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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

There is much evidence that G-proteins transduce the signal from receptors for Ca(2+)-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein alpha-subunits. It is proposed that this protein is the alpha-subunit of the G-protein that regulates the phospholipase in this tissue. Ca(2+)-mobilizing agonists and certain growth factors also promote the hydrolysis of phosphatidylcholine through the activation of phospholipases C and D in many cell types. This yields a larger amount of diacylglycerol for a longer time than does the hydrolysis of inositol phospholipids. Consequently phosphatidylcholine breakdown is probably a major factor in long-term regulation of protein kinase C. The functions of phosphatidic acid produced by phospholipase D are speculative, but there is evidence that it is a major source of diacylglycerol in many cell types. The regulation of phosphatidylcholine phospholipases is multiple and involves direct activation by G-proteins, and regulation by Ca2+, protein kinase C and perhaps growth factor receptor tyrosine kinases.
Mol Cell Biochem
PMID:Cell signalling through phospholipid breakdown. 165 98

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.
Mol Cell Biol 1991 Oct
PMID:v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine. 165 17

The role of membrane phospholipids in porcine testicular androgen and 16-androstene biosynthesis was examined by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Untreated (control) microsomes from immature pig testes converted pregnenolone to 17-hydroxypregnenolone and DHA to 5,16-androstadien-3 beta-ol (andien-beta) and 4,16-androstadien-3-one (dienone) in the 16-androstene pathway, these metabolites accounting for most (65%) of the pregnenolone converted. The 4-ene steroids in the androgen pathway (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) totalled less than 10% of the pregnenolone metabolites. No estrogens or 5 alpha-reduced metabolites were detected. Treatment with phospholipase A2 or C, decreased the conversion of pregnenolone to 4-ene-3-oxo steroids but did not decrease the quantities of 5-ene-3 beta-hydroxysteroids. Confirmation of these findings was obtained by measuring the individual enzymatic steps. Phospholipases A2 and C significantly reduced the conversion of DHA to androstenedione and andien-beta to dienone but did not affect 17-hydroxylase or 'andien-beta-synthetase'. However, when the C-17, 20 lyase step was measured alone, phospholipase C decreased the quantity of androstenedione produced indicating that the side-chain cleavage reaction may involve a lipid component. The different effects of phospholipases on these enzymes suggests that pregnenolone metabolism may be regulated by alterations in the membrane microenvironment.
J Steroid Biochem Mol Biol 1992 Jan
PMID:Phospholipases modulate immature pig testicular androgen and 16-androstene biosynthetic pathways in vitro. 173 40

We have investigated the mechanisms by which the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates prostaglandin E2 (PGE2) formation in the rat tracheal epithelial cell line EGV-6aigT, which can be grown in serum-free medium. The addition of TPA to cells that were prelabeled with [3H]arachidonic acid did not enhance the release of [3H]arachidonic acid and/or [3H]PGE2, indicating that TPA does not stimulate phospholipase activity. The addition of exogenous arachidonic acid to cells pretreated with TPA resulted in increased PGE2 formation, compared with basal levels, indicating an elevation in prostaglandin H synthase (PHS) activity. PHS activity was maximal at 4 hr and was dependent upon the concentration of TPA. Actinomycin D and cycloheximide blocked the TPA response. The recovery of PHS activity of cells in which the existing PHS was inhibited by aspirin was enhanced by TPA treatment. TPA treatment enhanced the expression of PHS mRNA, as measured by Northern analysis. The addition of actinomycin D and cycloheximide reduced the TPA enhancement of PHS mRNA, indicating that the increase in PHS activity required de novo RNA and protein synthesis. Furthermore, pretreatment of the cells with protein kinase C inhibitors reduced the TPA-dependent stimulation of PHS activity and the expression of PHS mRNA. The data suggest that TPA-stimulated de novo synthesis of PHS is mediated by protein kinase C.
Mol Pharmacol 1991 Feb
PMID:Stimulation of prostaglandin H synthase mRNA levels and prostaglandin biosynthesis by phorbol ester: mediation by protein kinase C. 189 4

