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)

Glycosyl-inositolphospholipid (glycosyl-PtdIns) anchors of proteins in mammalian cells which have been analyzed so far are exclusively of the alkylacyl type. However, little is known about the putative precursor of glycosyl-PtdIns, the alkylacyl derivative of glycerophosphoinositol (GroPIns), in these cells since it is generally believed that cellular GroPIns consists of diacyl-type molecular species only. In this report, we describe the isolation and identification of alkylacyl GroPIns molecular species in both human and bovine erythrocytes, and compare it with the molecular species compositions of the glycosyl-PtdIns anchors of human and bovine erythrocyte acetylcholinesterase. Diradyl GroPIns was isolated from lipid extracts of ghost membranes and treated with phospholipase C. Diradylglycerols of the glycosyl-PtdIns anchors of affinity-purified human and bovine erythrocyte acetylcholinesterase were generated by sequential treatment with glycoprotein phospholipase D and acidic phosphatase and by PtdIns-specific phospholipase C, respectively. Diradylglycerols were subsequently converted into benzoate derivatives and separated into diacyl, alkylacyl, and alkenylacylglycerol subclasses. The molecular species compositions were quantitated and determined by combined HPLC/mass spectrometry. We found that human and bovine erythrocyte membrane diradyl GroPIns consist of 1.5-4.8% alkylacyl GroPIns. Molecular species analysis showed a heterogeneous species composition for both human and bovine erythrocyte alkylacyl GroPIns. Their compositions are distinctly different from those of human and bovine erythrocyte acetylcholinesterase glycosyl-PtdIns anchors. The number of alkylacyl GroPIns molecules/cell is roughly equal with the number of glycosyl-PtdIns-anchored proteins in human erythrocytes.
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PMID:Alkylacyl glycerophosphoinositol in human and bovine erythrocytes. Molecular species composition and comparison with glycosyl-inositolphospholipid anchors of erythrocyte acetylcholinesterases. 139 75

Antineoplastic ether lipids have entered phase I clinical trial and, although their mechanism of action remains unclear, it is widely believed that the plasma membrane is the primary cellular drug target. In the present study the hypothesis was tested that metabolism of ether lipids acts as a detoxification process. [31P]-nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of the ether lipid SRI 62-834 (SRI) and the phosphate ester hexadecylphosphocholine (HPC) in the presence of both isolated phospholipases C and D and post-mitochondrial rat liver homogenate. Both SRI and HPC were slowly metabolised by phospholipase D to their alkyl phosphates and choline, and the alkyl phosphates were subsequently metabolised by phosphatase to yield the alcohols and inorganic phosphate. These studies failed to detect any metabolism of either SRI or HPC by phospholipase C, and the metabolism of platelet-activating factor (PAF) by this enzyme was not inhibited by the addition of either compound. The cytotoxicity of SRI, the related compound HPC and their metabolites was determined in vitro using three cell lines. Cytotoxicity was measured by analysis of cell growth kinetics, MTT assay and lactate dehydrogenase release. Closely similar results were obtained in the JB1 rat hepatoma cell line, in the non-transformed BL8 rat hepatocyte cell line, and in A549 human lung adenocarcinoma cells. SRI was the most toxic of the compounds analysed, the concentration required to produce 50% toxicity or growth inhibition (IC50) being 6-9 microM. The putative metabolite of SRI, 2,2'-bis(hydroxymethyl)tetrahydrofuran, and the known metabolites [2'-(octadecyloxymethyl)tetrahydrofuran-2'-yl]methyl phosphate and 2-hydroxymethyl-2-octadecyloxymethyltetrahydrofuran exhibited IC50 values of > 200, > 100 and 40-70 microM, respectively, consistent with metabolic detoxification. HPC was more cytotoxic (IC50, 37 microM) than its phosphate metabolite (IC50, 140 microM), but its toxicity was similar to that of its metabolite hexadecanol (IC50, 28 microM), suggesting that only the former metabolic route leads to detoxification.
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PMID:Is metabolism an important arbiter of anticancer activity of ether lipids? Metabolism of SRI 62-834 and hexadecylphosphocholine by [31P]-NMR spectroscopy and comparison of their cytotoxicities with those of their metabolites. 145 Dec 37

