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

Using DEAE-cellulose chromatography and Agarose gel filtration we have partially purified a low Km cyclic adenosine monophosphate (AMP) phosphodiesterase from the 100,000 X g supernatant of rat kidneys. The characteristics of this enzyme included a Km of approximately 4 muM a pH optimum of around 8.0 and a requirement for magnesium. This preparation should be suitable for investigation of possible effects of hormones, drugs and cellular constituents on the cyclic AMP pathway through any direct effects on the low Km enzyme. We have also demonstrated a nonspecific, high Km cyclic nucleotide phosphodiesterase and possibly a specific cyclic guanosine monophosphate (GMP) phosphodiesterase in the soluble fraction from rat kidneys.
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PMID:Multiple cyclic nucleotide phosphodiesterases in rat kidney. 0 May 37

The Lubrol-dispersed guanylate cyclase from sea urchin sperm was purified and isolated essentially free of detergent by GTP affinity chromatography, DEAE-Sephadex chromatography, and gel filtration. After removal of the detergent, the enzyme remained in solution in the presence of 20% glycerol. The specific activity of the purified enzyme was about 12 mumol of guanosine 3':5'-monophosphate (cyclic GMP) formed - min-1 - mg of protein-1 at 30 degrees, an activity about 4600 times that of a soluble guanylate cyclase purified recently from Escherichia coli (Macchia V., Varrone, S., Weissbach, H., Miller, D.L., and Pastan, I. (1975) J. Biol. Chem. 250, 6214-6217). The cyclic GMP phosphodiesterase activity was negligible and adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase was not detectable in the purified preparation. Cyclic AMP formation from ATP occurred at a rate of 0.002% of that of guanylate cyclase. In the absence of phosphodiesterase or guanosine triphosphatase inhibitors, 100% of the added GTP was converted to cyclic GMP. The purified enzyme required Mn2+ for maximum activity, the relative rates in the presence of Mg2+ or Ca2+ being less than 0.6% of the rates with Mn2+. The purified enzyme displayed classical Michaelis-Menten kinetics with respect to MnGTP (apparent Km is approximately equal to 170 muM) in contrast to the positively cooperative kinetic behavior displayed by the unpurified, detergent-dispersed, or particulate guanylate cyclase. The molecular weight of the purified enzyme was approximately 182,000 as estimated on Bio-Gel A-0.5m columns equilibrated in the presence or absence of 0.1 M NaCl. The unpurified, detergent-dispersed enzyme also migrated with an apparent molecular weight of 182,000 on columns equilibrated with 0.5% Lubrol WX and 0.1 M NaCl, but it migrated as a large aggregate (molecular weight is greater than 5 X 10(5)) on columns equilibrated in the absence of either the detergent of NaCl. After gel filtration, the unpurified, dispersed enzyme still yielded positive cooperative kinetic patterns as a function of MnGTP. Na dodecyl-SO4 gel electrophoresis of the enzyme after the DEAE-Sephadex or the gel filtration steps resulted in two major protein bands with estimated molecular weights of 118,000 and 75,000. Whether or not these protein bands represent the subunit molecular weights of guanylate cyclase is unknown at present.
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PMID:Sea urchin sperm guanylate cyclase. Purification and loss of cooperativity. 0 69

