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)

Purified rat liver nuclei were incubated in vitro in the presence of [adenylate-32P]nicotinamide adenine dinucleotide. The label was rapidly incorporated into trichloroacetic acid-insoluble material and also detected in particles carrying heterogeneous nuclear RNA. The particles were isolated by density gradient centrifugation, and their size determined to be 30-40 S from parallel experiments using nuclei labelled with [3H]uridine 5'-triphosphate under similar conditions. Treatment of the 30-40 S-particles with enzymes of different specificities showed that the label was tightly bound to proteins, not incorporated into nuclei acids and not utilized in phosphorylation of proteins. The label was detached by phosphodiesterase I from snake venom and identified as ADP-ribose and adenosine 5'-phosphate present at a ratio of 7.5 to 1 using thin layer chromatography on poly(ethyleneimine)-cellulose. Radioactively labelled (ADP-ribosylated) proteins were visualized by autoradiography following SDS-polyacrylamide gel electrophoresis. They included several major species of the ribonucleoprotein with molecular weights of 36000, 39000 and 42000, and a limited number of high molecular weight polypeptides.
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PMID:ADP-ribosylation of proteins associated with heterogeneous nuclear RNA in rat liver nuclei. 617 54

Two high-affinity monoclonal antibodies (ROS-1, ROS-2) have been produced to the rod outer segment phosphodiesterase (ROS PDE). These antibodies bind at different antigenic determinants. ROS-2 absorbs a subset of the total PDE activity from a detergent-solubilized retina extract, whereas ROS-1 adsorbs nearly all of the PDE activity. DEAE-cellulose chromatography separates two peaks of activity from a hypotonic extract of rod outer segments. Peak I activity is adsorbed only by ROS-1, whereas peak II activity is adsorbed by both ROS-1 and ROS-2. Both peaks of activity are activated by histone H2B and limited trypsin digestion, and both peaks of activity contain a heat-stable, trypsin-sensitive inhibitor. When analyzed by SDS gel electrophoresis, ROS-1 adsorbed a single peptide from peak I, which comigrated with phosphorylase B, whereas ROS-1 adsorbed two slightly faster migrating peptides from peak II. Histone H2B activated at least 80% of the PDE activity bound to ROS-2 but was less effective in activating the PDE bound to ROS-1. ROS-1 but not ROS-2 was an effective inhibitor of PDE activity, suggesting that ROS-1 may be a specific probe to study the effects of ROS PDE on the light response.
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PMID:Immunotitration analysis of the rod outer segment phosphodiesterase. 620 41

Calmodulin-dependent cyclic nucleotide phosphodiesterase was purified from bovine brain to apparent homogeneity by a new procedure involving DEAE-cellulose, Affi-Gel blue, calmodulin-Sepharose 4B, and Sephadex G-200 column chromatographies. The enzyme was purified more than 3,000-fold from the brain extracts with greater than 12% yield. The purified phosphodiesterase could be activated 10- to 15-fold by calmodulin and Ca2+ to a specific enzyme activity of more than 300 mumol of cAMP hydrolyzed/min/mg of protein. Molecular weight of the enzyme was determined to be 115,800 by the sedimentation equilibirum method or 124,000 from the sedimentation constant and Stokes radius of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight of 58,000. These results suggested that the calmodulin-dependent phosphodiesterase from bovine brain has a subunit structure of alpha2. Molecular weight of the complex of calmodulin and phosphodiesterase was the complex of calmodulin and phosphodiesterase was also calculated from the sedimentation constant and Stokes radius to be 159,000. Since calmodulin has a molecular weight of about 17,000, the result indicated that the stoichiometry of the complex is calmodulin2 alpha2. The catalytic subunit of cylic AMP-dependent protein kinase was found to catalyze the phosphorylation of the purified phosphodiesterase with the incorporation of 2 mol of phosphate/mol of the enzyme.
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PMID:Purification and properties of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase. 624 53

