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)

The challenge of 3T3-F442A fibroblasts with growth hormone led to both a decrease in the mobility on SDS/PAGE and activation of the PDE4A cyclic AMP-specific phosphodiesterase isoform PDE4A5. Activation was mediated by a JAK-2-dependent pathway coupled to the activation of phosphatidylinositol 3-kinase and p70S6 kinase. Activation was not dependent on the ability of growth hormone to stimulate ERK2 or protein kinase C or any effect on transcription. Blockade of activation of murine PDE4A5 ablated the ability of growth hormone to decrease intracellular cAMP levels. Antisense depletion of murine PDE4A5 mimicked the ability of rolipram to enhance the growth hormone-stimulated differentiation of 3T3-F442A cells to adipocytes. It is suggested that activation of PDE4A5 by growth hormone serves as a brake on the differentiation processes.
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PMID:Stimulation of p70S6 kinase via a growth hormone-controlled phosphatidylinositol 3-kinase pathway leads to the activation of a PDE4A cyclic AMP-specific phosphodiesterase in 3T3-F442A preadipocytes. 952 Apr 3

Rattlesnake venoms are complex biological products containing potentially autolytic components, and they provide a useful tool for the study of long-term maintenance of enzymes in a competent state, both in vivo and in vitro. To evaluate the stability of venom components, 15 aliquots of freshly extracted venom (from Crotalus molossus molossus) were subjected to 15 different temperature and storage conditions for 1 week and then lyophilized; conditions varied from storage at -80 degrees C (optimal preservation of activities) to dilution (1:24) and storage at 37 degrees C (maximal degradation potential). Effects of different storage conditions were evaluated using SDS-PAGE, metalloprotease zymogram gels, a cricket LD50 assay and enzyme assays (metalloprotease, serine proteases, phosphodiesterase, L-amino acid oxidase and phospholipase A2). Venom samples were remarkably refractive to widely varying conditions; enzyme activities of some samples were variable, particularly L-amino acid oxidase, and one sample treatment showed higher toxicity, but electrophoretic results indicated very little effect on venom proteins. This study suggests that most venom activities should remain stable even if stored or collected under potentially adverse conditions, and freezing samples is not necessarily advantageous. Proteins in the crude venom are not as labile as has been previously thought, and endogenous mechanisms present in the venoms likely inhibit autolysis during long-term storage that occurs in vivo in the gland.
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PMID:Effects of temperature and storage conditions on the electrophoretic, toxic and enzymatic stability of venom components. 953 Aug 14

Venoms from the scorpaeniformes Synanceja trachynis and Gymnapistes marmoratus were quantitatively analyzed for enzymic activity. S. trachynis venom displayed significantly higher hyaluronidase activity than G. marmoratus venom, and G. marmoratus venom displayed significantly higher levels of esterase, acid phosphatase, alkaline phosphatase and phosphodiesterase activity. No detectable quantities of phospholipase A2 activity were found in G. marmoratus venom. SDS-polyacrylamide gel electrophoresis of S. trachynis venom indicated the presence of 6 protein bands (20 kDa-295 kDa). G. marmoratus venom displayed 8 protein bands (11 kDa-109 kDa).
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PMID:Enzyme and biochemical studies of stonefish (Synanceja trachynis) and soldierfish (Gymnapistes marmoratus) venoms. 965 39

A poly(ADP-ribose) polymerase-like enzyme, detected in a crude homogenate from Sulfolobus solfataricus by means of activity and immunoblot analyses, was purified to electrophoretic homogeneity by a rapid procedure including two sequential affinity chromatographies, on NAD+-agarose and DNA-Sepharose. The latter column selected specifically the poly(ADP-ribosyl)ating enzyme with a 17% recovery of enzymic activity and a purification of more than 15000-fold. The molecular mass (54-55 kDa) assessed by SDS/PAGE and immunoblot was definitely lower than that determined for the corresponding eukaryotic protein. The enzyme was proved to be thermophilic, with a temperature optimum of approx. 80 degreesC, and thermostable, with a half-life of 204 min at 80 degreesC, in good agreement with the requirements of a thermozyme. It displayed a Km towards NAD+ of 154+/-50 microM; in the pH range 6.5-10.0 the activity values were similar, not showing a real optimum pH. The enzyme was able to bind homologous DNA, as evidenced by the ethidium bromide displacement assay. The product of the ADP-ribosylating reaction co-migrated with the short oligomers of ADP-ribose (less than 6 residues) from a eukaryotic source. Reverse-phase HPLC analysis of the products, after digestion with phosphodiesterase I, gave an elution profile reproducing that obtained by the enzymic digestion of the rat testis poly(ADP-ribose). These results strongly suggest that the activities of the purified enzyme include the elongation step.
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PMID:Purification and biochemical characterization of a poly(ADP-ribose) polymerase-like enzyme from the thermophilic archaeon Sulfolobus solfataricus. 976 45