The effects of the alpha-adrenergic agonist phenylephrine on the levels of adenosine 3':5'-monophosphate (cAMP) and the activity of the cAMP-dependent protein kinase in isolated rat liver parenchymal cells were studied. Cyclic AMP was very slightly (5 to 13%) increased in cells incubated with phenylephrine at a concentration (10(-5) M) which was maximally effective on glycogenolysis and gluconeogenesis. However, the increase was significant only at 5 min. Cyclic AMP levels with 10(-5) M phenylephrine measured at this time were reduced by the beta-adrenergic antagonist propranolol, but were unaffected by the alpha-blocker phenoxybenzamine, indicating that the elevation was due to weak beta activity of the agonist. When doses of glucagon, epinephrine, and phenylephrine which produced the same stimulation of glycogenolysis or gluconeogenesis were added to the same batches of cells, there were marked rises in cAMP with glucagon, minimal increases with epinephrine, and little or no changes with phenylephrine, indicating that the two catecholamine stimulated these processes largely by mechanisms not involving cAMP accumulation. DEAE-cellulose chromatography of homogenates of liver cells revealed two major peaks of cAMP-dependent protein kinase activity. These eluted at similar salt concentrations as the type I and II isozymes from rat heart. Optimal conditions for preservation of hormone effects on the activity of the enzyme in the cells were determined. High concentrations of phenylephrine (10(-5) M and 10(-4) M) produced a small increase (10 tp 16%) in the activity ratio (-cAMP/+cAMP) of the enzyme. This was abolished by propranolol, but not by phenoxybenzamine, indicating that it was due to weak beta activity of the agonist. The increase in the activity ratio of the kinase with 10(-5) M phenylephrine was much smaller than that produced by a glycogenolytically equivalent dose of glucagon. The changes in protein kinase induced by phenylephrine and the blockers and by glucagon were thus consistent with those in cAMP. Theophylline and 1-methyl-3-isobutylxanthine, which inhibit cAMP phosphodiesterase, potentiated the effects of phenylephrine on glycogenolysis and gluconeogenesis. The potentiations were blocked by phenoxybenzamine, but not by propranolol. Methylisobutylxanthine increased the levels of cAMP and enhanced the activation of protein kinase in cells incubated with phenylephrine. These effects were diminished or abolished by propanolol, but were unaffected by phenoxybenzamine. It is concluded from these data that alpha-adrenergic activation of glycogenolysis and gluconeogenesis in isolated rat liver parenchymal cells occurs by mechanisms not involving an increase in total cellular cAMP or activation of the cAMP-dependent protein kinase. The results also show that phosphodiesterase inhibitors potentiate alpha-adrenergic actions in hepatocytes mainly by a mechanism(s) not involving a rise in cAMP.
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PMID:Studies on the alpha-andrenergic activation of hepatic glucose output. II. Investigation of the roles of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in the actions of phenylephrine in isolated hepatocytes. 0 57

A phosphatidylinositol phosphodiesterase from the culture broth of Bacillus cereus, was purified to a homogeneous state as indicated by polyacrylamide gel electrophoresis, by ammonium sulfate precipitation and chromatography with DEAE-cellulose and CM-Sephadex. The enzyme (molecular weight: 29000 +/- 1000) was maximally active at pH 7.2-7.5, AND NOT INFLUENCED BY EDTA, ophenanthroline, monoiodoacetate, p-chloromercuribenzoate or reduced glutathione. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, under the conditions examined. The products from phosphatidylinositol of enzyme reaction were diacylglycerols and a mixture of myoinositol 1- and 1, 2-cyclic phosphates, suggesting that the enzyme was a phosphatidylinositol-specific phospholipase C. The enzyme released alkaline phosphatase quantitatively from rat kidney slices. A kinetic analysis was made on the release of alkaline phosphatase. The results suggest that phosphatidylinositol-specific phospholipase C can specifically act on plasma membrane of rat kidney slices.
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PMID:Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity. 1 Sep 86

The DNAase in human urine was purified about 30-fold with a recovery of 28%. This involved DEAE-cellulose and phosphocellulose chromatography steps and gel filtration on Sephadex G-75. The enzyme required divalent cations such as Co2+, Mg2+, Mn2+ and Zn2+ for activity, but Ca2+, Cu2+ and Fe2+ were ineffective. EDTA and G-actin inhibited the reaction. The maximum activity was observed at pH 5.5 in acetate buffer plus Co2+ or Mg2+ and Ca2+. It had a molecular weight of approximately 38 000, estimated by gel filtration on Sephadex G-75 and isoelectric point of around pH 3.9. The enzyme is an endonuclease which hydrolyzes native, double-stranded DNA about 3 to 4 times faster than thermally denatured DNA to produce 5'-phosphoryl- and 3'-hydroxyl-terminated oligonucleotides. The final preparation was free of non-specific acid and alkaline phosphatases, phosphodiesterase and ribonuclease activities.
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PMID:Purification and properties of deoxyribonuclease from human urine. 2 31

