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)

To study changes of junctional membrane permeability associated with transformation, the junctions and the nonjunctional membranes of quail embryo-, chick embryo- and mouse-3T3 cell cultures, infected with temperature-sensitive mutant Rous sarcoma virus, were probed with fluorescent-labelled glutamate. Junctional permeability fell in the transformed state. In the quail cells, the fall was detectable within 25 min of shifting the temperature down to the level (permissive) at which tyrosine-phosphorylation by the viral src gene product is expressed. This reduction of junctional permeability is one of the earliest manifestations of viral transformation. Normal permeability was restored within 30 min of raising the temperature to the nonpermissive level, a reversibility that could be displayed several times during the span of a cell generation. The reversal seems to reflect a reopening of cell-to-cell channels rather than a synthesis of new ones; it is not blocked by protein-synthesis inhibition. Treatments with cyclic AMP and phosphodiesterase inhibitor or with forskolin, which stimulate serine and threonine phosphorylation--the type of phosphorylation on which normal junctional permeability depends (Wiener & Loewenstein, 1983, Nature 305:433)--did not abolish, in general, the junctional effect of the virus; src tyrosine-phosphorylation apparently overrides the junctional upregulation mediated by cyclic AMP. Nonjunctional membrane permeability was not sensibly affected by the virus. It was affected, however, by temperature: lowering the temperature from the nonpermissive to the permissive level caused the nonjunctional permeability to fall, and vice versa. This change was unrelated to transformation. Its secondary effect on junctional transfer is in the opposite direction to that produced by the temperature-activated viral transformation.
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PMID:Intercellular communication and the control of growth: X. Alteration of junctional permeability by the src gene. A study with temperature-sensitive mutant Rous sarcoma virus. 609 20

Two new extracellular nucleases, nucleases SM1 and SM2, were purified from the culture fluid of S. marcescens kums 3958, a fresh clinical isolate. The purification was carried out by the following steps; ammonium sulfate precipitation, and DEAE-cellulose and Sephadex G-100 column chromatography. At the final step, nucleases SM1 and SM2 were purified about 3,700- and 1,000-fold, respectively. They were free from phosphomonoesterase and phosphodiesterase activities. The pIs were 8.1 and 7.5 for nucleases SM1 and SM2, respectively. The molecular weight was estimated to be 35,000 for both enzymes by SDS-polyacrylamide disc gel electrophoresis. The results of amino acid analyses showed that both the threonine and serine contents were higher in nuclease SM2 than in SM1. Furthermore, nuclease SM1 was more stable than nuclease SM2 at 4 degrees C. The other properties of the two enzymes were similar; pH optimum (8.0), Mg2+ or Mn2+ for activation, and inhibition by chemical reagents such as EDTA and pyrophosphate. No significant difference was found in base specificity between nucleases SM1 and SM2. Both enzymes specifically degraded double-stranded homopolymers, especially poly(I). poly(C), as well as yeast RNA and calf thymus DNA. They hardly degraded, however, single-stranded homopolymers such as poly(dA), poly(G), and poly(U).
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PMID:Isolation and characterization of nucleases from a clinical isolate of Serratia marcescens kums 3958. 635 Feb 76

Calmodulin is the name proposed for a multifunctional, calcium binding protein whose presence has been detected in a number of eukaryotic cells. In the studies summarized here, calmodulin has been isolated from spinach leaves (Spinacea oleracea), characterized, and compared to vertebrate calmodulins. Quantitative recovery data for a rapid-isolation protocol demonstrate that calmodulin is a major constituent of spinach leaves. Spinach calmodulin is indistinguishable from vertebrate calmodulins in phosphodiesterase activator activity using vertebrate brain phosphodiesterase and in quantitative immunoreactivity using antiserum made against vertebrate calmodulin. However, spinach calmodulin is really distinguished from vertebrate and invertebrate calmodulins in electrophoretic mobility and in amino acid composition. Spinach calmodulin, like vertebrate calmodulins, lacks tryptophan and contains 1 mol each of N epsilon-trimethyllysine and histidine per 17000 g of protein. In contrast to vertebrate calmodulins, spinach calmodulin has only one tyrosinyl residue and has a threonine/serine ratio of 1.3. While amino acid compositions indicate differences between spinach and vertebrate calmodulins, isolation and characterization of tryptic peptides containing the single histidinyl and N epsilon-trimethyllysyl residues and both prolinyl residues indicate that these regions in spinach calmodulin are similar to the corresponding regions in vertebrate calmodulin. These studies more fully define the general and specific characteristics of calmodulins and indicate that calmodulin structure is not as highly conserved among all eukaryotes as it is among vertebrates and invertebrates.
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PMID:Spinach calmodulin: isolation, characterization, and comparison with vertebrate calmodulins. 745 43

