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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Ectoenzyme release from kidney brush border membranes of Rattus norvegicus and Sus scrofa domesticus by phosphatidylinositol-specific phospholipase C (PIPLC) of Bacillus thuringiensis was studied. 2. The levels of specific activities of ectoenzymes in R. norvegicus kidney brush border membranes were higher than those in S. scrofa domesticus. About 10-fold higher values were found for specific activities of alkaline phosphatase and gamma-glutamyl transpeptidase in R. norvegicus. 3. Alkaline phosphodiesterase I, alkaline phosphatase and 5'-nucleotidase were released from both R. norvegicus and S. scrofa domesticus brush border membranes, while gamma-glutamyl transpeptidase and dipeptidyl peptidase IV were not solubilized. The enzyme release by the action of PIPLC was suppressed when purified anti-PIPLC antibody was added to the reaction mixture. This suggests that enzyme release must be due to the direct action of PIPLC on kidney brush border membranes. 4. The released alkaline phosphodiesterase I from kidney of S. scrofa domesticus had a molecular weight of 240,000 and was activated by Mg2+ and Ca2+, but strongly inhibited by EDTA.
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PMID:Proof of alkaline phosphodiesterase I as a phosphatidylinositol-anchor enzyme. 839 52

Membrane-bound liver alkaline phosphatase (Mem-LiALP, EC 3.1.3.1) is a high-molecular-mass liver alkaline phosphatase (ALP) present in metastatic, infiltrative and cholestatic liver disease. Shedding of hepatocyte plasma membrane fragments (LiPMF) is thought to be responsible for the appearance of Mem-LiALP in the circulation. Several other membrane-bound enzymes, such as gamma-glutamyltransferase (gamma-GT), leucine aminopeptidase (LAP), and 5'-nucleotidase (5'-Nu) are present in the membrane of the shedded LiPMF. By means of immunohistochemical and immunoassay procedures, we presently show that AD-1, a specific monoclonal antibody originally produced against Mem-LiALP, reacts with LAP, a constituent of the human liver plasma membrane. Using AD-1 as an immunosorbant, we isolated circulating LiPMF from cholestatic sera to a high level of purity and separated it from other high-molecular-mass material, such as liver ALP or similar lipoprotein-X complexes. These purified membrane fragments retained their biochemical characteristics. Glycosyl-phosphatidylinositol anchor bearing liver ALP (Anch-LiALP) could be released from the LiPMF by Triton X-100. Whereas ALP was released upon treatment of AD-1 purified LiPMF with phospholipase C, phospholipase D only cleaved the glycosyl-phosphatidylinositol anchor following detergent solubilization of the enzyme. Serum LiPMF from patients with different kinds of cholestatic liver disease were bound onto AD-1 coated nitrocellulose disks and the activity of four membrane-bound enzymes (LAP, ALP, 5'Nu, gamma-GT) was analyzed. A considerable interindividual variation of enzyme activities was observed, suggesting some heterogeneity in the membrane composition of these fragments.
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PMID:Purification of circulating liver plasma membrane fragments using a monoclonal antileucine aminopeptidase antibody. 861 23

A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000-g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low Km for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by alpha,beta-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of Mr = 54,300 +/- 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- Km "soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000-g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.
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PMID:A low-Km 5'-nucleotidase from rat brain cytosolic fraction: purification, kinetic properties, and description of regulation by a novel factor that increases sensitivity to inhibition by ATP and ADP. 876 9

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5'-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca(2+)-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the mental chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.
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PMID:Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes. 892 Sep 95

