SEARCH · PubMed Health

Results for “PHOSPHOLIPASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 865 records · Page 48Linked to original sources

PubMed1998

Activation of LA-N-2 cell phospholipase D by amyloid beta protein (25-35).

Amyloid beta protein is the major protein component of neuritic plaques found in the brain of Alzheimer's disease. The activation of phospholipase D by amyloid beta protein (25-35), quisqualate and phorbol 12, 13-dibutyrate was investigated in LA-N-2 cells by measuring phosphatidylethanol formation. The activation of phospholipase D by quisqualate and APP (25-35) was calcium-independent. The AbetaP (25-35) and quisqualate activation of phospholipase D appeared to be mediated through a pertussis toxin-sensitive GTP-binding protein. Phospholipase D activation by AbetaP (25-35), quisqualate and phorbol dibutyrate was not blunted by the protein kinase C inhibitors, staurosporine, H-7 and RO-31-8220. However, it was abolished by overnight exposure to phorbol dibutyrate. This activation of phospholipase D was prevented by the tyrosine kinase inhibitor, genistein but not by tyrophostin A. Several excitatory amino acid antagonists were tested for their ability to prevent the phospholipase D activation by quisqualate and AbetaP (25-35). Only NBQX was effective with an IC50 of 75 microM for AbetaP (25-35) and quisqualate. Activation of phospholipase D by AbetaP or quisqualate was absent in LA-N-2 cells previously desensitized by quisqualate or AbetaP (25-35), but the activation by phorbol dibutyrate was unaltered. The responsiveness to AbetaP and quisqualate in previously desensitized cells reappeared subsequent to a period of resensitization. The observations with the antagonist NBQX, and the desensitization and resensitization experiments, are consistent with a receptor occupancy mediated activation of phospholipase D by quisqualate and by AbetaP (25-35).

Amyloid beta-Peptides↗

PubMed1968

Lysosomal phospholipases A1 and A2 of bovine adrenal medulla.

1. [(32)P]Lecithin and [(32)P]phosphatidylethanolamine were prepared by incubating rat liver mince with [(32)P]phosphate. With these (32)P-labelled phospholipids conditions for the quantitative assay of phospholipase A activity were established. 2. The distribution of phospholipase A activity between subcellular fractions of the bovine adrenal medulla was determined. Phospholipases A(1) and A(2), with pH optima at 4.2 and 6.5 respectively, were found in the large-granule fraction. By means of sucrose-density-gradient centrifugation it was shown that both these phospholipases were localized in lysosomes. 3. Lysosomal phospholipase A(1) catalysed the hydrolysis of [(32)P]lecithin and [(32)P]phosphatidylethanolamine at the same rate. The enzymic activity was inhibited by 70% in the presence of 10mm-calcium chloride. 4. Lysosomal phospholipase A(2) catalysed the hydrolysis of [(32)P]phosphatidylethanolamine more rapidly than it hydrolysed [(32)P]lecithin. The hydrolysis of [(32)P]phosphatidylethanolamine, but not that of [(32)P]lecithin, by phospholipase A(2) was activated by 0.8mm-calcium chloride. However, the hydrolysis of both substrates was inhibited by 8mm-calcium chloride. 5. The significance of the presence of phospholipase activity in lysosomes is discussed in relation to the functions of lysosomes in general and in the adrenal medulla.

Adrenal Medulla↗

PubMed1988

Alcohol-induced stimulation of phospholipase C in human platelets requires G-protein activation.