Ever since the identification of two distinct Ang II receptor subtypes, the function of the AT2 receptor has been a subject of debate. As opposed to the AT1 subtype, this receptor does not interact with G-proteins in most cell lines and tissues. We show here that, in intact PC12W cells which express only AT2 receptors, Ang II significantly decreases basal and atrial natriuretic peptide (ANP)-stimulated cGMP concentration. This effect is mimicked by the AT2 selective agonist CGP 42112, and is not prevented by the AT1 selective antagonist losartan, indicating that this is an AT2 receptor mediated response. The lack of effect of the phosphodiesterase (PDE) inhibitor IBMX shows that this mechanism does not involve PDE stimulation. This is confirmed by the finding that neither Ang II or CGP 42112 affect the Ca++/calmodulin dependent cGMP PDE activity. Furthermore Ang II and CGP 42112 have no effect on nitroprusside-stimulated cGMP levels in these cells, thus ruling out interactions between the AT2 receptor and soluble guanylate cyclase. These data indicate that the AT2 receptor mediated decrease of cGMP is due to the selective inhibition of particulate guanylate cyclase (pGC) activity. In an accompanying paper we report that interaction of Ang II with the AT2 receptor in the same cells results in the stimulation of phosphotyrosine phosphatase (PTPase) activity. Interestingly, the PTPase inhibitors sodium orthovanadate and phenylarsine oxyde, but not the Ser/Thr phosphatase inhibitor okadiac acid, inhibitthe Ang II and CGP 42112 induced decreases in cellular cGMP concentration. These findings suggest that stimulation of PTPase activity may be involved in the regulation of pGC activity via AT2 receptors.
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PMID:Angiotensin AT2 receptor mediated inhibition of particulate guanylate cyclase: a link with protein tyrosine phosphatase stimulation? 752 2

The plasma cell differentiation antigen PC-1 was purified to homogeneity from rat liver membranes. Denaturing electrophoresis revealed polypeptides of 118 and 128 kDa, which were both recognized by antibodies against recombinant murine PC-1. During gel filtration PC-1 migrated as a protein of about 500 kDa, suggesting a tetrameric structure. Purified PC-1 displayed a phosphodiesterase-I/nucleotide pyrophosphatase activity that could be completely blocked by EDTA, dithiothreitol and acidic fibroblast growth factor (extrapolated Ki = 1.3 nM). Purified PC-1 was also capable of threonine autophosphorylation and of phosphorylation of histone IIa. The autophosphorylation of PC-1 was inhibited by addition of histone IIa, and it was blocked by phosphodiesterase-I inhibitors (acidic fibroblast growth factor, dithiothreitol), by nucleotides (ATP, ADP, AMP), and by vanadate. When added to autophosphorylated PC-1, these compounds caused a prompt dephosphorylation. However, the same agents did not affect the (de)phosphorylation of histone IIa, which is not a substrate for the PC-1 phosphatase. These data indicate that phosphodiesterase-I inhibitors, nucleotides and vanadate affect the (de)phosphorylation of PC-1 by stimulating the PC-1 phosphatase and/or by shielding the autophosphorylation site from the PC-1 kinase. The rate of dephosphorylation of PC-1 was independent of the dilution, suggesting an autocatalytic intramolecular process. We propose that the autophosphorylation of PC-1 serves to block its nucleotide pyrophosphatase activity when extracellular ATP becomes scarce.
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PMID:Regulation of purified hepatic PC-1 (phosphodiesterase-I/nucleotide pyrophosphatase) by threonine auto(de)phosphorylation and by binding of acidic fibroblast growth factor. 753 98