At the neuromuscular junction and possibly also at the synaptic level in the brain, the main sequence of events (see Fig. 5) that involves purines in modulation of ACh release includes the following observations: (1) storage of ATP and its release either together with, or independently of acetylcholine. ATP is also released from the post-junctional component. Adenosine as such is released either from the motor nerve terminals or from the post-junctional component. (2) There is extracellular hydrolysis of ATP to adenosine, which is the active substance to modulate transmitter release. The key enzyme in the conversion of AMP into adenosine is the ecto 5'-nucleotidase. When ecto-5'-nucleotidase is not available (e.g. in cholinergic nerve terminals of the cerebral cortex) ATP as such exerts the neuromodulatory role normally fulfilled by adenosine. (3) Both the inhibition and the excitation induced by adenosine on ACh release in the rat is inactivated through up-take and deamination. (4) Adenosine-induced inhibition of ACh release is mediated via A1 receptors and the excitation via A2a receptors. The A2a receptors are positively coupled to the adenylate cyclase/cyclic AMP system, whereas the presynaptic A1 receptors (a) may be negatively linked to adenylate cyclase and (b) to phospholipase C, and, upon stimulation, (c) increase potassium conductance and (d) decrease calcium conductance. (5) Activation of A2a receptors is essential for substances that facilitate ACh release (e.g. CGRP, forskolin) to exert their effects, as well as for induction of nicotinic autofacilitatory receptor desensitization. (6) There are interactions between A1 and A2a receptors. Thus, the net adenosine neuromodulatory response is the resultant, at each moment, of the relative degree of activation of each one of these receptors. This relative activation depends upon the intensity (frequency, pulse duration) of stimulation of the motor nerve terminals. (7) Adenosine released as such seems to preferentially activate A1 receptors, whereas the adenosine formed from metabolism of adenine nucleotides prefers to activate the A2a receptors. In conclusion, to find out precisely what occurs with ACh in transmitting its message at the synaptic level, one has to consider the subtle ways used by purines to modulate the ACh response. It therefore appears of interest that pharmacological and therapeutic strategies use this knowledge to approach cholinergic transmission deficiencies based upon reduction of ACh release.
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PMID:Purinergic regulation of acetylcholine release. 900 12

The effects of temperature on the three-dimensional organization and on the secondary structure of GPI-anchored 5'-nucleotidase from bull seminal plasma and of its anchor-less form (solubilized ecto-5'-nucleotidase), obtained after GPI anchor removal by phosphatidylinositol-specific phospholipase C were investigated in parallel by circular dichroism and fluorescence spectroscopy. The structural features of the two enzymes were correlated to their functional properties in the temperature range of 25-90 degrees C. The kinetic data indicated that the enzyme activities were temperature dependent, showing the maximal values at 60 degrees C. The relevant Arrhenius plots were linear in the temperature range of 20-60 degrees C and the activation energies were 44.4 and 51.8 kJ/mol for the solubilized and GPI-anchored 5'-nucleotidase, respectively. The time-course measurements of enzyme activity, in the temperature range of 25-55 degrees C, revealed that the two enzymes were of different thermal stability, the solubilized ectoenzyme showing lower thermal deactivation constants and longer half lives. Fluorescence and near UV circular dichroism spectroscopy showed that temperature increases induced remarkable changes in the protein tertiary structure of the two enzymes, whereas far-UV circular dichroism analysis revealed only a small temperature effect on the protein secondary structure content.
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PMID:Temperature effects on the structural and functional properties of GPI-anchored and anchor-less bull seminal plasma ecto-5'-nucleotidase. 953 2

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5'-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.
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PMID:Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer. 957 57

The chicken T-tubule Mg2+-ATPase is an integral membrane glycoprotein that presents properties different from those of other ATPases located in skeletal muscle cells and exhibits ATP-hydrolysing activity on the extracellular side of the transverse tubule (TT) membranes. In this study we demonstrate that TT vesicles purified from chicken skeletal muscle possess ecto-ADPase and ecto-5'-nucleotidase activities that, along with ecto-ATPase, are able to sequentially degrade extracellular ATP to ADP, AMP and adenosine. Characterization studies of these TT ectonucleotidases revealed remarkable differences between ecto-ATPase and ecto-ADPase activities with respect to thermal stability, temperature dependence of the hydrolytic activity, effect of ionic strength, kinetic behaviour, divalent cation preference and responses to azide, N-ethylmaleimide, NaSCN, Triton X-100 and concanavalin A. Ecto-ATPase, but not ecto-ADPase, was inhibited by a polyclonal antibody against the chicken TT ecto-ATPase. On the basis of these results we propose that ATP and ADP hydrolysis are accomplished by two distinct enzymes and therefore the TT ecto-ATPase is not an apyrase. 5'-Nucleotidase activity was inhibited by adenosine 5'-[alpha,beta-methylene]diphosphate and concanavalin A, followed simple Michaelis-Menten kinetics and was released from the membranes by treatment with phosphatidylinositol-specific phospholipase C, indicating that AMP hydrolysis in T-tubules is catalysed by a typical ecto-5'-nucleotidase. Results obtained from electrophoresis experiments under native conditions suggest that ecto-ATPase, ecto-ADPase and 5'-nucleotidase might be associated, forming functional complexes in the T-tubule membranes. The TT ectonucleotidases constitute an enzymic cascade for the degradation of extracellular ATP that might be involved in the regulation of purinergic signalling in the muscle fibre.
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PMID:T-tubule membranes from chicken skeletal muscle possess an enzymic cascade for degradation of extracellular ATP. 958 72