In previous studies we have demonstrated that ethanol activates hormone-sensitive phospholipase C in intact human platelets, resulting in the mobilization of intracellular Ca2+ and platelet shape change. The present study aims to localize further this effect of ethanol by examining its interaction with the regulation of phospholipase C in a permeabilized cell system. In platelets permeabilized with a minimal concentration (18 micrograms/ml) of saponin, ethanol by itself did not activate phospholipase C. However, ethanol potentiated the activation of phospholipase C in response to the non-hydrolysable GTP analogue GTP[S] (guanosine 5'-[gamma-thio]triphosphate), an effect similar to that observed with thrombin. Ethanol also potentiated the response to fluoride, which acts directly on G-proteins. Other short-chain alcohols also stimulated phospholipase C in a synergistic manner with GTP[S]. The ability of specific alcohols to stimulate phospholipase C was directly related to their respective lipid-solubilities, as determined by their partition coefficients. Moreover, the potencies of each alcohol correlated with their ability to elicit Ca2+ mobilization and shape change in intact platelets. These effects of ethanol were eliminated by a disruption of receptor-phospholipase C coupling induced by the addition of higher concentrations of saponin. These data indicate that the activation of phospholipase C by ethanol may occur by affecting protein-protein interactions in the signal-transduction complex involving GTP-binding regulatory proteins.

1-Butanol↗

PubMedDate not supplied

Exogenous phospholipase C stimulates epithelial cell migration and integrin expression in vitro.

Phospholipase C secreted by bacterial pathogens has been identified as a virulence factor in several human diseases and has been implicated in impeding wound healing. The role of phospholipase C in the intracellular signal control of epithelial growth was studied in normal human skin keratinocytes cultured in conditions simulating aspects of wound healing. Bacillus cereus phospholipase C decreased cell-cell contact and increased cell migration resulting in disruption of the advancing epithelial sheet. Phospholipase C-induced migration was blocked by inhibitor of the phosphoinositol signal transduction pathway neomycin sulfate and protein kinase C inhibitor RO-31-8220. Induced migration was associated with elevated levels of matrix metalloproteinase-9 which, when blocked by tissue inhibitor of metalloproteinase-1, was accompanied by a loss of migration. Adhesion studies showed that phospholipase C treatment enhanced cell binding to fibronectin, vitronectin and collagen IV. Immunostained phospholipase C-stimulated cells cultured on fibronectin showed enhanced expression and relocation of the integrin subunits alpha(v), alpha5 and beta1. Confocal microscopy showed that phospholipase C-induced levels of integrin subunit beta1 were predominantly deposited on the basal surface of the cell apparently in focal contacts and associated with actin stress fibers. These results indicate that exogenous phospholipase C signaling from a bacterial source may play an important role in perturbing normal reepithelialization via altered expression of integrins and matrix metalloproteinase-9.

Bacillus cereus↗

PubMed2001

Protein expression of phospholipase C in pregnant and nonpregnant rat uterine tissue.

OBJECTIVE: The phosphatidylinositol signaling pathway appears to play a significant role in the intracellular events leading to agonist-stimulated phasic myometrial contractions. The studies described in this report were performed to characterize phospholipase C isoform expression at the protein level and to confirm histologic localization of these proteins within the myometrial smooth muscle layers of the uterus. METHODS: For these studies, uterine tissue was obtained from timed- pregnant and spontaneously cycling adult female Sprague-Dawley rats. After isolation of myometrial cell membranes and cytosolic proteins, Western blots were performed by using phospholipase C isoform-specific antibodies. Tissue cross-sections of near-term pregnant rat uterus were used with the phospholipase C isoform-specific antibodies for immunohistochemical studies. RESULTS: The Western blot studies confirmed expression of the phospholipase C-beta3, -gamma1, -gamma2, and -delta1 proteins in both the membrane and cytosolic fractions of rat myometrium; in contrast, only trace amounts of the phospholipase C-beta1 protein was observed in this tissue. The immunohistochemical studies demonstrated localization of the phospholipase C-beta3, -gamma1, -gamma2, -delta1 and to a lesser degree phospholipase C-beta1 isoforms within the longitudinal and circular smooth muscle layers of the near-term pregnant rat uterus. CONCLUSION: These studies have confirmed the simultaneous expression of several phospholipase C proteins within the smooth muscle cells of the pregnant and nonpregnant rat uterus, thereby providing support for the possible redundant role of these signal transduction enzymes during the generation of cytosolic calcium oscillations and phasic myometrial contractions.