PC-1 is an ecto-enzyme possessing alkaline phosphodiesterase I (EC 3.1.4.1) and nucleotide pyrophosphatase (EC 3.6.1.9) activities. It has also been proposed to be an ecto-protein kinase capable of phosphorylating itself as well as exogenous proteins. We have investigated the phosphorylation capability of PC-1 and have developed a novel method for its detection and characterization based on autophosphorylation, which allows detection without the use of antibodies. When cells expressing membrane PC-1 were held on ice with [gamma-32P]ATP, SDS/PAGE of whole cell lysates showed a single band which was PC-1; this band was absent in cells not expressing PC-1. Immunoprecipitates of soluble PC-1 isolated from culture supernatants of cells expressing PC-1 were also capable of autophosphorylation, and the size of the labeled protein was the same as previously reported for soluble PC-1. PC-1 was also labeled with [alpha-32P]ATP and [35S]dATP[alpha S]. We found no evidence that PC-1 was capable of phosphorylating proteins other than itself, and conclude that it is not a true kinase, and that the observed labeling with [gamma-32P]ATP, [alpha-32P]ATP and [35S]dATP[alpha S] reflect transient covalent adducts that are part of the catalytic cycle of phosphodiesterase/pyrophosphatase activity rather than intrinsic kinase activity. Mutation of the active-site threonine to tyrosine, serine or alanine reduced the 5'-nucleotide phosphodiesterase activity of PC-1 and its ability to autophosphorylate to undetectable levels. Together, these data suggest that both activities depend on the same site.
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PMID:Autophosphorylation of PC-1 (alkaline phosphodiesterase I/nucleotide pyrophosphatase) and analysis of the active site. 773 62

Drosophila Rrp1 has several tightly associated enzymatic activities, including double-strand DNA 3'-exonuclease, apurinic/apyrimidinic endonuclease, 3'-phosphatase, and 3'-phosphodiesterase. The carboxyl-terminal third of Rrp1, homologous to Escherichia coli exonuclease III, is sufficient to repair oxidative and alkylation-induced DNA damage in vivo. Using a screen for partial complementation of repair-deficient E. coli, we isolated three mutants of the nuclease domain of Rrp1: T462A, K463Q, and L484P, that protect against methyl methanesulfonate (MMS)-induced but not t-BuO2H-induced DNA damage. Thr-462 and Lys-463 are highly conserved residues found in a cluster of 5 conserved amino acids (LQETK), while Leu-484 is poorly conserved. Gln-460 Glu-461, Thr-462, and Lys-463 and Leu-484 were altered by site-directed mutagenesis using a plasmid including the entire Rrp1 gene and mutant proteins were purified. Mutants of the three residues Glu-461, Thr-462, and Lys-463 demonstrate 8-200-fold lower phosphodiesterase specific activity than wild-type Rrp1. E461A has a 30-fold reduction in AP endonuclease and is MMS-sensitive, but all other mutants have near-normal AP endonuclease and are MMS-resistant. Glu-461 appears to be essential for the nuclease function for Rrp1. Lys-463 and, to a lesser extent, Thr-462 influence the substrate specificity of the Rrp1 nuclease.
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PMID:Single amino acid changes alter the repair specificity of Drosophila Rrp1. Isolation of mutants deficient in repair of oxidative DNA damage. 779 76