Triethyl lead chloride (Et3PbCl) was found to induce a shift of fatty acids from membrane phospholipids to triacylglycerols in the human promyelocytic leukemia cell line HL-60. High concentrations of Et3PbCl (greater than 10 microM) caused a substantial liberation of [14C]arachidonic acid within 10 to 20 min in dimethyl sulfoxide-differentiated cells, comparable to the effect of the calcium ionophore A23187 (10 microM). Following liberation of arachidonic acid, its metabolites could be detected. Prolongation of the incubation time and reduction of Et3PbCl concentration resulted in a shift of fatty acids from phospholipids to triacylglycerols. Deacylation of phospholipids and reacylation into phospholipids and triacylglycerols were in equilibrium when the cells were treated with Et3PbCl at concentrations of less than or equal to 10 microM for 5 hr or less than or equal to 1 microM for 24 hr; no increase of free fatty acids could be observed, and the loss of fatty acids within the phospholipids was equivalent to the increase of fatty acid content within the triacylglycerols. Moreover, under these conditions, no loss of viability was seen after 24 hr, as compared with untreated differentiated cells. This concentration- and time-dependent effect of Et3PbCl might be due to a stimulated liberation of fatty acids via phospholipase A2, because this stimulation could be totally prevented by the phospholipase inhibitors quinacrine and p-bromophenacylbromide. Additionally, pretreatment of differentiated HL-60 cells with pertussis toxin resulted in a drastic reduction of [14C]arachidonic acid liberation when cells were stimulated with Et3PbCl. These results suggest the involvement of a pertussis toxin-sensitive GTP-binding protein and of a signal transduction mechanism during stimulated fatty acid release; release does not seem to be via a direct stimulation of phospholipase activity by the lead compound.
Mol Pharmacol 1991 Apr
PMID:Directed shift of fatty acids from phospholipids to triacylglycerols in HL-60 cells induced by nanomolar concentrations of triethyl lead chloride: involvement of a pertussis toxin-sensitive pathway. 190 39

Exposure to oxidants permeabilizes cell membranes and liberates unesterified fatty acids (UFA) in a variety of cell types, including endothelial cells. Products of phospholipase activity, particularly UFA and lysophosphatides, possess potent detergent-like properties, and we postulated that oxidant injury might be mediated by the accumulation of these toxic phospholipase products. Several radiolabels were incorporated into defined positions in the phospholipids of cultured, confluent bovine pulmonary endothelial cells (BPAEC). The release of radiolabeled fatty acids and the accumulation of cell-associated phospholipase products were measured and compared to a standard cytotoxicity assay (51Cr release) in response to an oxidant stress, in this case 0.1 to 10 mM hydrogen peroxide (H2O2). H2O2 caused time- and dose-dependent 51Cr release as well as liberation of saturated ([14C]stearic acid) and unsaturated ([3H]arachidonic acid) fatty acids and the accumulation of phospholipase A2 and C products. The ability of BPAEC to incorporate UFA into complex phospholipids was shown to be severely impaired in the presence of H2O2. Further studies showed that H2O2 caused depletion of BPAEC adenosine triphosphate (ATP) content to undetectable levels, and that the depletion of cellular ATP by iodoacetic acid induced substantial release of [3H]arachidonic acid but not [14C]stearic acid from BPAEC. This finding suggests that release of UFA in response to an oxidant stress may be due in part to a defect in ATP-dependent reacylation pathways and need not reflect any increase in phospholipase activities. Also unsaturated fatty acids were found to be toxic to BPAEC upon adding them to supernatants of cultured monolayers.
Am J Respir Cell Mol Biol 1991 May
PMID:Relationship of oxidant-mediated cytotoxicity to phospholipid metabolism in endothelial cells. 202 79

Oxidative stress induced by cumene hydroperoxide was studied in cultured neonatal rat myocytes. A progressive increase of irreversible cell injury as determined by leakage of the cytoplastic enzyme alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) from the cells was noted at concentrations ranging from 25-100 microM cumene hydroperoxide (incubation time 90 min). Cumene hydroperoxide-induced damage was reduced or prevented by several compounds: the application of Trolox C, a water-soluble vitamin E analogue, and of phospholipase A2 inhibitors chlorpromazine and (to a lesser extent) quinacrine prevented alpha-HBDH release. ICRF-159, a chelator of divalent cations, ascorbic acid, a potent antioxidant, and the cysteine protease inhibitor leupeptin did not reduce the cumene hydroperoxide-induced cytotoxicity. Detoxification of hydroperoxides by the glutathione peroxidase system results in an increased flux through the pentose phosphate shunt and loss of NADPH. Glucose inhibited the cumene hydroperoxide-induced alpha-HBDH release, probably by replenishing NADPH. These results indicate that cumene hydroperoxide, after exhaustion of the glutathione system, induces irreversible injury in cultured myocytes by a mechanism that depends to a large extent on deterioration of cellular membranes caused by lipid peroxidation and phospholipase activation.
J Mol Cell Cardiol 1990 Oct
PMID:Prevention of cumene hydroperoxide induced oxidative stress in cultured neonatal rat myocytes by scavengers and enzyme inhibitors. 209 37

