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)

A shift in the incubation temperature of rabbit alveolar macrophages (0 degree C leads to 37 degrees C leads to 0 degree C) resulted in a 40-60% reduction in the ability of cells to bind alphamacroglobulin. 125I-trypsin complexes (alphaM. 125I-T). The reduction in binding activity did not reflect a disruption of cell integrity since the levels of intracellular components (lactate dehydrogenase, beta-N-acetyl-hexosaminidase) or other plasma membrane components (alkaline phosphodiesterase) were unaltered. Analysis of receptor-ligand interaction indicated that the temperature shift effected a decline in receptor number rather than an alteration in ligand-receptor affinity. Studies indicated that a temperature shift resulted in the loss of unoccupied receptors, and that ligand bound to receptors was not lost. However, after ligand internalization, receptors were removed by the temperature shift. The rate of receptor loss was maximal when cells were incubated at temperatures greater than 24 degrees C. Receptor loss was not prevented by treatment of cells with colchicine, cytochalasin B, or N-ethylamaleimide, but was prevented by treatment with the cross-linking agent paraformaldehyde. Data indicate that the reduction in alphaM. 125I-T binding activity resulted from shedding of receptors into the media since media obtained from temperature-shifted cells contained material that competed with cell-bound receptors for alphaM. 125I-T. Additionally, binding of alphaM. 125I-T was diminished on membrane fragments obtained from temperature-shifted cells. Incubation with Triton X-100, of cells whose receptors were occupied with alphaM. 125I-T, led to the extraction of 40% of cell-bound activity. However, no radioactivity was extracted from cells labeled with alphaM. 125I-T after a temperature shift. Measurement of ligand accumulation by control and temperature-shifted cells incubated at 20 degrees C indicated that control cells exhibited a subpopulation of receptors capable of binding ligand but only slowly internalizing it. This subpopulation was not present on temperature-shifted cells. These results indicate that surface receptors for alphamacroglobulin . protease complexes are heterogeneous and that the temperature shift resulted in the selective loss of membrane components.
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PMID:Temperature shifts induce the selective loss of alveolar-macrophage plasma membrane components. 618 Oct 76

As a basis for attempts to define the structures of the proteins within myelin, methods have been developed for their extraction and isolation in solutions of non-denaturing detergents. With use of solutions of deoxycholate or Triton X-100, up to 90% of the protein has been extracted from bovine CNS myelin, along with most of the phospholipid. The proteolipid protein has been purified in deoxycholate solutions by chromatography on a blue dye-ligand column, which retained all of the basic protein and 2',3'-cyclic nucleotide-3'-phosphodiesterase, and then on Sephacryl S300, which separated proteolipid protein from phospholipid and high-molecular-weight proteins. The proteolipid protein was isolated from Triton X-100 extracts of myelin by adsorption onto phosphocellulose resin, with subsequent elution by 0.5 M sodium chloride. Gel permeation chromatography was used as the final purification step. Sedimentation equilibrium experiments gave a monomer molecular weight of 134,000 +/- 8000 in deoxycholate and 145,000 +/- 17,000 in Triton X-100 solutions. On the basis of an apparent subunit molecular weight of 23,500 it was deduced that the native protein is probably hexameric. Above 0.2 gL-1 in Triton X-100 solutions and 0.5 gL-1 in deoxycholate solutions the protein aggregated. In deoxycholate solutions the protein adopts the highly helical conformation expected for an intrinsic membrane protein.
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PMID:Structure of the proteolipid protein extracted from bovine central nervous system myelin with nondenaturing detergents. 619 60