Norepinephrine (NE) plus guanosine triphosphate (GTP) increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized smooth muscle. We used alpha-toxin-permeabilized rabbit mesenteric arteries to determine the temporal relationships among force, myosin light chain (MLC) phosphorylation, stiffness, and shortening velocity during contractions in response to Ca2+ alone and to the same [Ca2+] in the presence of NE plus GTP. The addition of NE plus GTP caused a marked increase in the tonic contraction but only transiently elevated the level of MLC phosphorylation over that observed in the presence of Ca2+ alone. NE plus GTP induced similar increases in force and stiffness, but shortening velocity depended solely on the [Ca2+]. A regulated MLC phosphatase could explain the initial increase in force and MLC phosphorylation, but not the maintenance of enhanced force while MLC phosphorylation levels fell to values similar to those in response to Ca2+ alone. Therefore, additional elements must be involved in the maintenance of the receptor and G protein-dependent increase in myofilament Ca2+ sensitivity.
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PMID:Transient myosin phosphorylation at constant Ca2+ during agonist activation of permeabilized arteries. 151 96

In Pseudomonas aeruginosa, the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipase C. Although the natural substrate of this enzyme is phosphorylcholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p-nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorylcholine phosphatase) mediated by divalent cations Mg2+, Zn2+ and Cu2+ revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Km values obtained for p-NPP in the presence of Mg2+, Zn2+ and Cu2+ were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The KA values for the same ions were 1.25 mM, 0.05 mM and 0.03 mM, respectively. The Vmax obtained in the presence of Cu2+ was about twofold higher than that obtained in the presence of Mg2+ or Zn2+. The inhibition observed with Al3+ seems to be a multi-site inhibition. The K'app and n values, from the Hill plot, were about 0.25 mM and 4.0 mM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may exert its action under physiological conditions, depending on the availability of either one of these metal ions.
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PMID:Pseudomonas aeruginosa acid phosphatase. Activation by divalent cations and inhibition by aluminium ion. 154 81

Whereas bacteria in the genus Legionella have emerged as relatively frequent causes of pneumonia, the mechanisms underlying their pathogenicity are obscure. The legionellae are facultative intracellular pathogens which multiply within the phagosome of mononuclear phagocytes and are not killed efficiently by polymorphonuclear leukocytes. The functional defects that might permit the intracellular survival of the legionellae have remained an enigma until recently. Phagosome-lysosome fusion is inhibited by a single strain (Philadelphia 1) of Legionella pneumophila serogroup 1, but not by other strains of L. pneumophila or other species. It has been found that following the ingestion of Legionella organisms, the subsequent activation of neutrophils and monocytes in response to both soluble and particulate stimuli is profoundly impaired and the bactericidal activity of these cells is attenuated, suggesting that Legionella bacterial cell-associated factors have an inhibitory effect on phagocyte activation. Two factors elaborated by the legionellae which inhibit phagocyte activation have been described. First, the Legionella (cyto)toxin blocks neutrophil oxidative metabolism in response to various agonists by an unknown mechanism. Second, L. micdadei bacterial cells contain a phosphatase which blocks superoxide anion production by stimulated neutrophils. The Legionella phosphatase disrupts the formation of critical intracellular second messengers in neutrophils. In addition to the toxin and phosphatase, several other moieties that may serve as virulence factors by promoting cell invasion or intracellular survival and multiplication are elaborated by the legionellae. Molecular biological studies show that a cell surface protein named Mip is necessary for the efficient invasion of monocytes. A possible role for a Legionella phospholipase C as a virulence factor is still largely theoretical. L. micdadei contains an unusual protein kinase which catalyzes the phosphorylation of eukaryotic substrates, including phosphatidylinositol and tubulin. Since the phosphorylation of either phosphatidylinositol or tubulin might compromise phagocyte activation and bactericidal functions, this enzyme may well be a virulence factor. Administration of the L. pneumophila exoprotease induces lesions resembling those of Legionella pneumonia and kills guinea pigs, suggesting that this protein plays a role in the pathogenesis of legionellosis. However, recent work with a genetically engineered strain has convincingly shown that the protease is not necessary for intracellular survival or virulence. As might be expected with a complex process like intracellular parasitism, it appears that the capability of Legionella strains to invade and multiply in host phagocytes is multifactorial and that no single moiety which is responsible for the virulence phenotype will be found.
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PMID:Virulence factors of the family Legionellaceae. 157 12

