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)

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

The fatty acid selectivity of cytosolic phospholipase C (phosphatidylinositol phosphodiesterase) from pig and human platelets towards phosphatidylinositol was evaluated. For this purpose, the relative conversion of rat liver phosphatidylinositol (enriched in stearate and arachidonate) and sheep liver phosphatidylinositol (enriched in stearate plus oleate and containing linoleate and arachidonate) was compared and, in addition, the fatty acid compositions of the diacylglycerol products were determined by gas-liquid chromatography. The cytosolic enzyme exhibited essentially complete specificity for phosphatidylinositol when choline-, ethanolamine-, serine-, or inositol-containing phospholipids labelled with [14C]stearate were tested as substrates. Similar percentage conversions of rat and sheep liver phosphatidylinositols to 1,2-diacylglycerol were found with phospholipase C from either pig or human platelets. Furthermore, the newly formed diacylglycerols and the unreacted phospholipid had fatty acid compositions which were very similar to the corresponding substrates. These results suggest that the phospholipase C from isolated platelet cytosol is highly selective towards phosphatidylinositol, but not with respect to the fatty acid composition of naturally occurring phosphatidylinositol. They also suggest that any preferential release of arachidonoyl diacylglycerol in stimulated human platelets is more likely controlled by compartmentation of the corresponding phosphatidylinositol precursor within platelet membranes and its availability, rather than directly by a marked enzyme preference for arachidonate-containing species.
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PMID:Evaluation of the fatty acid selectivity of a phosphatidylinositol-specific cytosolic phospholipase C from pig and human platelets. 651 12

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.
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PMID:Specific localization of scallop gill epithelial calmodulin in cilia. 708 52

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

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

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

Using recombinant baculovirus vectors, the three subunits of mouse rod photoreceptor cGMP phosphodiesterase (PDE) (alpha beta gamma 2) have been expressed in insect cells. The recombinant alpha,beta subunits accumulate to 5 mg/liter culture, but most (98%) of the expressed polypeptides are insoluble. In the soluble fraction, individually expressed alpha and beta subunits showed insignificant PDE activity, but coexpression (by coinfection) of alpha beta subunits elevated PDE activity 7-fold and coexpression of alpha beta gamma up to 15-fold. The soluble expressed holoenzyme associated with ROS membranes under isotonic, but not hypotonic, conditions. The Km of the soluble holoenzyme was 11-16 microM both for coexpressed alpha beta subunits and for alpha beta gamma subunits, similar to the Km (6-80 microM) of native PDE. Site-directed mutagenesis of cysteine to serine in the C-terminal CAAX box of both alpha and beta subunits substantially decreased the protein expression level, abolished post-translational isoprenylation, and prevented subunit binding to the rod outer segment (ROS) membranes. The mutant holoenzyme, however, showed a cGMP hydrolytic activity comparable with that of the normal recombinant enzyme. These results suggest that both alpha and beta subunits are required for the formation of a functional enzyme and that isoprenylation of the subunits is essential for membrane association and stability of PDE.
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PMID:Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase. 810 63

Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Manganiello, V.C., and Belfrage, P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [32P]cGI PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with the phosphopeptide released by the same treatment of M297FRRPS(P)LPCISREQ310. This peptide was synthesized based on the deduced sequence of the cloned rat adipocyte cGI PDE and phosphorylated by cAMP-dependent protein kinase (protein kinase A). Radiosequencing of authentic and synthetic tryptic 32P-peptides showed that a single site in cGI PDE (Ser302) was phosphorylated in adipocytes incubated with isoproterenol and/or insulin. The more than additive phosphorylation and activation of cGI PDE in response to the two hormones found in this report and previously (Smith, C.J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V.C. (1991) J. Biol. Chem. 266, 13385-13390) is proposed to reflect cross-talk between their respective signal transduction pathways at the level of the cGI PDE serine protein kinase or upstream regulatory component(s).
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PMID:Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol. 862 20

cGMP phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells that regulates the level of the second messenger, cGMP. PDE consists of catalytic alpha and beta subunits (Palpha and Pbeta) and two inhibitory gamma subunits (Pgamma) that block PDE activity in the dark. The major inhibitory region has been localized to the C terminus of Pgamma. The last C-terminal residues -IleIle form an important hydrophobic domain critical for the inhibition of PDE activity. In this study, mutants of Pgamma were designed for cross-linking experiments to identify regions on Palpha and Pbeta subunits that bind to the Pgamma C terminus. In one of the mutants, the cysteine at position 68 was substituted with serine, and the last four C-terminal residues of Pgamma were replaced with a single cysteine. This mutant, Pgamma83Cys, was labeled with photoprobe 4-(N-maleimido) benzophenone (MBP) at the cysteine residue. The labeled Pgamma83CysMBP mutant was a more potent inhibitor of PDE activity than the unlabeled mutant, indicating that the hydrophobic MBP probe mimics the Pgamma hydrophobic C terminus. A specific, high-yield cross-linking of up to 70% was achieved between the Pgamma83CysMBP and PDE catalytic subunits. Palpha and the N-terminally truncated Pbeta (lacking 147 aa residues) cross-linked to Pgamma83CysMBP with the same efficiency. Using mass spectrometric analysis of tryptic fragments from the cross-linked PDE, we identified the site of cross-linking to aa residues 751-763 of Palpha. The corresponding region of Pbeta, Pbeta-749-761, also may bind to the Pgamma C terminus. Our data suggest that Pgamma blocks PDE activity through the binding to the catalytic site of PDE, near the NKXD motif, a consensus sequence for interaction with the guanine ring of cGMP.
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PMID:Mechanism of photoreceptor cGMP phosphodiesterase inhibition by its gamma-subunits. 864 88


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