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)

By sequential acid treatment, gel filtration and KM-cellulose sorption a 18--20-fold purified preparation of ribonuclease with a yield of 50--60% was obtained from the culture liquid filtrate of Actinomyces rimosus 994. The preparation had a high specific activity of 450,000--600,000 units/mg protein, contained 85--98% protein, insignificant amounts of carbohydrates and hydroxytetracycline, and no quantities of DNase, phosphomonoesterases, phosphodiesterase or proteases. In RNA degradation (preparation of the total yeast RNA of the Sigma Co.) optimal results were obtained at 50 degrees C and pH 7.0--7.2 in phosphate buffer and 7.6--8.0 IN Tris-HCl buffer. The preparation was stable at high temperatures (80--100 degrees) in the wide pH range and during storage in the lyophilized form and in buffer solutions. RNase effect was inhibited by zinc, copper, iron and cobalt cations and activated by beta-mercaptoethanol, citrate and EDTA. Protamine sulphate and urea in low concentrations (0.01% and 1--4 M, respectively) accelerated and in high concentrations (1% and 8 M, respectively) terminated the enzyme reaction. With respect to many properties RNase from Act. rimosus 994 was similar to extracellular RNases, produced by other actinomycetes and fungi.
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PMID:[Preparation of extracellular ribonuclease form Actinomyces rimosus 994]. 3 16

Cyclic AMP and cyclic GMP phosphodiesterase activities (3' : 5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca2+ in the presence of the activator. However, the activity of the cyclic AMP phosphodiesterase was not significantly stimulated by Ca2+ up to 100 muM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60-70%) of the media enzyme was found in 105 000 times g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 times g supernatant the main kinetic parameters were specified as follows: 1) The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg2+ was achieved at 4mM of MgC12. 2) High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3) Activity-substrate concentration relationship revealed a high affinity (Km equals 1.0 muM) and low affinity (Km equals 45 muM) for cyclic AMP. The homogenate and 105 000 times g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent Km values for cyclic AMP hydrolysis were 1.2 muM and 36-40 muM and an enzyme extracted by sonication from 105 000 times g precipitate also exhibited non-linear kinetics (Km equals 5.1 muM and 70 muM). 4) Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I5 o at 0.62 muM, I5 o of the media at 0.62 muM and I5 o of the adventitia at 1.0 muM) than on the brain (I5 o at 8.5 muM) and serum (I5 o at 20 muM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.
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PMID:Cyclic 3',5'-AMP phosphodiesterase of rabbit aorta. 16 19

Frog (Rana catesbiana) rod outer segment disc membranes contain a cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which is activated by light in the presence of ATP. This enzyme is firmly bound to the disc membrane, but can be eluted from the membrane with 10 mM Tris-HCl buffer, pH 7.4 and 2 mM EDTA. The eluted phosphodiesterase has reduced activity, but can be activated approximately 10-fold by polycations such as protamine and polylysine. The eluted phosphodiesterase can no longer be activated by light in the presence of ATP, that is, activation by light apparently depends on the native orientation of phosphodiesterase in relationship to other disc membrane components. The eluted phosphodiesterase was purified to homogeneity as judged by analytical polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The over-all purification from intact retina was approximately 925-fold. The purification of phosphodiesterase from the isolated rod outer segment preparation was about 185-fold with a 28% yield. Phosphodiesterase accounts for approximately 0.5% of the disc membrane protein. The eluted phosphodiesterase (inactive form) has a sedimentation coefficient of 12.4 S corresponding to an approximate molecular weight of 240,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates the purified phosphodiesterase into two subunits of 120,000 and 110,000 daltons. With cyclic 3':5'-GMP (cGMP) as substrate the Km for the purified phosphodiesterase is 70 muM. Protamine increases the Vmax without changing the Km for cGMP. The isoelectric point (pI) of the native dimer is 5.7. Limited exposure of the eluted phosphodiesterase (inactive form) to trypsin produces a somewhat greater activation than is obtained with 0.5 mg/ml of protamine. The trypsin-activated phosphodiesterase has a sedimentation coefficient of 7.8 S corresponding to an approximate molecular weight of 170,000. The 110,000-dalton subunit is much less sensitive to trypsin hydrolysis and the 120,000-dalton subunit is rapidly replaced by smaller fragments. On the basis of the molecular weight of the purified phosphodiesterase (240,000) and the concentrations of phosphodiesterase and rhodopsin in the rod outer segment, it is estimated that the molar ratio ophosphodiesterase to rhodopsin in the rod outer segment is approximately 1:900. Since all of the disc phosphodiesterase molecules are activated when 0.1% of the rhodopsins are bleached, we conclude that in the presence of ATP 1 molecule of bleached rhodopsin can activate 1 molecule of phosphodiesterase.
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PMID:Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments. 16 36