Gustin, a zinc-metalloprotein constituting about 3% of human parotid saliva protein was previously isolated and characterized as a single polypeptide chain of 37kDa with one mole of zinc tightly bound to the protein. It exhibited biological activity activating calmodulin dependent bovine brain cAMP phosphodiesterase and was decreased in saliva of patients with loss of taste in whom taste buds showed a specific pathological morphology. Determination of its primary structure by amino acid sequence revealed it was identical with carbonic anhydrase (CA) [EC 4.2.1.1] VI and had two N-linked glycosylation sites. Analysis by reverse phase HPLC and SDS-PAGE before and after deglycosylation confirmed a single peak with molecular weight of the purified protein being 37kDa, the deglycosylated protein, 33kDa. N-linked carbohydrate chains contained N-acetyl glucosamine, galactose, mannose, and fucose interior to di, tri and tetra sialyated termini. By isoelectric focusing five increasingly acidic pI values were determined consistent with addition of sialic acid as the terminal carbohydrate residue on the N-linked glycoforms of the protein. Gustin was found to exhibit CA activity but was inhibited by known CA inhibitors in a different manner than CA I or II. These findings, consistent with analysis of previous investigators, indicate that parotid saliva gustin is CA VI.
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PMID:Gustin from human parotid saliva is carbonic anhydrase VI. 978 98

The purification and characterization of a novel phosphodiesterase (PDE) is presented. The activity was detected in the extracellular medium of Tetrahymena thermophila cultures, by the release of p-nitrophenol from p-nitrophenylphosphocholine (PNPPC) with an acidic pH optimum. In cell homogenates, it is sedimentable, shows a latency similar to that of acid phosphatase and is co-secreted with this enzyme, indicating that it is a lysosomal hydrolase. PNPPC-PDE was purified to homogeneity from the extracellular medium, yielding a single band of 58 kD on SDS polyacrylamide gel electrophoresis. It catalyzed the release of glycerol from glycerophosphocholine (GPC) and GPC competitively inhibits degradation of PNPPC. We present further evidence indicating that the natural substrate for PNPPC-PDE is GPC. Thus, Tetrahymena becomes the first eukaryote in which a lysosomal GPC-PDE is observed. This finding provides a new pathway for the complete breakdown of phosphatidylcholine in a lysosomal medium.
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PMID:Lysosomal glycerophosphocholine phosphodiesterase in Tetrahymena. 1020 74

A novel N-glycosylated, mannose-rich protein has been purified approx. 4000-fold from human liver in a seven-step procedure including ion-exchange chromatography and fractionation on concanavalin A-Sepharose, Sephadex G-75 and oligo(dT)-cellulose matrices. The molecular mass of the protein is 46 kDa when measured by gel filtration (i.e. under non-denaturing conditions) and 60 kDa by SDS/PAGE (i.e. under denaturing conditions). The protein possesses two DNA backbone-incising activities, namely, the random introduction of single-strand breaks in native DNA and the rupture of the phosphodiester linkage internal to cyclobutyl pyrimidine dimers, the major class of DNA lesions induced by solar UV rays. Both activities are optimal at pH 5.0 in vitro, although the non-specific nuclease displays appreciable activity at neutral pH, depending on the buffer composition. The protein has been named acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase (AN/IDP). As a nuclease, the protein 'prefers' a linear DNA structure over a covalently closed circular molecule and is more proficient at digesting single-stranded than double-stranded DNA. The polynucleotide cleavage products of the nuclease contain 5'-OH and 3'-PO(4) termini, which are refractory to direct rejoining by DNA ligases. Depending on the substrate, the nuclease activity exhibits a temperature optimum of 50 degrees C or greater, and is neither stimulated by Mg(2+) or Ca(2+) nor inhibited by Zn(2+). AN/IDP is present in human liver and in cultured human cells of both fibroblastic and lymphocytic origins. Intracellularly, the protein can be readily detected in both the cytosolic and nuclear fractions, although much more (approx. 3-fold) is found in the latter fraction. We propose that this bifunctional enzyme may be involved in both apoptotic DNA digestion and metabolism of cyclobutyl pyrimidine dimers in UV-irradiated human cells.
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PMID:Purification and characterization of a novel human acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase. 1064 17

The cyclic AMP phosphodiesterase (PDE) activity in Leishmania mexicana is mainly located (>95%) in the soluble fraction of the cell. The intact parasite, as well as plasma membranes, showed PDE activity, probably indicating that at least part of the activity in the particulate fraction resides on the parasite cell surface, with its catalytic domain facing the extracellular moiety. For the first time, a highly specific cAMP phosphodiesterase (PDE) was purified from the soluble fraction to apparent homogeneity after a single step 2239-fold purification using pseudo-affinity chromatography on Cibacron Blue 3GA agarose. The enzyme was identified as a 61-kDa protein on SDS-PAGE, with a K(m) of 277 microM at 30 degrees C (optimum temperature). The native enzyme protein showed an apparent molecular size of approximately 200000 estimated by molecular sieve chromatography on Sephacryl S-300. Further characterization of the PDE activity present in the soluble fraction shows that the enzyme requires Mg(2+) for maximal activity. Furthermore, no activity was detected when assayed at pHs below 6.0, but above this value it increased dramatically, reaching the optimum at pH 7.2. On the basis of the K(m) and PDE activity in presence of specific drugs or modulators such as rolipram, OPC-3911, cGMP, IBMX, zaprinast, theophylline, caffeine and Ca(2+)/calmodulin, this enzyme does not seem to conform to any of the ten previously described Class I PDE families but to the PDE class II (or non-mammalian PDEs) similar to the those found in Candida albicans, Dictyostelium discoideum, Saccharomyces cerevisiae or Vibrio fischeri.
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PMID:Characterization of cyclic AMP phosphodiesterases in Leishmania mexicana and purification of a soluble form. 1069 57