Calmodulin has been purified from cell bodies of the green alga Chlamydomonas by Ca++-dependent affinity chromatography on fluphenazine-Sepharose 4B. Calmodulin from this primitive organism closely resembles that from bovine brain in a number of properties, including (a) binding to fluphenazine in a Ca++-dependent, reversible manner, (b) functioning as a heat-stable, Ca++-dependent activator of cyclic nucleotide phosphodiesterase, and (c) electrophoretic mobility in SDS-polyacrylamide gels in both the presence and absence of Ca++, which causes a shift in the relative mobility of calmodulin. Calmodulin has also been identified by the criteria of phosphodiesterase activation and electrophoretic mobility in both the detergent soluble "membrane plus matrix" and the axoneme fractions of Chlamydomonas flagella. Calmodulin is not associated with the partially purified 12S or 18S dynein ATPases of Chlamydomonas. The presence of calmodulin in the flagellum suggests that it is involved in one or more of the Ca++-dependent activities of this organelle.
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PMID:Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella. 625 28

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP, EC 3.1.4.37) has been isolated from rat brain myelin by chromatography on successive columns of phenyl-Sepharose CL-4B, CM-Sepharose CL-6B, and 8-(6-aminohexyl) amino-2'AMP-Sepharose 4B. From 15 g of rat brain, approximately 400 micrograms of pure CNP was obtained, with a specific activity of 1,200 (2',3'-cyclic AMP) units/mg protein. The Km of the rat enzyme was 3.7 mM, using 2',3'-cAMP as the substrate. Isoelectric focusing of the enzyme indicated a broad isoelectric range of 8.5-9.0. On SDS polyacrylamide gels, rat CNP appears as two protein bands of approximately 48,000 and 50,000 M.W., with an upper band intensity of about 1/10 that of the lower band. The relative intensities of the bands for CNP and the molecular weights correspond to the Wolfgram proteins W1 and W2 described by other investigators. The amino acid analysis of the purified rat enzyme compared favorably with reported determinations for the bovine enzyme and also with reported values for the rat Wolfgram proteins W1 and W2.
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PMID:Purification of rat 2',3'-cyclic nucleotide 3'-phosphodiesterase. 625 58

1. The ability of a range of phenothiazines to inhibit activation of brain phosphodiesterase by purified calmodulin was studied. Trifluoperazine, prochlorperazine and 8-hydroxyprochlorperazine produced equipotent dose-dependent inhibition with half-maximum inhibition at 12mum. When tested at 10 or 50mum, 7-hydroxyprochlorperazine was a similarly potent inhibitor. However, trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were ineffective at concentrations up to 50mum, and produced only a modest inhibition at 100mum. 2. The same phenothiazines were tested for their ability to inhibit activation of brain phosphodiesterase by boiled extracts of rat islets of Langerhans. At a concentration of 20mum, 70-80% inhibition was observed with trifluoperazine, prochlorperazine, 7-hydroxyprochlorperazine or 8-hydroxyprochlorperazine, whereas trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were less effective. 3. The effect of these phenothiazines on insulin release from pancreatic islets was studied in batch-type incubations. Insulin release stimulated by glucose (20mm) was markedly inhibited by 10mum-trifluoperazine or -prochlorperazine and further inhibited at a concentration of 20mum. 8-Hydroxyprochlorperazine (20mum) was also a potent inhibitor but 7-hydroxyprochlorperazine (20mum) elicited only a modest inhibition of glucose-stimulated insulin release; no inhibition was observed with trifluoperazine-5-oxide or N-methyl-2-(trifluoromethyl)phenothiazine. 4. Trifluoperazine (20mum) markedly inhibited insulin release stimulated by leucine or 4-methyl-2-oxopentanoate in the absence of glucose, and both trifluoperazine and prochlorperazine (20mum) decreased insulin release stimulated by glibenclamide in the presence of 3.3mm-glucose. 5. None of the phenothiazines affected basal insulin release in the presence of 2mm-glucose. 6. Trifluoperazine (20mum) did not inhibit islet glucose utilization nor the incorporation of [(3)H]leucine into (pro)insulin or total islet protein. 7. Islet extracts catalysed the incorporation of (32)P from [gamma-(32)P]ATP into endogenous protein substrates. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis resolved several phosphorylated bands, but incorporation was slight. However, calmodulin in the presence of Ca(2+) greatly enhanced incorporation: the predominant phosphorylated band had an estimated mol.wt. of 55000. This enhanced incorporation was abolished by trifluoperazine, but not by cyclic AMP-dependent protein kinase inhibitor protein. 8. These results suggest that islet phosphodiesterase-stimulating activity is similar to, although not necessarily identical with, calmodulin from skeletal muscle; that islet calmodulin may play an important role in Ca(2+)-dependent stimulus-secretion coupling in the beta-cell; and that calmodulin may exert part at least of its effect on secretion via phosphorylation of endogenous islet proteins.
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PMID:Evidence for the participation of calmodulin in stimulus-secretion coupling in the pancreatic beta-cell. 626 64