Calmodulin (CaM) was purified from bovine brain and identified on the basis of its phosphodiesterase activity. Its purity was further tested by electrophoretic migration in polyacrylamide gels in the presence of sodium dodecyl sulfate. Apo-CaM was prepared from holo-CaM using hydroxyapatite chromatography. The Ca2+ binding sites on CaM and the pKa of each of the functional groups bound to Ca2+ were identified from the dependence of Ca2+ interaction with the functional group as a function of pH. EGTA was found to diminish the peaks corresponding to the pKa values of the groups bound to Ca2+. The use of bromophenacyl bromide, a modifier for aspartate and glutamate residues in proteins, diminished the peaks at pH = 3.4 and 4.3. Diethyl pyrocarbonate, a modifier for histidine residues, reduced the peak at pH = 6.2, corresponding to the pKa of the imidazole group in histidine. Furthermore, the peak at pH = 11.6 was eliminated using the specific tyrosine modifier, N-acetylimidazole. Diethylpyrocarbonate also eliminated four small peaks at pH = 7.2, 7.8, 8.2 and 8.8. This effect could be attributed to the binding of threonine and serine residues. The crystallographic results for parvalbumin, which has a similar molecular structure, suggest identical Ca2+ binding sites.
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PMID:Elucidation of pKa values for Ca2+ binding sites in calmodulin by spectrofluorometry. 784 65

Human cell lines express two genetically distinct isoforms of DNA topoisomerase (topo II) II: topo II alpha (p170) and topo II beta (p180). We detected a higher molecular weight form with an apparent molecular mass of about 190 kDa in M phase-arrested HeLa cells (Kimura, K., Saijo, M., Ui, M., and Enomoto, T. (1994) J. Biol. Chem. 269, 1173-1176). In this study we confirmed, using anti-topo II alpha and topo II beta monoclonal antibodies, that this higher molecular weight form is topo II beta and consists of doublet bands around 190 kDa. We confirmed that the doublet bands constituted an M phase-specific phenomenon and were not an artifact of the procedure used to accumulate mitotic cells. Digesting the immunoprecipitated materials from mitotic cell extracts with alkaline phosphatase resulted in the disappearance of the doublet bands and the appearance of the 180-kDa band with the concomitant disappearance of 32P label in the region of the doublet bands. Neither heat-inactivated alkaline phosphatase nor phosphodiesterase affected the doublet bands and the 32P label. Topo II beta in interphase cells was also phosphorylated, but the shift in apparent molecular weight was very slight after alkaline phosphatase digestion. Analysis of the labeled phosphoamino acids present in topo II beta from M phase and logarithmically growing cells indicated that phosphorylation occurred mainly on serine and fairly on threonine residues in both topo II beta isoforms. These results indicated that topo II beta is phosphorylated at specific sites in M phase, resulting in the formation of the doublet bands.
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PMID:Identification of the nature of modification that causes the shift of DNA topoisomerase II beta to apparent higher molecular weight forms in the M phase. 792 18

CaM[3 TnC] is a calmodulin-cardiac troponin C chimeric protein containing the first, second, and fourth calcium-binding domains of calmodulin (CaM) and the third calcium-binding domain of cardiac troponin C (cTnC) (George, S. E., Su, Z., Fan, D., and Means, A. R. (1993) J. Biol. Chem. 268, 25213-25220). CaM[3 TnC] shows altered activation of phosphodiesterase (PDE) and is a potent competitive inhibitor of smooth muscle myosin light chain kinase (smMLCK) activation by CaM. To determine why CaM[3 TnC] exhibits altered target enzyme interactions, we constructed a series of domain 3 CaM mutants. We began with subdomain substitutions, replacing most of CaM's helix 5, Ca2+ binding loop 3, and helix 6 with the corresponding subdomains of cTnC. Only CaM[helix 6-TnC] exhibited significant impairment of smMLCK and PDE activation. We then individually substituted the residues in the region of CaM's helix 6 with the corresponding cTnC residue. This revealed that CaM residues Thr-110, Leu-112, and Lys-115 were critical for full smMLCK activation and could not be substituted by the corresponding cTnC residue (Gln, Thr, and Thr, respectively). In contrast, only the L112T substitution significantly affected PDE activation. The CaM-smMLCK peptide structure (Meador, W. E., Means, A. R., and Quiocho, F. A. (1992) Science 257, 1251-1255) suggests a relationship between the proposed helix 6 smMLCK-activating residues and those previously described in helix 2 (VanBerkum, M. F. A., and Means, A. R. (1991) J. Biol. Chem. 266, 21488-21495).
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PMID:Role of domain 3 of calmodulin in activation of calmodulin-stimulated phosphodiesterase and smooth muscle myosin light chain kinase. 820 99


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