The ability of acute insulin treatment to elicit a redistribution of the liver insulin-like growth factor-II/ mannose 6-phosphate (IGF-II/M6P) receptor has been studied in rats, using cell fractionation. Injection of insulin (0.4-50 microg) led to a time- and dose-dependent decrease in IGF-II binding activity in Golgi-endosomal (GE) fractions, along with an increase in activity in the plasma membrane (PM) fraction; only receptor number was affected. Quantitative subfractionation of the microsomal fraction on sucrose density gradients showed that IGF-II binding activity distributed similarly to galactosyltransferase (a Golgi marker), at slightly higher densities than in vivo internalized (125)I-insulin, and at lower densities than 5' nucleotidase and alkaline phosphodiesterase (two plasma membrane markers). Insulin treatment led to a slight time-dependent and reversible shift of IGF-II binding activity toward higher densities. Subfractionation of the GE fraction on Percoll gradients showed that IGF-II binding activity was broadly distributed, with about 60% at low densities coinciding with galactosyltransferase and early internalized (125)I-insulin and with 40% at high densities in the region of late internalized (125)I-insulin. Insulin treatment caused a time-dependent and reversible shift of the distribution of IGF-II binding activity toward low densities. On SDS-PAGE, the size of the affinity-labeled IGF-II/M6P receptor was comparable in GE and PM fractions (about 255 kDa), but on Western blots receptor size was slightly lower in the latter (245 kDa) than in the former (255 kDa). Insulin treatment did not affect the size, but modified the abundance of the IGF-II/M6P receptor in a manner similar to that of IGF-II binding. In vivo chloroquine treatment fully suppressed the changes in IGF-II binding activity in liver GE and PM fractions observed in insulin-treated rats. We conclude that insulin elicits a time-dependent and reversible redistribution of liver IGF-II receptors from Golgi elements and endosomes to the plasma membrane, presumably via early endosomes.
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PMID:Insulin-induced redistribution of the insulin-like growth factor II/mannose 6-phosphate receptor in intact rat liver. 1072 96

R.E. Hill and S.P. Mackessy. Characterization of venom (Duvernoy's secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins. Toxicon XX, xx-yy, 2000. - Venomous colubrids, which include more than 700 snake species worldwide, represent a vast potential source of novel biological compounds. The present study characterized venom (Duvernoy's gland secretion) collected from twelve species of opisthoglyphous (rear-fanged) colubrid snakes, an extremely diverse assemblage of non-venomous to highly venomous snakes. Most venoms displayed proteolytic activity (casein), though activity levels varied considerably. Low phosphodiesterase activity was detected in several venoms (Amphiesma stolata, Diadophis punctatus, Heterodon nasicus kennerlyi, H. n. nasicus and Thamnophis elegans vagrans), and acetylcholinesterase was found in Boiga irregularis saliva and venom, but no venoms displayed hyaluronidase, thrombin-like or kallikrein-like activities. High phospholipase A(2) (PLA(2)) activity was found in Trimorphodon biscutatus lambda venom, and moderate levels were detected in Boiga dendrophila and D. p. regalis venoms as well as B. dendrophila and H. n. nasicus salivas. Non-reducing SDS-PAGE revealed 7-20 protein bands (3.5 to over 200 kD, depending on species) for all venoms analyzed, and electrophoretic profiles of venoms were typically quite distinct from saliva profiles. Components from A. stolata, Hydrodynastes gigas, Tantilla nigriceps and T. e. vagrans venoms showed protease activity when run on gelatin zymogram gels. N-terminal protein sequences for three 26 kD venom components of three species (H. gigas, H. torquata, T. biscutatus) and one 3.5 kD component (T. nigriceps) were also obtained, and the 3.5 kD peptide showed apparent sequence homology with human vascular endothelial growth factor; these data represent the first sequences of colubrid venom components. Protease, phosphodiesterase and PLA(2) activities are also common to elapid and viperid snake venoms, but it is apparent that numerous other (as yet undescribed) components make up the majority of colubrid venom proteins. The complex nature of venoms produced by most species surveyed, and the high levels of protease or phospholipase A(2) activity of some venoms, suggest that many colubrids could become an important source of human health concern as encounters with these snakes increase.
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PMID:Characterization of venom (Duvernoy's secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins. 1085 9


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