The effect of albuterol and terbutaline on the cyclic 3',5'-adenosine monophosphate (cAMP) system was studied in rat uterus, aorta and myocardium and in dog bronchus, and was compared to that of isoproterenol in order to determine whether the tissue specificity observed in their functional effects is reflected in their effect on the cAMP system. Tissue specimens were either homogenized in Tris buffer for enzyme activity measurements or incubated in Krebs-Ringer-bicarbonate medium with the test drugs. Both albuterol and terbutaline produce an increase in cAMP content in the tissues due to a direct effect on adenylate cyclase. This effect can be potentiated by a phosphodiesterase inhibitor and antagonized by a beta adrenergic blocking compound. The cAMP response to each beta adrenergic agonist differs in the tissues examined: in uterus and aorta where the maximal effects are idenitcal, the ED50 values may reflect differences in affinity which may account for the different cAMP response to the compounds at the lower concentrations. In bronchus and myocardium, both the maximum effect and ED50 values of the compounds are different. Albuterol and terbutaline increases cAMP content in bronchus significantly and have only a small effect on cAMP cont in myocardium, whereas isoproterenol increases cAMP level significantly in both tissues. The results indicate that the tissue specificity of albuterol and terbutaline may have its origin at the level of the cAMP system.
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PMID:Effect of albuterol and terbutaline, synthetic beta adrenergic stimulants, on the cyclic 3',5'-adenosine monophosphate system in smooth muscle. 17 25

Rabbit anterior mesenteric-portal vein was shown to exhibit a positive correlation between relaxation and cyclic AMP levels using different concentrations of isoproterenol for varying exposure times. No consistent relationship was found between cyclic AMP and relaxation using a series of phosphodiesterase inhibitors (papaverine, SC2964, or RA233). In sodium-free (Tris-substituted) Kreb's solution, the addition of isoproterenol still increased cyclic AMP levels, but there was no relaxation. Dibutyryl cyclic AMP was also ineffective in sodium-free solution. Under identical conditions, both relaxation and increases in cyclic AMP produced by papaverine and SC2964 were found to be independent of extracellular sodium. We conclude, therefore, that increased cyclic AMP may be involved in isoproterenol-induced relaxation possibly acting via increased Na+-Ca2+ exchange, but plays only a small role, if any, in smooth muscle relaxation produced by papaverine, RA233, or SC2964.
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PMID:Quantitative aspects of cyclic AMP and relaxation in the rabbit anterior mesenteric-portal vein. 17 62

Mutants of Salmonella typhimurium with defects in the heptose region of the lipopolysaccharide (LPS) molecule (heptose-deficient, chemotype Re) leak periplasmic enzymes (acid phosphatase (EC 3.1.3.2), cyclic phosphodiesterase, ribonuclease I (EC 3.1.4.22), and phosphoglucose isomerase (EC 5.3.1.9) (PGI is at least partially periplasmic in E. coli and S. typhimurium; see below)) and do not leak an internal enzyme (glucose-6-phosphate dehydrogenase) into the growth medium. The extent of this leakage is markedly increased at higher temperature (42 degrees C). Leakage of periplasmic enzymes from the strains lacking units distal to heptose I in the LPS molecule (chemotype Rd2) occurs only at 42 degrees C, and not at 30 or 37 degrees C. The extent of leakage of these enzymes from smooth strain and mutants of other LPS chemotypes (Rc, Rd1) is not significant, and is not influenced by growth temperatures. The kinetics of leakage of periplasmic enzymes after shift to 42 degrees C in nutrient broth reveal an accelerated release into the medium from heptose-deficient strains of cyclic phosphodiesterase and ribonuclease I after 30 min at 42 degrees C, and phosphoglucose isomerase after 60 min at 42 degrees C; at 30 degrees C the rate of release of cyclic phosphodiesterase and ribonuclease I is relatively slower. After 60 min at 42 degrees C in nutrient broth, growth of these strains has either slowed down or stopped. In L-broth, which permits the growth of the heptose-deficient strain (SA1377) at 42 degrees C, leakage of cyclic phosphodiesterase and phosphoglucose isomerase occurs, whereas there is no detectable leakage of these enzymes from the isogenic smooth strain (SA1355). Thus, leakage of the periplasmic enzymes from the heptose-deficient strain occurs with or without growth. Mg2+ (0.75 mM), sodium chloride (50 mM), and sucrose (100 mM) in nutrient broth at 42 degrees C prevent the leakage of these enzymes. The shedding of LPS from the heptose-deficient as well as the smooth strains is enhanced by high temperature (42 degrees C), whereas considerable leakage of protein occurs only in the heptose-deficient strain at 42 degrees C and not in the smooth strain. The smooth and heptose-deficient strains are equally sensitive to osmotic shock although a significant proportion of acid phosphatase and cyclic phosphodiesterase activities from the heptose-deficient cells grown at 42 degrees C comes off in the Tris-NaCl wash step suggesting a rather loose attachment of these enzymes onto the cell surface.
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PMID:Leakage of periplasmic enzymes from lipopolysaccharide-defective mutants of Salmonella typhimurium. 18