Pneumocystis carinii pneumonia is a hallmark disease associated with AIDS. An abundant glycoprotein, termed gpA, on the surface of P. carinii is considered an important factor in host-parasite interactions. The primary structure of ferret P. carinii gpA contains a carboxyl-terminal sequence characteristic of a signal for glycosylphosphatidylinositol (GPI) anchors. Here we report the capacity for this gpA carboxyl sequence to direct attachment of a secreted protein, human growth hormone (hGH), to the membranes of COS cells. A control fusion protein (hGHDAF37) was obtained which, under the direction of the GPI signal from decay accelerating factor, directs hGH cell surface expression. A construct (phGH2-1A30) was created similar to hGHDAF37 by fusing hGH to the putative GPI signal sequence encoded in the terminal 30 residues from a ferret P. carinii gpA cDNA clone. By indirect immunofluorescent staining, hGH was detected on the surface of COS cells transfected with phGH2-1A30; this surface location was confirmed by confocal laser cytometry. Metabolic labeling with [3H]ethanolamine and subsequent immunopurification of hGH from cells transfected with phGH2-1A30 confirmed that a lipid moiety characteristic of a conventional GPI anchor was linked covalently to hGH, and cell surface hGH2-1A30 fusion protein was sensitive to enzymatic cleavage by phosphatidylinositol-phospholipase C. Furthermore, hGH2-1A30 recombinant protein cofractionated with 5'-nucleotidase, a classical GPI-anchored membrane marker. Together, these results indicate that the carboxyl-terminal residues of ferret P. carinii gpA constitute a biologically functional GPI consensus domain, thus providing a potential mechanism for antigenic variation of P. carinii gpA during P. carinii pneumonia.
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PMID:The carboxyl terminus of Pneumocystis carinii glycoprotein A encodes a functional glycosylphosphatidylinositol signal sequence. 974 3

Bacterial phospholipases C are known to act on biomembranes, since they can cleave the phosphodiester linkage between the polar head and the hydrophobic moiety of each phospholipid in these membranes. These enzymes have been classified into three groups; phosphatidylcholine (PC)-, sphingomyelin (SM)- and phosphatidylinositol (PI)-degrading phospholipases C. Enzymatic properties and toxicities of these phospholipases C are reviewed, in relation to author's research. Studies on the hemolytic phospholipases of Clostridium sp., Bacillus cereus etc., revealed that hydrolysis of choline-containing phospholipids such as PC and SM was responsible for the hemolysis of mammalian erythrocytes by these enzymes in the presence of Ca2+ and/or Mg2+. Also, the studies on a structure-activity correlation of SM-hydrolyzing phospholipase C from B. cereus disclosed the similarity of active sites between this enzyme and bovine pancreatic DNase I. By action of PI-degrading phospholipases C, several membrane proteins such as alkaline phosphatase, 5'-nucleotidase, VSG (protozoal surface glycoprotein) etc., were shown to be released from the plasma membranes of eucaryotic cells. From structural analysis, these proteins have been revealed to be glycosylphosphatidylinositol (GPI)-anchored proteins bound to the plasma membranes with carboxyl terminal-attached glycolipid. Biochemistry and molecular biology of GPI-anchored proteins, including the structures and biosynthetic routes of GPI glycolipids as well as the process of GPI attachment to proteins, requirements of C-terminal signal peptide for the protein modification by GPI, and distribution of GPI-anchored proteins in living world, are described in relation to our studies.
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PMID:[Development from actions of bacterial phospholipases C on eucaryotic plasma membranes to molecular biology of GPI-anchored proteins]. 1043


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