The content and molecular species composition of 1,2-diacylglycerol (DAG) in rat sciatic nerve was determined and compared with the molecular species profiles for glycerophospholipid classes in order to gain information concerning the metabolic pathways of DAG formation. The level of DAG in freshly dissected epineurium-free nerve (44 +/- 2 pmol/mg wet weight) was 10-40% of that in other tissues and cultured cells. The predominant DAG molecular species were 18:0/20:4 (30%) and 16:0/18:1 (17%). In comparison with phospholipid molecular species patterns, DAG was characterized by a substantial but lower proportion of the 18:0/20:4 species than was found in phosphoinositides, and a significant fraction of saturated species such as those found in phosphatidylcholine. In nerve from diabetic rats, both the content and arachidonoyl-containing molecular species of DAG were reduced. These species were also decreased in individual glycerophospholipids, except for phosphatidylinositol. The distribution of molecular species in phosphatidic acid (PA) did not resemble that of any other phospholipid. A large rise in DAG content occurred when nerve was incubated in vitro. Molecular species analysis indicated that phosphoinositides were the main source, especially during the initial period. This process was virtually abolished in a Ca(2+)-free medium and probably reflects a response to tissue injury. Evidence was obtained for the isomerization of DAG to 1,3-diacylglycerol during incubation. PA content and molecular species composition of incubated nerve did not change. However, inclusion of propranolol, a PA phosphatase inhibitor, caused a 40% accumulation of PA within 10 min, suggesting that formation of this phospholipid is continuous. These findings support the conclusion that DAG is principally derived from phosphoinositides by phospholipase C hydrolysis, but a minor fraction could be derived from phosphatidylcholine either by the action of phospholipase C or via phospholipase D and PA phosphatase. The metabolic origins of PA appear to be diverse.
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PMID:Diacylglycerol composition and metabolism in peripheral nerve. 163 7

A phospholipase C was solubilized and purified from membranes of porcine brain cortex. Simultaneously, a phospholipase C was purified from a cytosolic fraction of porcine brain cortex. The enrichment of phospholipase C from either fraction was about 1000-fold as determined by hydrolysis of phosphatidylinositol 4,5-bisphosphate. Phospholipases C purified from membranes or from cytosol were indistinguishable with regard to the following properties: The enzyme activities copurified with a protein of 145 kDa. The standard sedimentation coefficients (s20,w values) of the purified enzymes were 6.2 S in the absence or presence of 0.3% (w/v) sodium cholate; Stokes' radii, estimated by gel filtration on a Superose 6 HR 10/30 column in the presence of 0.3% sodium cholate, were 4.5 nm; calculated molecular masses were about 120 kDa; no significant hydrolysis of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine by preparations of purified phospholipase C was observed; adenine and guanine nucleotides affected the activity of purified enzymes in a complex manner. Thus, the enzymes purified from membraneous and from cytosolic fractions exhibited properties of the phospholipase C-beta form. The enzymes purified from either fraction required Ca2+ at a low concentration (100 nM to 10 microM) for maximal activity. The advantage of the present purification procedure is that the purified enzymes were free of phosphatidylinositol 4,5-bisphosphate 5-phosphatase, inositol 1,4,5-trisphosphate 5-phosphatase and guanine nucleotide-binding proteins after three chromatographic steps. The purified enzymes may, therefore, prove useful for studying the hormonal regulation of phospholipase C in reconstituted systems and for the preparation of [5-32P]inositol 1,4,5-trisphosphate from [5-32P]phosphatidylinositol 4,5-bisphosphate.
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PMID:Improved purification and characterization of membraneous and cytosolic inositol phospholipid-specific phospholipases C from porcine brain cortex. 165 Jan 89