Animals↗

PubMed2005

Requirement of phospholipase D1 activity in H-RasV12-induced transformation.

The ability of the Ras oncogene to transform normal cells has been well established. One downstream effector of Ras is the lipid hydrolyzing enzyme phospholipase D. Recent evidence has emerged indicating a role for phospholipase D in cell proliferation, membrane trafficking, and migration. To study the potential importance of phospholipase D in the oncogenic ability of Ras, we used Rat-2 fibroblasts with reduced phospholipase D1 activity (Rat-2V25). Here, we show that H-Ras transformation of Rat-2 fibroblasts requires normal phospholipase D1 activity. WT Rat-2 fibroblasts transfected with the H-RasV12 oncogene grew colonies in soft agar and tumors in nude mice. However, Rat-2V25 cells when transfected with the H-RasV12 oncogene did not form colonies in soft agar or produce tumors when xenografted onto nude mice. Interestingly, in the presence of phosphatidic acid, the product of phospholipase D, growth in soft agar and tumor formation was restored. We also observed a dramatic increase in the expression of phospholipase D1 in colorectal tumors when compared with adjacent normal mucosa. Our studies identify phospholipase D1 as a critical downstream mediator of H-Ras-induced tumor formation.

Animals↗

PubMed1993

Distinctive subtypes of bovine phospholipase C that have preferential expression in the retina and high homology to the norpA gene product of Drosophila.

The Drosophila norpA gene encodes a phospholipase C involved in phototransduction. However, phospholipase C apparently is not directly involved in phototransduction in vertebrate photoreceptors, although light-activated phospholipase C activity has been reported in vertebrate rod outer segments. Conserved regions of norpA cDNA were used to isolate bovine cDNAs that would encode four alternative forms of phospholipase C of the beta class that are highly homologous to the norpA protein and expressed preferentially in the retina. Two of the variants are highly unusual in that they lack much of the N-terminal region present in all other known phospholipases C. The sequence conservation between these proteins and the norpA protein is higher than that between any other known phospholipases C. GTPase sequence motifs found in proteins of the GTPase superfamily are found conserved in all four variants of the bovine retinal protein as well as the norpA protein but not in other phospholipases C. Results suggest that these proteins together with the norpA protein constitute a distinctive subfamily of phospholipases C that are closely related in structure, function, and tissue distribution. Mutations in the norpA gene, in addition to blocking phototransduction, cause light-dependent degeneration of photoreceptors. In view of the strong similarity in structure and tissue distribution, a defect in these proteins may have similar consequences in the mammalian retina.

Amino Acid Sequence↗

PubMed1983

Combination chemotherapy with Clostridium perfringens phospholipase C and cytosine antimetabolites: complementary inhibition directed at membrane lipids.

