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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)

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

The effect of preincubation of preparations of the outer segments of optic rods with the nonhydrolyzed analog GTP-guanilyl-5'-imidodiphosphate (Gpp(NH)p) and NaF, the combined effect of these agents as well as the action of (NH4)2SO4 (10-800 mM), MgSO4 (2-50 mM) and induction of peroxide oxidation of lipids are studied as applied to the catalytic activity of phosphodiesterase of cyclic nucleotides. Gpp(NH)p and NaF are shown to be tightly bound to GTP-binding proteins (G-proteins) of outer segments of optic rods, additional activation of phosphodiesterase in the presence of Gpp(NH)p being observed after preincubation with NaF and subsequent washing of the membrane. A problem on different binding sites of the ion F and Gpp(NH)p on G-proteins is discussed. It is found that (NH4)2SO4 does not affect the basal activity of phosphodiesterase but inhibits the activating effect of Gpp(NH)p and NaF on the enzyme. Induction of peroxide oxidation of lipids prevented by the addition of ionol (antioxidant) in a dose of 5.10(-4) M has the same effect. Changes in the concentration of Mg2+ in the medium influence insignificantly the basal activity of phosphodiesterase but are necessary for manifestation of the activating effect of Gpp(NH)p and NaF.
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PMID:[Influence of effectors of hormone-sensitive adenylate cyclase on the activation system of photostimulated cyclic nucleotide phosphodiesterase from outer rod segments]. 301 May 21

The effect of cyclic nucleotide analgoues upon the immediate induction of the guinea pig acrosome reaction (AR) was studied. Dibutyryl (dB) CGMP and 8-bromo-cGMP, when added to sperm suspensions after varying periods of preincubation in glucose-free BWW medium (NaCl 94.59 mM, KCl 4.7 mM, CaCl2 1.71 mM, KH2PO4 1.19 mM, MgSO4 1.19 mM, NaHCO3 25.07 mM, pyruvate 0.25 mM, lactate 21.58 mM, and bovine seru albumen 1 g/liter), induced the AR in a large proportion of spermatozoa relative to controls. The proportion of ARs induced upon the addition of dB cGMP or 8-bromo-cGMP (10mM) at 1 h was equivalent to that obtained after a 5-h incubation in glucose-free BWW alone. The effect of cGMP analogues was concentration dependent over the tested range of 2-12 mM (less than 1-20%). The simultaneous addition if imidazole (10 mM), a cAMP phosphodiesterase stimulator, potentiated the effect (imidazole + 12 mM 8-bromo-cGMP: 73%). cAMP analogues were without effect. The presence of extracellular Ca++ was required, and it is suggested that a rise in the cGMP/CAMP ratio triggers Ca++ influx and the AR.
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PMID:Induction of the acrosome reaction in guinea pig spermatozoa by cGMP analogues. 624 66

The unicellular eukaryote Tetrahymena pyriformis secretes significant amounts of cyclic AMP into its external medium. Cells transferred from growth medium into any of the following three different non-nutrient media: (a) 5 mM phosphate buffer containing 47 mM NaCl and 1 mM MgSO4, (b) 10 mM Tris, or (c) 1.3 mM Tris containing 1 mM citrate and 1 mM Ca(OH)2, released to the outside almost 60--80% of the total cyclic AMP produced during 2--5 h of incubation. Tris-citrate-Ca+2 medium was chosen for further experiments because of its minimal nonspecific interference in the cyclic AMP radioimmunoassay. The identity of the secreted material recognized as cyclic AMP by radioimmunoassay was confirmed by demonstrating its almost complete hydrolysis with commerical beef heart phosphodiesterase. Furthermore, the radioimmunoassay-active material in the concentrated medium co-chromatographed on paper with [3H]cyclic AMP, as judged by assay of the eluted material. After resuspending cells in Tris-citrate-Ca2+ medium, the extracellular concentration of cyclic AMP rose steadily over a 5-h period, reaching a level equvalent to approximately 35--50 pmol cyclic AMP/10(6) cells vs. an internal cyclic AMP quantity at 5 h of 8--10 pmol/10(6) cells. After 5 h, the level of extracellular cyclic AMP reached a plateau. There was no degradation or uptake of external cyclic AMP by the cells during this period.
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PMID:Evidence for a pronounced secretion of cyclic AMP by Tetrahymena. 624 69