Vicia faba root cells contain several nucleolytic activities: phosphomonoesterase and phosphodiesterase (which however were not studied in details), one nuclease and four ribonucleases. These results were obtained by separating the extracted proteins into anionic and cationic species by chromatography on CM-cellulose at pH 5.5 and analysing each kind of proteins. Anionic species were subjected to chromatography on DEAE-cellulose which lead to isolation of one nuclease (A1) and two RNAases (A2, A3), the properties of which were studied. It was shown that the RNAases pH optima are near 6; A2 is more thermolabile than A3; both are endonucleases unable to attack double-stranded structure; studies with homopolymers, i.e. poly(A), poly(I), poly(C), poly(U), showed that their base specificities were analogous to that of already known plant RNAases. The cationic proteins, analysed with CM-cellulose, contain two RNAases (C1, C2). The pH optima were near 6 and 7, respectively; C1 is much more thermolabile than C2; both were endonucleases inactive on double-stranded structures. C1 and C2 hydrolysed poly(C) and poly(U) but not poly(A) and poly(U).
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PMID:Study on plant RNAases. Isolation and properties of several activities from Vicia faba root cells. 2 32

Phosphodiesterase activity of cultured cells was determined with bis-(4-methylumbelliferyl) phosphate as substrate. In the presence of Triton X-100 an acid component was evident and results indicated that this enzyme was identical with sphingomyelinase. Acid phosphodiesterase activity was specifically inhibited by sphingomyelin. In fibroblasts from patients with Niemann-Pick diseases types A, B and C, acid phosphodiesterase activity was deficient whereas neutral activity was normal. Neutral activity could, however, be removed by acid precipitation or by binding to DEAE-cellulose. Hence a simple and sensitive fluorimetric method is described for the assay of sphingomyelinase activity in the diagnosis of Niemann-Pick disease.
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PMID:Diagnosis of Niemann-Pick disease using a simple and sensitive fluorimetric assay of sphingomyelinase activity. 3 94

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

By means of DEAE-Sephadex A-50 Column chromatography, Trimeresurus gramineus venom was separated into twelve fractions. The fibrinogenolytic activities were distributed in Fractions 1 and 10. These enzymes were further purified by gel filtration and were homogeneous as judged by cellulose acetate membrane, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugal analysis. Both of them were single peptide chains. The sedimentation constants of alpha- (Fraction 1) and beta-fibrinogenases (Fraction 10) were 2.20 and 3.60, respectively. The molecular weights of alpha- and beta-fibrinogenases were 23 500 and 25 000 respectively. The contents of proline and glycine were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha-fibrinogenase and beta-fibrinogenase were pH greater than 10 and 4.5, respectively. The optimal pH of alpha-fibrinogenase was approx. 7.4 and that of beta-fibrinogenase was approx. 9.0. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.4, 7.4 and 9.0, while that of beta-fibrinogenase was much less affected by the same treatment. The specific fibrinogenolytic activity alpha-fibrinogenase was 31 mg fibrinogen/min per mg protein, while that of beta-fibrinogenase was 9 mg fibrinogen/min per mg protein. alpha-Fibrinogenase cleaved specifically the alpha(A) chain of monomeric fibrinogen without cleaving the beta(B) chain and gamma-chain. beta-fibrinogenase preferentially cleaved the beta(B) chain, and the alpha(A) chain was also partially cleaved by beta-fibrinogenase, if the incubation time was prolonged. Both enzymes showed proteolytic activities toward fibrinogen, fibrin and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities found in the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 14 times that of crude venom, while alpha-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of alpha-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of beta-fibrinogenase was inhibited markedly by phenylmethanesulfonylfluoride. alpha- and beta-fibrinogenases exert their fibrinogenolytic activity by a direct action on fibrinogen or fibrin without activation of plasminogen.
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PMID:Alpha and beta-fibrinogenases from Trimeresurus gramineus snake venom. 4 82


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