Tumor cell membranes were susceptible to the action of Clostridium perfringens phospholipase C, and this was reflected by inhibition of cellular replication in culture. The differential susceptibility of two neoplastic cell lines to this enzyme was studied in detail. The growth of sarcoma 180 cells cultured in Fischer's medium was markedly inhibited by phospholipase C; whereas, in contrast, cultured L1210 leukemia cells were relatively resistant to the cytotoxic effects of this enzyme. The differential sensitivity of these two neoplastic cell lines to phospholipase C was corroborated by dye-exclusion tests. Thus, leukemia L1210 cells exposed to a concentration of 0.2 mg of phospholipase C per ml of Fischer's medium for 30 min at 37 degrees C were able to exclude Trypan Blue; whereas, only about 21% of sarcoma 180 cells treated under identical conditions were able to exclude the dye. That the cytotoxicity of phospholipase C to sarcoma 180 was the result of hydrolysis of phospholipids of the plasma membrane was supported by measurements of the rate of hydrolysis of radioactivity from the phospholipid of neoplastic cells prelabeled with [3H]choline. Eighty-two percent of incorporated radioactive choline was released from sarcoma 180 cells treated with phospholipase C in Fischer's medium, whereas, only 20% of the label from [3H]choline was solubilized from L1210 leukemia cells treated with the enzyme under similar conditions. Scanning electron microscopy revealed significant damage to sarcoma 180 cells exposed to phospholipase C in Fischer's medium, which was characterized by alterations in size and shape of cells, disappearance of microvilli, and appearance of fistulas in cell membranes; relatively resistant L1210 leukemic cells did not appear to be markedly damaged by comparable enzyme treatment. Exposure of leukemia L1210 cells to phospholipase C in Puck's saline A increased the sensitivity of these cells to enzymatic action. Under these conditions, a comparable amount of phospholipid was hydrolyzed from surface membranes of sarcoma 180 and leukemia L1210 cells, and the degree of membrane damage appeared to be similar, as measured by the capacity of the tumor cell lines to exclude Trypan Blue and by scanning electron microscopy. The extensive damage to membranes by hydrolysis of phospholipids was not accompanied by a change in the degree of specific binding of [3H]concanavalin A(ConA).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1997

Phospholipase activity in Cryptococcus neoformans: a new virulence factor?

Fifty isolates of Cryptococcus neoformans were examined for extracellular phospholipase production after inoculation onto egg yolk agar; 49 produced a pericolonial precipitate indicative of phospholipase activity. Phospholipase B (PLB), lysophospholipase, and lysophospholipase-transacylase activities were identified by radiometric analysis in supernatants from 4 clinical isolates. The ratio of colony diameter to colony plus precipitate on agar (Pz) correlated with PLB activity. Phospholipase production was similar in 12 environmental and 13 clinical isolates of C. neoformans var. gattii. Environmental strains of C. neoformans var. neoformans (n = 8) produced more phospholipase at 72 h than did 17 clinical isolates (mean Pz, 0.57 vs. 0.72; P < .01); however, Pz values were similar at 96 h. Quantitation of cryptococci in the lungs and brains of BALB/c mice inoculated intravenously with 4 strains expressing high, intermediate, or low phospholipase activity revealed a correlation between phospholipase activity and virulence. Phospholipases secreted by C. neoformans may be implicated in virulence.

Acyltransferases↗

PubMed1979

Phospholipase A of sea snake Laticauda semifasciata venom. Isolation and properties of novel forms lacking tryptophan.

The venom gland extracts of the sea snake Laticauda semifasciata contained at least four forms of phospholipase A separable on a CM-cellulose column. They were designated as phospholipases A I-IV in the order of elution from the column. Phospholipases A I, III, and IV were isolated in a homogeneous state. They were similar to one another in amino acid composition and molecular weight (14,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phospholipase A I contained one tryptophan residue. whereas III and IV did not. Although all these forms had the same A2-type positional specificity, they were classified into two groups (I, and III and IV) on the basis of enzymic properties. Phospholipase A I had a higher specific activity and showed normal kinetics, whereas III and IV had approximately one-tenth of the specific activity of I and showed biphasic kinetics due to their activation by the reaction products. Phospholipase A I, the major form, seems to be identical with phospholipase A reported previously (Tu, A.T., Passey, R.B., & Toom, P.M. (1970) Arch. Biochem. Biophys. 140, 96-106), whereas the other two, III and IV, are new. Phospholipase A I became more like III and IV in enzymic properties on modification with N-bromosuccinimide.

Amino Acids↗

PubMed1985

Intracellular phospholipase activities of Tetrahymena pyriformis.