Cyclic-AMP phosphodiesterase (PDE) accumulates during the aggregation stage of Dictyostelium where it functions in maintaining extracellular levels of cyclic AMP (cAMP). The activity decreases during the subsequent multicellular slug stage and then accumulates again during sorocarp construction, but the enzyme is active only in the developing stalk. Because of the possible significance of this localized activity in only one of the two cell types, we have purified the enzyme from the multicellular stage in order to understand its mode of regulation in vivo. We find that the enzyme which is localized in the prestalk cells is similar in many respects to the extracellular PDE which is active at the aggregation stage. The enzyme from both stages is inhibited by a low molecular weight protein. The mechanism of this inhibition is through a shift in the apparent Km for cAMP from micromolar to millimolar levels. The inhibited form of the enzyme can be activated by preincubation with MgSO4 and dithiothreitol (DTT). This activation treatment releases the inhibitor from the enzyme, thus restoring the low Km form, changes the molecular weight of the culmination stage enzyme from 95 000-100 000 to 68 000 by releasing the Mr 35 000-40 000 inhibitor protein, and causes irreversible loss of inhibitor activity. Although the inhibitor could be obtained in high yield from the aggregation stage by simply heating the extracellular fluid, it could not be detected from culmination stage extracts when prepared by this method. However, inclusion of calcium in the extraction buffer resulted in release of inhibitor from both heated and nonheated samples. The results indicate that the stalk cell specific PDE is regulated similarly to the aggregation stage PDE and opens the possibility of differential regulation of PDE in the two cell types.
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PMID:Purification and properties of a cyclic-AMP phosphodiesterase that is active in only one cell type during the multicellular development of Dictyostelium discoideum. 630 42

A sensitive method for separation and fluorometric quantification of 2',3'-cyclic nucleotide-3'-phosphodiesterase (EC 3.1.4.37) (CNPase) reaction products with 2',3'-cyclic adenosine monophosphate as substrate is presented. The 2'-AMP product was separated by cellulose thin-layer chromatography in 4 M MgSO4-0.5 M sodium acetate-2-propanol (80:18:2, v/v/v). After reaction with glyoxal dihydrate, the amount of reaction product was determined fluorometrically using excitation at 328 nm and emission at 382 nm. These results correlated well with those obtained using Kurihara and Tsukada's [(1967) J. Neurochem. 14: 1167-1174] paper chromatographic method (r = 0.96). With this fluorometric method, amounts as low as 0.20 nmol of 2'-AMP can be determined, and its sensitivity is comparable to that of the radiochemical method. The method is easy to use and sensitive enough for measuring CNPase activity in tissue with low enzyme activity.
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PMID:A method for chromatographic separation and fluorometric quantification of 2',3'-cyclic nucleotide-3'-phosphodiesterase reaction products. 632 31

Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5'-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO4 as an activator, 5 mM theophylline as an inhibitor of 3' 5'-AMP-phosphodiesterase and 2 mm lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10(-4) M adrenaline in the presence of 5'-guanylylimido-diphosphate (GMP-PNP) as well as with 10(-2) M NaF. In the cells incubated in a medium devoid of theophylline and containing 5'-AMP instead of AMP-PNP, 5'-nucleotidase activity was observed in the same cell structures as AC activity, Hydrolysis of 5'-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5'-AMP in all cell structures. No staining was observed with 2 mM beta -glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5'-AMP or p-nitrophenyl phosphate, but not beta -glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 303nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.
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PMID:Ultracytochemical localization of adenylate cyclase activity in rat thymocytes. 729 93