax was 3500 mumol/(min.mg protein) with a Km of 1.6 microM for the transferase substrate. Leukotriene C and prostaglandin E2 were found to be specific noncompetitive inhibitors of the rat galactosyltransferase-associated kinase.
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PMID:Isolation and characterization of a new Ca2+/calmodulin-dependent protein kinase from isoproterenol-stimulated proliferating rat parotid acinar cells. 138 38

Binding of the bronchodilators N3-alkylxanthine and N3-alkyl-N1-methylxanthine derivatives to guinea pig serum albumin was investigated in vitro using the ultrafiltration method. A marked difference in the binding parameters of xanthine derivatives was observed, and binding was shown to be concentration dependent. Significant relations were observed among their binding parameter, dissociation constant (Kd), and hydrophobicity (log PC). The extent of binding of xanthine derivatives was increased both when a N3-methyl group was replaced by a longer alkyl chain and when a N3-alkylxanthine molecule was additionally replaced by a methyl group. Reversed-phase HPLC retention, as an index of hydrophobicity of xanthine derivatives, was also determined. Significant relationships were found between the adjusted retention time data for each xanthine derivative and their hydrophobicity or biological activities, such as their abilities to cause muscle relaxation and cyclic AMP phosphodiesterase (PDE) inhibition. These findings indicate that the difference in the extent of binding among xanthine derivatives is related to hydrophobicity, which is an important determinant of their biological activities.
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PMID:Protein binding of xanthine derivatives to guinea pig serum albumin. 165 Aug 23

We hypothesized that Iloprost, a long-acting prostacyclin analog, would inhibit neutrophil (PMN)-induced lung injury and decrease PMN adherence to vascular endothelium. Human PMNs infused into isolated buffer-perfused rat lungs subsequently stimulated with phorbol myristate acetate (PMA) resulted in lung injury as assessed by the accumulation of [125I]bovine serum albumin (125I-BSA) in lung parenchyma and alveolar lavage fluid. Addition of Iloprost to the lung perfusate, prior to activation of the PMNs, reduced lung injury as assessed by a decrease in the accumulation of 125I-BSA in the lung. This protective effect was not due to the vasodilatory effect of Iloprost. Protection by Iloprost was not linked to a reduction in PMA-induced PMN superoxide production since Iloprost did not reduce the amount of superoxide released into lung perfusate. In vitro, Iloprost caused a dose-dependent inhibition of PMA-stimulated PMN adherence to endothelial cells. Iloprost did not affect the number of Mo1 adhesion molecules constitutively expressed or the number of receptors expressed on the PMNs following PMA. Addition of cAMP or dibutyryl cAMP to the endothelial cells mimicked the effects of Iloprost, diminishing PMA-stimulated PMN adhesion. In separate experiments, addition of the phosphodiesterase inhibitor IBMX to Iloprost resulted in a greater inhibition of PMA-stimulated PMN adherence, while addition of an adenylate cyclase inhibitor, SQ 22,536, or cAMP antibodies with the Iloprost abolished Iloprost's inhibitory effect on PMN adhesion. Thus, Iloprost inhibits PMA-activated PMN-induced lung injury despite continued superoxide production. Iloprost inhibition of PMN adhesion is dependent on cAMP.
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PMID:Iloprost inhibits neutrophil-induced lung injury and neutrophil adherence to endothelial monolayers. 169 99

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.
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PMID:Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin. 184 37

Previous studies in our laboratory showed that ANP inhibits increases in endothelial monolayer permeability to macromolecules induced by thrombin. In this present study, we investigated the second messenger system involved in the influence of ANP on monolayer permeability. In bovine aortic endothelial cells (BAEC), ANP (100 nM) caused increased cGMP levels which were measurable at 30 sec and maximal at 3 min. Addition of 8-bromo cGMP (1 mM) to BAEC monolayers mimicked the actions of ANP by inhibiting thrombin- mediated increases in permeability to [125I]-labeled bovine serum albumin. Inhibition of increases in permeability by lower concentrations of ANP was enhanced by the cGMP-selective phosphodiesterase inhibitor, M&B 22948 (100 microM). The use of ANP structural analogs which stimulate cGMP production (AP III or BNP) prevented thrombin-induced increases in monolayer permeability, whereas AP-I, which does not increase cGMP levels, was ineffective.
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PMID:Atrial natriuretic peptide regulation of endothelial permeability is mediated by cGMP. 217 80

ADvF11 cells are a CHO adhesion variant which, unlike wild type (WT) cells, are not able to adhere to fibronectin (Fn) coated substrata or to be aggregated by Fn-beads. However, ADvF11 cells bind Fn-beads to the same extent as WT cells, thus suggesting that the defect(s) associated with ADvF11 cells are distal to the initial receptor-ligand binding event (Cheung and Juliano, Exp. Cell Res. 152:127, 1984). In this communication we report that cAMP analogs such as dibutyryl-cAMP (dbcAMP) and 8-bromo-cAMP are able to correct defect(s) associated with ADvF11 cells enabling them to adhere to Fn-coated dishes and to aggregate in the presence of Fn-beads. However, only approximately 40% of ADvF11 cells were found to be responsive to dbcAMP suggesting heterogeneity in the cell population with respect to dbcAMP sensitivity. Further analysis of this partial response led us to isolate a subclone of ADvF11 cells, F11CA11, which is highly responsive to dbcAMP treatment. Induction of Fn-mediated cell adhesion and aggregation in F11CA11 by dbcAMP is both time and dose dependent. Optimal responses were obtained after overnight incubation in alpha-MEM containing, 1% fetal calf serum, 4% bovine serum albumin, 0.5 mM dbcAMP and 0.2 mM methyl-isobutyl-xanthine (MIX), a phosphodiesterase inhibitor. Under these conditions, 70-80% of F11CA11 cells were found to be adherent, compared to 5-7% of untreated F11CA11 cells and 95-100% of WT cells. Aggregation of dbcAMP-MIX treated F11CA11 cells induced by Fn-beads also approached that of WT cells. In addition, treatment with dbcAMP-MIX markedly increased the ability of F11CA11 cells to internalize Fn-beads. The maintenance of the adherent phenotype required the constant presence of dbcAMP-MIX. Removal of dbcAMP-MIX from the incubation medium resulted in return to the original nonadhesive phenotype. Thus, elevation of cAMP levels can dramatically modify the behavior of F11CA11 cells with respect to fibronectin mediated adhesion, aggregation and endocytosis, in effect causing a phenotypic reversion of all three parameters to wild type status. This suggests that the mechanisms for adhesion, aggregation and endocytosis may each involve regulation by cyclic AMP-protein kinase systems.
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PMID:cAMP-induced phenotypic reversion of adhesion, aggregation, and endocytosis in adhesion-defective CHO cell variants. 241 52


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