Bloodstream forms of Trypanosoma brucei have been screened for the presence of enzymes that could serve as markers for the plasma membrane, flagellar pocket, lysosomes, endoplasmic reticulum and Golgi apparatus in order to study the subcellular organization of the digestive system of the parasite. Acetylesterase, acid DNase, acid phosphatase, acid phosphodiesterase, acid proteinase, acid RNase, alanine aminotransferase, galactosyl transferase, alpha-glucosidase, inosine diphosphatase and alpha-mannosidase were partially characterized and their assays optimized for pH-dependent activity, linearity of reaction with respect to incubation time and enzyme concentration, and the effect of inhibitors and activators. The association of these enzymes with particulate material and the presence of structural latency were investigated. Acid proteinase and alpha-mannosidase are particle-bound and latent in cytoplasmic extracts; they can be activated and solubilized in part by Triton X-100. Similar results were obtained for acid phosphatase, acid phosphodiesterase and inosine diphosphatase. Neutral alpha-glucosidase, though partly sedimentable, does not show latency and is readily solubilized by the detergent. Galactosyl transferase is firmly membrane-bound even in the presence of 0.1% Triton X-100. Cell fractionation by differential centrifugation and density equilibration on sucrose gradients revealed that both alpha-mannosidase and acid proteinase are associated with organelles that band at a density of about 1.20 g/cm3. Inosine diphosphatase, galactosyl transferase, acid phosphatase and acid phosphodiesterase sediment predominantly as microsomal constituents equilibrating at densities between 1.13 and 1.15 g/cm3. In addition, inosine diphosphatase and galactosyl transferase exhibit considerable activity at higher densities (1.18-1.25 g/cm3). Neutral alpha-glucosidase is mainly recovered in the nuclear and microsomal fraction; its particulate part equilibrates as a single band at rho = 1.22 g/cm3. Acetylesterase and acid DNase are largely soluble, whereas acid RNase does not produce distinct sedimentation and banding profiles. In intact cells, neutral alpha-glucosidase and acid phosphatase appear to be highly accessible to their substrates. It is tentatively concluded that (a) acid proteinase and alpha-mannosidase are lysosomal enzymes, (b) acid phosphatase and acid phosphodiesterase are associated with the flagellar pocket and part of the former enzyme probably with the endoplasmic reticulum, (c) galactosyl transferase is a constituent of the Golgi apparatus, and (d) alpha-glucosidase may serve as a marker for the plasma membrane. Inosine diphosphatase may also be derived from the latter structure.
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PMID:Subcellular fractionation of Trypanosoma brucei bloodstream forms with special reference to hydrolases. 624 76

A phosphodiesterase activity is shown to copurify with the plasma membrane fraction prepared by the two-phase partition method. The enrichment in phosphodiesterase parallels that of alkaline phosphatase, which is thought to be a typical membranous enzyme. Up to 66% of the phosphodiesterase activity can be solubilized by a treatment with 0.2% Triton X-100. Higher doses were ineffective in solubilizing more activity. Analysis by native gel electrophoresis showed that an activity extracted by 2 M NaCl migrated at the same position as 'soluble' phosphodiesterase of cytosolic or extracellular origin. In contrast, the Triton-solubilized enzyme had an apparently higher molecular weight. When subjected to charge shift electrophoresis on agarose gels in the presence of an ionic detergent, the Triton-solubilized phosphodiesterase displayed a hydrophobic character. This behaviour contrasts with that of 'soluble' phosphodiesterases, the electrophoretic mobility of which is unaffected by the presence of an anionic detergent. The hydrophobic character of the membranous enzyme was lost after gentle hydrolysis by papain.
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PMID:The hydrophobic character of the membrane-bound phosphodiesterase from Dictyostelium discoideum. 626 Feb 58

Placental sphingomyelinase has been purified to apparent homogeneity by a procedure that makes extensive use of hydrophobic interaction chromatography on sphingosylphosphocholine-CH-, octyl-, hexyl- and Blue-Sepharoses. Enzyme purification is about 10000- 14000-fold over starting extract with excellent yield (usually greater than 28%). Purification of bis-4-methylumbelliferyl phosphate phosphodiesterase activity generally paralleled that of sphingomyelinase during the final stages of the procedure. The enzyme also hydrolysed bis-p-nitrophenyl phosphate, but at a lower rate compared with bis-4-methylumbelliferyl phosphate. A single major protein was observed under non-denaturing conditions. Sphingomyelinase, denatured by reduction and alkylation, is composed of a major polypeptide chain with an apparent molecular weight of 89 100 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two minor lower-molecular-weight components were consistently obtained at 47 500 and 30 700. These results were also obtained after maleoylation of the reduced and alkylated sample. The enzyme contains a blocked-N-terminal amino acid. An extensive search for contaminating enzymes revealed the presence of minor amounts of acid phosphatase, which were removed from the final enzyme sample. The highly purified enzyme is stable for several weeks when stored with Triton X-100 at 4 degrees C. The pure enzyme aggregates under denaturing and electrophoretic conditions and special care must be taken to ensure that hydrophobic bonding of the protein is decreased as much as possible. The reproducibility and large scale of this procedure should facilitate further study on the structure and kinetic properties of the enzyme.
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PMID:Purification of sphingomyelinase to apparent homogeneity by using hydrophobic chromatography. 627 5