The isolated, brush-border membrane of Hymenolepis diminuta contained an enzyme which hydrolyzed phosphodiester bonds. This enzyme appeared to be a Type I phosphodiesterase (E. C. 3.1.4.1) (produces nucleoside 5'-phosphates) and had no activity against synthetic, Type II phosphodiesterase substrates (mononucleotides substituted at the 3' position). The effects of various potential inhibitors of enzymatic activity, and cation requirements of this enzyme, demonstrated a distinct difference between the phosphodiesterase and alkaline phosphatase activities of the isolated, brush-border membrane. SDS-polyacrylamide gel electrophoresis of the isolated membrane preparation, followed by localization of phosphodiesterase activity in the gels, indicated the enzyme had a molecular weight of approximately 87,000. Thus, the phosphodiesterase activity represents a previously undescribed, membrane-bound enzyme of the brush-border of Hymenolepis diminuta.
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PMID:Type I phosphodiesterase in the isolated, brush-border membrane of Hymenolepis diminuta. 627 42

Extruded trichocysts are composed of a family of proteins with molecular weights between 15,000 and 20,000. We have used heat treatment and affinity chromatography on fluphenazine-Sepharose to purify calmodulinlike proteins from whole cells and from extruded trichocysts. The purified protein from trichocysts is indistinguishable from that of whole cells; it is heat-stable, activates brain phosphodiesterase in a Ca++-dependent fashion, changes mobility on SDS polyacrylamide gels in the presence of Ca++, contains 1 mol of trimethyllysine/17 kdaltons, and has the amino acid composition characteristic of calmodulins. Calmodulin is a major component of purified, extruded trichocysts, of which it represents between 1 and 10% by mass. The other proteins of the trichocyst also resemble calmodulin in several properties. Possible roles for calmodulin in the Ca++-activated extrusion of trichocysts is discussed.
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PMID:Calmodulin is a major component of extruded trichocysts from Paramecium tetraurelia. 627 12

A specific protein associated with rod-outer-segment disc membranes binds GTP only in the presence of bleached rhodopsin. Once formed the protein-GTP complex becomes a soluble activator of cGMP phosphodiesterase. It is shown that this activator complex can be completely separated from rhodopsin and retain its ability to activate phosphodiesterase when added to a pool of totally dark (unilluminated) disc membranes. The photoreactive GTP analogue p3-(4-azidoanilido)-5' GTP (AAGTP) is shown to be a more effective substrate than GTP, Gpp(NH)p or 8-azido GTP. [8, 5' 3H] AAGTP was used to specifically covalently label the GTP-binding protein. The protein labeled exhibits a mass of 40,000 daltons when analyzed by SDS-PAGE.
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PMID:A GTP-protein activator of phosphodiesterase which forms in response to bleached rhodopsin. 627 4

Extracellular phosphodiesterase for adenosine 3':5'-monophosphate [EC 3.1.4.17] was purified from the supernatant of aggregation phase culture of Dictyostelium discoideum, and two types (type I and type II) of the enzyme were found. The type I enzyme was not absorbed on DEAE-Sephacel at pH 8.5 and had an apparent molecular weight of about 67,000 daltons. In contrast, the type II enzyme was adsorbed on DEAE-Sephacel and had an apparent molecular weight of about 120,000 daltons. The Km values of the two types were similar (2-4 microM). Upon SDS polyacrylamide gel electrophoresis analyses, however, both types produced the same bands with molecular weights of 55,000 and 57,000, indicating that they are two different forms composed of common constituents. During the growth phase, the two types of the enzyme were present in culture supernatant in roughly equal amounts, but type II accumulated predominantly in the aggregation phase, suggesting that the ratio of activity of the two forms is under developmental control. Rabbit antiserum prepared against purified type II enzyme cross-reacted with type I as well as membrane-bound enzyme, indicating that the three classes of the enzyme possess some common sequence.
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PMID:Purification and characterization of the extracellular cyclic AMP phosphodiesterase of Dictyostelium discoideum. 630 95


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