Phospholipase activity was studied in the protozoan Tetrahymena pyriformis NT-1 by using exogenous phosphatidylethanolamine and phosphatidylcholine. Several phospholipase activities were found in Tetrahymena homogenates. They were distinguished with respect to pH optimum, activity dependence on Ca2+, substrate specificity and positional specificity. Ca2+-Dependent phospholipase activity had an optimal pH around 9 and gave rise to free fatty acid and lysophospholipid. This enzyme hydrolyzes phosphatidylethanolamine but not phosphatidylcholine. The alkaline phospholipase with A1 activity was located mainly in the surface membrane (pellicle fraction). The enzyme activity had a pH optimum ranging from 8 to 9, and required 2 mM CaCl2 for the maximal activity. All detergents tested inhibited the enzyme activity. Ca2+-Independent phospholipase activity had an optimal pH from 4 to 5 and gave rise to free fatty acid, lysophospholipid, diacylglycerol, and monoacylglycerol. We concluded that there are at least three phospholipase in Tetrahymena homogenates, i.e., alkaline phospholipase A and acidic phospholipases A and C.

Animals↗

PubMed1988

Regulation of platelet phospholipase C.

We have investigated factors affecting the activation of phospholipase C in human platelets. Prior exposure of platelets to phorbol esters that stimulated protein kinase C inhibits the activation of phospholipase C in response to a variety of receptor-directed agonists, including alpha- and gamma-thrombin and thromboxane A2 analogues. Such activation has been assayed by measurements of accumulated InsP3 (including Ins(1,4,5)P3 and Ins(1,3,4)P3) and PtdOH. Inhibition is not overcome by Ca2+ ionophores, and substances that block or mimic Na+-H+ exchange neither block nor mimic these inhibitory effects. Cyclic AMP and cyclic GMP, other agents known to inhibit phospholipase C activation, do not accumulate in platelets exposed to phorbol esters. Although a portion of the effects of phorbol ester on InsP3 accumulation may be explained by 5-phosphomonoesterase activity, it is likely that more direct effects on phospholipase C are being exerted as well, and contribute the major inhibitory route. We have examined the susceptibility of adenylyl cyclase-associated Gi and 'Gp'-activated phospholipase C to inhibitory ADP-ribosylation by pertussis toxin-derived enzyme (S1 protomer) administered to saponin-permeabilized platelets. The effects of alpha-thrombin on adenylyl cyclase can be inhibited by up to 50% by S1, at which point inhibition of phospholipase C is barely detectable. Thromboxane A2 analogues, which do not affect adenylyl cyclase (Gi), stimulate phospholipase C; this effect is not impaired by S1. We therefore propose that the inhibitory effects of phorbol esters on the activation of phospholipase C are not mediated primarily by effects on Gi.

Blood Platelets↗

PubMed1991

Characterization of fMet-Leu-Phe-stimulated phospholipase C in streptolysin-O-permeabilised cells.

Phospholipase C (specific for inositol lipids) is known to be present both in membranes and cytosol. Receptor-mediated activation of this enzyme occurs via a guanine nucleotide regulatory protein (G-protein), designated Gp. We have compared the stimulation of this enzyme by fMet-Leu-Phe via the G-protein in HL60 membranes and in permeabilised cells. fMet-Leu-Phe stimulated phospholipase C in membranes at 2 min and the response was dependent on exogenously added GTP. GTP alone also stimulated phospholipase C activity such that at 10 min the response to fMet-Leu-Phe was minimal. In comparison, the response to fMet-Leu-Phe in permeabilised cells was greater in extent but did not require added GTP. However, it was antagonized by GDP analogues (GDP[beta S] greater than GDP greater than dGDP) and by pertussis toxin pretreatment, indicating that fMet-Leu-Phe-stimulated phospholipase C activity was also mediated via Gp. GTP and its analogue GTP[gamma S] also stimulated phospholipase C and their effects were strictly additive to the stimulation obtained with fMet-Leu-Phe. Such additivity was also observed when two receptor-directed agonists, fMet-Leu-Phe and ATP, were used to stimulate intact cells. It is concluded that (a) the size of the response with fMet-Leu-Phe in membranes is limited by the loss of a component, possibly phospholipase C, and (b) stoichiometry and physical organisation of multiple species of G-proteins and/or phospholipases C may explain the independent nature of phospholipase C activation by fMet-Leu-Phe, ATP and guanine nucleotides.