The location of 2',3'-cyclic nucleotide 2',3'-phosphodiesterase in human erythrocyte membranes was determined. This was accomplished by comparing the enzyme's accessibility with that of glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic surface marker) and acetylcholinesterase (external marker) in sealed and unsealed ghosts and normal and inverted membrane vesicles. The results showed that 2',3'-cyclic nucleotide 3'-phosphodiesterase, like glyceraldehyde-3-phosphate dehydrogenase, meets several criteria for an inner (cytoplasmic) membrane location: (1) the enzyme was accessible to substrate in unsealed ghosts and inside-out vesicles but not in sealed or right-side-out vesicles, (2) latent activity in sealed ghosts could be exposed with detergent (Triton X-100), (3) activity in unsealed ghosts was gradually sequestered during resealing and could be re-exposed with detergent, and (4) the enzyme was susceptible to trypsin proteolysis only in unsealed ghosts. These results demonstrate that the active site of 2',3'-cyclic nucleotide 3'-phosphodiesterase faces the cytoplasm of erythrocytes and that the enzyme may not span the lipid bilayer of the membrane. The localization of the phosphodiesterase on the inner membrane surface of erythrocytes suggests that the similar enzyme of myelin may be embedded within the major dense line of the compact lamellae.
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PMID:Localization of 2',3'-cyclic nucleotide 3'-phosphodiesterase in human erythrocyte membranes. 627 4

1. Incubation of Schistosoma mansoni for 5 min in a phosphate-buffered medium, pH 7.4, released tegumental material containing the following phosphohydrolase activities: alkaline phosphatase, 5'-nucleotidase, glycerol-2-phosphatase, glucose 6-phosphatase, phosphodiesterase and ATPase. 2. Maximum activity of these enzymes was measured at pH 9.5; however, the phosphodiesterase and ATPase activities were also appreciable at pH 7.0. 3. Solubilization of the released tegumental material in 1% Triton X-100 followed by gel filtration distinguished three peaks of enzyme activity: an ATPase (mol.wt. greater than 1000 000), a phosphodiesterase (mol.wt. 1 000 000) and an alkaline phosphomonoesterase with broad specificity (mol.wt. 232 000). 4. The ATPase activity was highly activated by 10 mM-Mg2+ or 1 mM-Ca2+ and was inhibited by chelating agents. Ouabain, Na+ and K+ had little effect on enzyme activity, whereas activity was increased by 50% in the presence of calmodulin. The phosphodiesterase activity was highest in the presence of 100 mM-Na+ or -K+, and 10 mM-Mg2+ or -Ca2+. Alkaline phosphatase activity was also stimulated by 100 mM-Na+ or -K+, and 10 mM-Mg2+; however Ca2+ inhibited at greater than 1 mM. 5. Surface iodination of parasites followed by detergent solubilization and gel filtration of the released tegumental membranes indicated that these enzymes were not accessible. A major surface component, apparent mol.wt. 80 000, was iodinated. 6. Rabbit anti-(mouse liver 5'-nucleotidase) antibodies did not inhibit the phosphohydrolase activities. However, an immunoglobulin G fraction from sera of mice chronically infected with S. mansoni partially inhibited alkaline phosphatase activity, but was without effect on the phosphodiesterase and ATPase activities. 7. The location of the enzymes in the double membrane of the tegument and their significance in host-parasite interactions is discussed.
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PMID:Properties of a series of tegumental membrane-bound phosphohydrolase activities of Schistosoma mansoni. 627 49