Fat cells particulate phosphodiesterase activity can be solubilized in high yield (80--100%) in a buffer system (30 mM Tris - HCl, pH 8.0) containing non-ionic detergents (0.1% Brij 30, 1.0% Triton X-100), salt (3.0 mM MgSO4, 5.0 mM NaBr) and dithiothreitol (5.0 mM). Polyacrylamide gel electrophoresis of the solubilized enzyme activity indicated the presence of two bands of activities of different electrophoretic mobilities, both of which hydrolyzed cyclic AMP and cyclic GMP. The solubilized activity eluted from DEAE Bio-Gel columns as a somewhat broad profile with at least two peaks of activity. Activity against both cyclic AMP and cyclic GMP eluted in similar but not identical patterns. The solubilized enzyme and DEAE column eluates wxhibited low (less than 1 micronM) Michaelis constants for cyclic AMP and cyclic GMP. In addition, the increases in phosphodiesterase activity induced by incubation of intact fat cells with insulin or adrenocorticotropic hormone are maintained in the solubilized state.
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PMID:Solubilization and characterization of hormone- responsive phosphodiesterase activity of rat fat cells. 19 14

1. The hydrolysis of guanosine triphosphate (GTP) and the consequent formation of guanosine diphosphate (GDP) and phosphate (P1) are activated by light in a suspension of broken retinal rods: the hydrolysis rate with GTP in the micrometer concentration range is 2.5-3.5 n-mole/min per mg of rhodopsin in the preparation. 2. The ionic composition of the medium suspending the rods is not critical: the hydrolysis is present in NaCl saline solution with MG2+ as well as in Tris-HC1 buffer solution, and with the chelating agent EDTA. 3. The ionic strength is critical: the effect is reduced when the broken rods are suspended in a low salt mannitol solution, and is altogether abolished when they are separated from the mannitol solution; it reappears when the mannitol solution is added again in the presence of salts. An element essential for the effect is thus reversibly released in the mannitol solution. No hydrolytic activity on GTP, however, is found in the mannitol soluble fraction. 4. The cyclic nucleotide phosphodiesterase is eluted from the rods in the mannitol solution, and is reaggregated to the rods in the presence of salts; once recombined with the rods, it can be activated by light. 5. The activation of the phosphodiesterase by light is present in the absence of added nucleotide triphosphates.
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PMID:Light-activated hydrolysis of GTP and cyclic GMP in the rod outer segments. 20 80

An enzyme capable of hydrolyzing 4-methylumbelliferyl phenylphosphonate to 4-methylumbelliferone and phenylphosphonic acid has been detected in human serum. It has a Km value of 1.72 x 10(-4) mol/L, has an optimum pH of 8.8-9.1 in Tris buffer, and shows maximum activity at 60 degrees C (30 min). The enzymic activity can be inhibited by Na3PO4, EDTA, and cysteine. We saw no effect of CuSO4, adenosine, thymidine, NaN3, diethyl p-nitrophenyl phosphate, p-chloromercuribenzoate, isopropyl fluorophosphate, or eserine on the enzymic activity. The enzyme cannot hydrolyze substrates of phosphodiesterase I or alkaline phosphatase. The enzyme is considered a phosphonate esterase.
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PMID:Specific detection and properties of enzyme hydrolyzing phosphonate ester in serum. 154 54

The cAMP-dependent regulation of ion channels was studied by using the whole-cell configuration of the patch clamp technique. In isolated cardiac ventricular myocytes, the beta-adrenergically regulated Cl- current (ICl) exhibited an unusual dependence on Na+, such that replacement of extracellular Na+ with compounds such as tetramethylammonium, choline, Tris, or N-methyl-D-glucamine resulted in a reduction in current amplitude without changing the reversal potential. Replacement of extracellular Na+ with tetramethylammonium also reduced the magnitude of the beta-adrenergically enhanced Ca2+ current and delayed rectifier K+ current, suggesting that removal of Na+ was affecting the cAMP pathway that regulates all three currents. Replacement of extracellular Na+ also reduced ICl that was stimulated by (i) direct activation of adenylate cyclase with forskolin, (ii) inhibition of phosphodiesterase with 3-isobutyl-1-methylxanthine, (iii) exposure to the membrane-permeable cAMP derivative 8-bromoadenosine 3',5'-cyclic monophosphate, or (iv) direct phosphorylation of the channel with protein kinase A catalytic subunit. This suggests that the Na+ dependence is at a point beyond the activation of protein kinase A. The Na+ dependence of ICl regulation could not be explained by changes in intracellular Ca2+. However, the sensitivity of the ICl to changes in extracellular Na+ depended significantly on the intracellular Na+ concentration, suggesting that intracellular Na+ plays an important role in the cAMP-dependent regulation of ion channels.
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PMID:Intracellular Na+ modulates the cAMP-dependent regulation of ion channels in the heart. 171 81


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