The mechanism of G protein-mediated sensitization of the contractile apparatus of smooth muscle to Ca2+ was studied in receptor-coupled alpha-toxin-permeabilized rabbit portal vein smooth muscle. To test the hypothesis that Ca2+ sensitization is due to inhibition of myosin light-chain (MLC) phosphatase activity, we measured the effect of guanosine 5'-[gamma-thio]triphosphate and phenylephrine on the rate of MLC dephosphorylation in muscles preactivated with Ca2+ and incubated in Ca(2+)- and ATP-free solution containing 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) to block MLC kinase activity. Guanosine 5'-[gamma-thio]triphosphate alone (300 microM) or in combination (3 microM) with phenylephrine decreased the rates of relaxation and dephosphorylation of MLC to about half of control values; this inhibition is sufficient to account for maximal G protein-mediated Ca2+ sensitization of MLC phosphorylation. The rate of thiophosphorylation of MLC with adenosine 5'-[gamma-thio]-triphosphate was not affected by guanosine 5'-[gamma-thio]triphosphate. We suggest that inhibition of protein phosphatase(s) by G protein(s) may have important regulatory functions.
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PMID:G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. 165 67

Choline, acetylcholine and betaine used as the sole carbon, nitrogen or carbon and nitrogen source increase cholinesterase activity in addition to phosphorylcholine phosphatase and phospholipase C activities in Pseudomonas aeruginosa. The cholinesterase activity catalyses the hydrolysis of acetylthiocholine (Km approx. 0.13 mM) and propionylthiocholine (Km approx. 0.26 mM), but not butyrylthiocholine, which is a pure competitive inhibitor (Ki 0.05 mM). Increasing choline concentrations in the assay mixture decreased the affinity of cholinesterase for acetylthiocholine, but in all cases prevented inhibition raised by high substrate concentrations. Considering the properties of these enzymes, and the fact that in the corneal epithelium there exists a high acetylcholine concentration and that Pseudomonas aeruginosa produces corneal infection, it is proposed that these enzymes acting coordinately might contribute to the breakdown of the corneal epithelial membrane.
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PMID:Pseudomonas aeruginosa cholinesterase and phosphorylcholine phosphatase: two enzymes contributing to corneal infection. 165 99

Receptor tyrosine kinases couple to multiple intracellular effector molecules that are crucial for normal cell growth and transformation. Stimulation of membrane phospholipid hydrolysis by receptor tyrosine kinases is one such pathway for generating intracellular second messengers that may be important for mitogenesis. Certain receptor tyrosine kinases tyrosine phosphorylate a phosphoinositide-specific phospholipase C that hydrolyses the membrane phospholipid phosphatidylinositol 4,5-bisphosphate. In contrast, the glycoprotein receptor for colony stimulating factor 1, a transmembrane tyrosine kinase, does not utilize this pathway, but rather stimulates the hydrolysis of phosphatidylcholine. Here we show that eluates of antiphosphotyrosine affinity purified lysates of colony-stimulating factor 1-stimulated cells contain elevated levels of phosphatidylcholine-specific phospholipase C activity. The affinity-purified activity is sensitive to tyrosine-specific T-cell phosphatase, and is detected in the membrane fraction of stimulated cells. Recovery of phospholipase C activity in the antiphosphotyrosine protein fraction is reduced by pertussis toxin pretreatment of cells. The phosphatidylcholine phospholipase C activity in isolated membranes of colony-stimulating factor 1-treated cells was also reduced by pertussis toxin treatment and stimulated by guanosine 5'-3-O-(thio)triphosphate. These results indicate that colony stimulating factor 1 receptor-mediated stimulation of phosphatidylcholine-specific phospholipase C requires tyrosine phosphorylation, and might be affected by a G-protein coupled pathway.
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PMID:Activation of a phosphatidylcholine-specific phospholipase C by colony stimulating factor 1 receptor requires tyrosine phosphorylation and a guanine nucleotide-binding protein. 147 33


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