Adenosine Triphosphate↗

PubMed1994

Differential effects of P2-purinoceptor antagonists on phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors.

1. Stimulation of P2Y-purinoceptors on turkey erythrocytes and many other cell types results in activation of phospholipase C. In contrast, we have observed recently that P2Y-purinoceptors on C6 rat glioma cells are not coupled to phospholipase C, but rather, inhibit adenylyl cyclase. 2. In this study we investigated the pharmacological selectivity of the P2-purinoceptor antagonists, suramin, reactive blue 2, and pyridoxal phosphate 6-azophenyl 2',4'-disulphonic acid (PPADS) for phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors. 3. In C6 glioma cells, suramin and reactive blue 2 competitively antagonized the inhibitory effect of 2MeSATP on adenylyl cyclase (pKB = 5.4 +/- 0.2 and 7.6 +/- 0.1, respectively), whereas PPADS at concentrations up to 100 microM had no effect. 4. In contrast, in the turkey erythrocyte preparation, PPADS at concentrations up to 30 microM was a competitive antagonist of P2Y-purinoceptor-stimulated phospholipase C activity (pKB = 5.9 +/- 0.1). Suramin and reactive blue 2 produced both a shift to the right of the concentration-effect of 2MeSATP for the activation of phospholipase C and a significant decrease in the maximal inositol phosphate response. 5. Turkey erythrocytes also express a phospholipase C-coupled beta-adrenoceptor. Concentrations of PPADS that competitively inhibited the P2Y-purinoceptor-mediated response had only minimal effects on the activation of phospholipase C by beta-adrenoceptors. In contrast, suramin and reactive blue 2 produced a non-competitive inhibition, characterized by decreases in the maximal response to isoprenaline with no change in the potency of this beta-adrenoceptor agonist. 6. The differential effect of PPADS on P2Y-purinoceptors of C6 glioma cells and turkey erythrocytes adds further support to the idea that different P2Y-purinoceptor subtypes mediate coupling to adenylylcyclic and phospholipase C.

Adenylyl Cyclase Inhibitors↗

PubMed1991

Somatostatin inhibition of phospholipase C activity in isolated rat pancreatic islets.

We have assessed the effect of somatostatin on the phospholipase C activity in isolated rat pancreatic islets. The phospholipase C activity was measured as the generation of inositol 1,4,5-trisphosphate and its metabolite inositol 1,3,4-trisphosphate from the hydrolysis of polyphosphoinositides. Inositol phosphates were measured using anion-exchange fast protein liquid chromatography analysis of extracts from islets prelabelled with myo-[3H]inositol. Somatostatin (1-1000 nmol l-1) significantly inhibited the glucose-induced (12 mmol l-1) phospholipase C activity in a concentration-dependent manner. The Ca2+ channel blocker verapamil (25 mumol l-1) also inhibited the glucose-induced (12 mmol l-1) phospholipase C, whereas the combination of somatostatin and verapamil did not induce any additional inhibition. At 3.3 mmol l-1 glucose, the hypoglycaemic sulphonylurea, tolbutamide (1 mmol l-1), increased the phospholipase C activity. This effect was reversed by somatostatin (100 nmol l-1). Tolbutamide did not further increase the glucose-induced (12 mmol l-1) phospholipase C activity. However, the somatostatin inhibition of glucose-induced (12 mmol l-1) phospholipase C was reversed by tolbutamide. The activator of adenylyl cyclase, forskolin (20 mumol l-1), did not exert any effect on the PLC-inhibition of somatostatin, whereas forskolin alone inhibited the phospholipase C activation at 12 mmol l-1 glucose. Our study demonstrates that somatostatin inhibits the hydrolysis of polyphosphoinositides in pancreatic islets, apparently via a mechanism dependent on Ca2+ and not on cAMP.