Human genomic DNA and the HSV tk gene were cotransfected into mouse Ltk- cells and assayed for the acquisition of a Gs-coupled receptor to obtain cell lines expressing human receptors that are so far unavailable. The transfected cells were distributed into 96-well microtitration plates at a density such that after HAT (100 microM hypoxanthine, 1 microM aminopterin, and 10 microM thymidine) selection each well contained, on the average, two to three tk+ cell clones. After replication, half of them were tested for expression of a new phenotype: an adenylyl cyclase stimulatory receptor not normally expressed in the Ltk- recipient cell. The screen yielded a positive result on testing cells arising from the third transfection, the newly expressed receptor is that for arginine vasopressin, commonly referred to as type 2 or V2. DNA from primary transformants (HTB-1 cells) served to obtain secondary transformants by the same technique (HTB-2 cells). Pharmacological properties confirmed that this new receptor, which stimulates adenylyl cyclase activity 7- to 10-fold, is the human V2 receptor and not the activated homologous murine gene. The new cell line provides a permanent accessible source to study the human receptor, by-passing the need for human kidneys. The V2 receptor was susceptible to homologous down-regulation in the HTB-2 cell, but no down-regulation of the cell authentic prostaglandin E1 receptor was observed. The vasopressin receptor did not modify phospholipase-C activity in these cells as expected from V2 receptors. Thus, we successfully applied genomic DNA-mediated gene transfer and were able to develop a cell line expressing a Gs-coupled human receptor of low abundance and poor accessibility.
Mol Endocrinol 1990 Feb
PMID:Development and characterization of a mouse cell line expressing the human V2 vasopressin receptor gene. 213 94

In Torpedo electric organ much of the acetylcholinesterase is a 'globular' dimer (G2), anchored to the plasma membrane via covalently attached phosphatidylinositol and solubilized by a bacterial phosphatidylinositol-specific phospholipase C. This suggested that selective solubilization with phosphatidylinositol-specific phospholipase C, coupled with immunocytochemistry, might be used to localize G2 acetylcholinesterase in excitable tissues of Torpedo. Cryostat sections of electric organ, electromotor nerve, electric lobe and back muscle from Torpedo ocellata were labelled, using three different antibody preparations to Torpedo acetylcholinesterase, followed by a fluorescent second antibody, before and after exposure to the phospholipase. Sites of innervation on electrocytes and myofibers were labelled selectively, as were motor and electromotor nerves. In all these cases labelling was substantially diminished by prior exposure to the phospholipase. The results support our previous assignment, based on biochemical evidence, for a neuronal and synaptic localization of the G2 acetylcholinesterase in Torpedo. Electric lobe acetylcholinesterase appears insensitive to the phospholipase treatment and lacks certain epitopes present in both electric organ and electromotor nerve enzyme. This suggests that substantial processing of the G2 form occurs concomitantly with its movement from the electric lobe into the electromotor nerve.
Brain Res Mol Brain Res 1990 Aug
PMID:Immunocytochemical localization of phosphatidylinositol-anchored acetylcholinesterase in excitable membranes of Torpedo ocellata. 217 Jul 99

The crystal structure of an engineered phospholipase A2 with enhanced activity has been refined to an R-factor of 18.6% at 2.1 A resolution using a combination of molecular dynamics refinement by the GROMOS package and least-squares refinement by TNT. This mutant phospholipase was obtained previously by deleting residues 62 to 66 in porcine pancreatic phospholipase A2, and changing Asp59 to Ser, Ser60 to Gly and Asn67 to Tyr. The refined structure allowed a detailed comparison with wild-type porcine and Crotalus atrox phospholipase A2. The conformation of the deletion region appears to be intermediate between that in those two enzymes. The residues in the active center are virtually the same. An internal hydrophobic area occupied by Phe63 in the wild-type porcine phospholipase A2 is kept as conserved as possible by local rearrangement of neighboring atoms. In the mutant structure, this hydrophobic pocket is now occupied by the disulfide bond between residues 61 and 91. A detailed description of the second binding site for a calcium ion in this enzyme is given.
J Mol Biol 1990 Nov 20
PMID:Structure of an engineered porcine phospholipase A2 with enhanced activity at 2.1 A resolution. Comparison with the wild-type porcine and Crotalus atrox phospholipase A2. 225 38


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