The ability of nonionic detergents to solubilize the membrane-bound enzymes of the brush-border plasma membrane of Hymenolepis diminuta was investigated. Of the detergents tested (Triton X-100, Tween 80, Brij 35, Lubrol PX and WX, W-1, and beta-octyl-D-glucoside), only Triton was an effective solubilizing agent. Optimal solubilization was achieved by incubating an isolated fraction of the brush-border membrane in the presence of 1% Triton X-100 for 60 min at 37 C, followed by centrifugation at 100,000 g for 60 min at 25 C. This treatment resulted in solubilization of 94% of the alkaline phosphohydrolase, 91% of the phosphodiesterase and ribonuclease, and 88% of the 5'-nucleotidase activities. The pH optima for enzymes solubilized in nonionic and ionic detergents (Triton and sodium dodecyl sulfate, respectively) did not differ. Isoelectric focusing of the Triton-solubilized material demonstrated the presence of at least 14 polypeptides, a majority of which had isoelectric points below pH 7.
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PMID:Solubilization of the membrane-bound enzymes of the brush-border plasma membrane of Hymenolepis diminuta (Cestoda) using nonionic detergents. 628 6

Phosphomonoesterase and diesterase that cleave phosphatidylinositol-4-phosphate (diphosphoinositide, DPI) and phosphatidylinositol-4,5-bisphosphate (triphosphoinositide, TPI) were detected in three subfractions of purified rat brain myelin, and some properties of the enzymes were studied. Monoesterase activity was stimulated by KCl, maximally at a concentration of 25 mM, and inhibited at KCl concentrations above 50 mM. Addition of boiled pH 5 supernatant of rat brain homogenate doubled the enzymic activity; EDTA was inhibitory. The specific activities were nearly equal in the "low density", "medium density", and "heavy density" myelin fractions but about 30% lower than in whole brain homogenate. The monophosphatase could be solubilized by extraction with 0.2% Triton X-100. The phosphodiesterase activity was inhibited by EDTA and EGTA and not stimulated by KCl or pH 5 supernatant. Specific activities were nearly equal in whole brain and myelin but were by about 60 percent elevated in the "heavy density" over the "low density" myelin fractions. These results show that hydrolases operative in the fast turnover of the inositide phosphate groups are distributed over the entire myelin structure.
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PMID:Polyphosphoinositide mono- and diphosphoesterases of three subfractions of rat brain myelin. 628 49

Isolated rat kidney proximal tubule brush border membrane vesicles exhibit an increase in diacylglycerol levels (20- to 30-fold) and a concomitant decrease in phosphatidylinositol when incubated with [3H]arachidonate-labeled lipids, Ca2+, and deoxycholate. Levels of free arachidonate, triglyceride, and noninositol phospholipids are not altered. These results suggest phosphatidylinositol phosphodiesterase activity is associated with rat proximal tubule brush border membrane. Presence of both deoxycholate and certain divalent cations was necessary to demonstrate enzyme activity. Optimum pH ranged from 7.0 to 8.5. Ca2+, Mg2+, and Mn2+ stimulated diglyceride production while Ba2+, Zn2+, Hg2+, and K+ were ineffective. HgCl2 inhibited Ca2+-stimulated phosphatidylinositol phosphodiesterase. Mg2+ and deoxycholate-dependent enzyme activity was shown to be phosphatidylinositol specific. Sodium lauryl sulfate, tetradecyltrimethylammonium bromide, and Triton X-100 did not activate phosphatidylinositol phosphodiesterase in the presence of Ca2+. In combination with deoxycholate, diglyceride formation was not affected by sodium lauryl sulfate, partially inhibited by Triton X-100, and completely abolished by tetradecyltrimethylammonium bromide. Diglyceride kinase activity was not found associated with brush border membrane phosphatidylinositol phosphodiesterase. ATP (1-5 mM) inhibited Ca2+- or Mg2+-stimulated, deoxycholate-dependent phosphatidylinositol hydrolysis by chelating the required divalent cation.
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PMID:Characterization of rat kidney proximal tubule brush border membrane-associated phosphatidylinositol phosphodiesterase. 630 56


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