Animals↗

PubMed1997

Molecular characterization of pldA, the structural gene for a phospholipase A from Campylobacter coli, and its contribution to cell-associated hemolysis.

A gene (pldA) encoding a 35.0-kDa protein with significant homology to the Escherichia coli outer membrane phospholipase was identified upstream of an operon encoding an enterochelin transport system in Campylobacter coli. The results of this study suggest that this gene encodes an outer membrane phospholipase A in C. coli. First, expression of the pldA gene product in a PldA-deficient mutant of E. coli led to the restoration of phospholipase A activity. The recombinant product also partitioned to the outer membrane, suggesting that it may be similarly located in C. coli. Second, heterologous overexpression in E. coli, followed by in vitro folding and purification of C. coli PldA, resulted in pure protein which displayed calcium-dependent lysophospholipase and phospholipase A activities in vitro. Finally, mutants of C. coli in which the pldA gene had been inactivated by allelic exchange were deficient in phospholipase A activity. Phospholipases are associated with lysis of erythrocytes by a number of bacterial pathogens. The pldA mutant was shown to have a reduced hemolytic activity compared to the wild-type strain, suggesting a role for the phospholipase A in the lysis of erythrocytes by C. coli. Since hemolysins are intimately associated with the disease-causing potential of a number of bacterial pathogens, it is likely that the phospholipase A plays some role in Campylobacter virulence.

Amino Acid Sequence↗

PubMed2002

Similarities in the induction of post-Golgi vesicles by the vaccinia virus F13L protein and phospholipase D.

Intracellular mature vaccinia virions are wrapped by cisternae, derived from virus-modified trans-Golgi or endosomal membranes, and then transported via microtubules to the cell periphery. Two viral proteins, encoded by the F13L and B5R open reading frames, are essential for the membrane-wrapping step. Previous transfection studies indicated that F13L induces the formation of post-Golgi vesicles that incorporate the B5R protein and that this activity depends on an intact F13L phospholipase motif. Here we show that the F13L protein has a general effect on the trafficking of integral membrane proteins from the Golgi apparatus, as both the vaccinia virus A36R protein and the vesicular stomatitis virus G protein also colocalized with the F13L protein in vesicles. In addition, increased expression of cellular phospholipase D, which has a similar phospholipase motif as, but little amino acid sequence identity with, F13L, induced post-Golgi vesicles that contained B5R and A36R proteins. Butanol-1, which prevents the formation of phosphatidic acid by phospholipase D and specifically inhibits phospholipase D-mediated vesicle formation, also inhibited F13L-induced vesicle formation, whereas secondary and tertiary alcohols had no effect. Moreover, inhibition of phospholipase activity by butanol-1 also reduced plaque size and decreased the formation of extracellular vaccinia virus without affecting the yield of intracellular mature virus. Phospholipase D, however, could not complement a vaccinia virus F13L deletion mutant, indicating that F13L has additional virus-specific properties. Taken together, these data support an important role for F13L in inducing the formation of vesicle precursors of the vaccinia virus membrane via phospholipase activity or activation.

Alcohols↗

PubMed1992

Use of [1-14C]oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity.

Autoclaved Escherichia coli labelled with [1-14C]oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro. The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity. Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of [1-14C]oleate labelled, autoclaved E. coli optimally at pH 7.0-8.0 to generate [14C]phosphatidic acid in the presence of 5 mM added Ca2+. Other divalent cations would not substitute for Ca2+. Activity was linear with time and protein up to 30% of the hydrolysis of substrate. Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100. The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E. coli by human synovial fluid phospholipase A2. Accumulation of [14C]diglyceride was observed after 10 min of incubation. This accumulation was inhibited by NaF (IC50 = 18 microM) or propanolol (IC50 = 180 microM) suggesting the S. chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase. Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner. These results demonstrate that [1-14C]oleate labelled, autoclaved E. coli can be used to measure phospholipase D activity by monitoring accumulation of either [14C]phosphatidic acid or [14C]phosphatidylethanol.

Brassica↗