UNIPROT:O95477 - FACTA Search

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Query: UNIPROT:O95477 (membrane-bound)
29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have characterized a membrane-bound phosphatidylcholine (PC) specific phospholipase C (PC-PLC) in plasma membranes from rat cardiac muscle, and have investigated the role of PC-PLC and PC-specific phospholipase D (PC-PLD) activities in the mechanism of action of atrial natriuretic factor (ANF). In purified sarcolemma, ANF stimulated over a wide range of concentrations with a maximum at 10(-11) M the hydrolysis of phosphatidylcholine through PC-PLD giving phosphatidate and choline, whereas higher concentrations of ANF (10(-10) M) preferentially stimulated PC breakdown through PC-PLC to form diacylglycerol and phosphocholine. To confirm the involvement of the PC-PLD in the mechanism of ANF action, we measured the transphosphatidylation reaction, a specific assay for this phospholipase which in the presence of ethanol catalyses the phosphatidylethanol formation from PC. ANF stimulated phosphatidylethanol formation with the same dose-response behavior as phosphatidate formation. The significant diacylglycerol increase at 10(-10) M ANF, in the presence of propranolol, a potent inhibitor of phosphatidate phosphatase which can hydrolyse phosphatidate to give diacylglycerol, suggested a direct involvement of PC-PLC. The use of GTP-gamma-S, a non hydrolysable analog of GTP, and of pertussis toxin showed the involvement of a pertussis toxin insensitive G protein in PC-PLC mediated ANF signal transduction. We suggest a differential effect of ANF on PC breakdown by phospholipases C and D depending on the concentration of the peptide.
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PMID:Selective activation by atrial natriuretic factor of phosphatidylcholine-specific phospholipase activities in purified heart muscle plasma membranes. 773 Oct 62

N-acylphosphatidylethanolamine (NAPE) was recently shown to be synthesized in vitro in cottonseed microsomes by the direct N-acylation of phosphatidylethanolamine (PE) with unesterfied fatty acids (K.D. Chapman and T. S. Moore, 1993, Plant Physiol. 102, 761-769). Here we examine the relationship of the synthesis and turnover of NAPE in cottonseed microsomes to the O-acylation of other membrane phospholipids. PE was N-acylated in a time-dependent manner with [1-14C]palmitic acid independent of exogenously supplied ATP. O-Acylation of PE and phosphatidylcholine (PC) with [1-14C]palmitic acid proceeded only in the presence of ATP. Further radiolabeling experiments with [1-14C]palmitoylCoA and phosphatidyl(N-[1-14C]-palmitoyl)ethanolamine indicated that O-acylation of phospholipids occurred via an acylCoA intermediate and not via an NAPE intermediate. [1-14C]palmitic acid was released from PC[1-14C-dipalmitoyl] in cottonseed microsomes in a Ca(2+)-dependent manner and this [14C]-FFA was incorporated into [14C]NAPE in a linear fashion. Cottonseed NAPE was selectively hydrolyzed to N-acylethanolamine (NAE) and N-acyl lysophosphatidylethanolamine (NAlysoPE) by Ca(2+)-independent, membrane-bound phospholipase D and A activities, respectively. NAlysoPE was not hydrolyzed to NAE, indicating that the phospholipase D that was active toward NAPE did not recognize NAlysoPE; instead NAlysoPE was converted to NAPE in the presence of Ca2+. Collectively, our results indicate that NAPE synthesis and the O-acylation of other phospholipids occur by two separate pathways and that microsomal NAPE is selectively turned over by membrane-bound phospholipase activities. A pathway for the metabolism of cottonseed NAPE is outlined.
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PMID:Metabolism of cottonseed microsomal N-acylphosphatidylethanolamine. 773 69

Regulation of phospholipase D (PLD) activation by protein kinase C (PKC) was studied in membranes isolated from human promyelocytic leukemia HL60 cells. The activation of membrane-bound PLD by PKC partially purified from rat brain was most effectively induced with phorbol 12-myristate 13-acetate (PMA) and Ca2+ (1 microM) which caused translocation of PKC to membranes. Ro31-8425, a potent inhibitor of PKC, suppressed the catalytic activity of PKC in a concentration-dependent manner, with complete inhibition at 5 microM. However, the PKC-mediated PLD activation was not affected by Ro31-8425. It was thus suggested that membrane-bound PLD of HL60 cells was activated by PKC translocation but probably via a phosphorylation-independent mechanism. Furthermore, addition by guanosine 5'-3-O-(thio)trisphosphate (GTP gamma S) potentiated the PKC-mediated PLD activation and this potentiating effect was abolished by Rho GTPase dissociation inhibitor (RhoGDI). The suppressed PLD activation by RhoGDI was completely restored by addition of recombinant RhoA. These results indicate that the PKC-mediated PLD activation can be synergistically potentiated by RhoA in HL60 membranes.
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PMID:Activation of membrane-bound phospholipase D by protein kinase C in HL60 cells: synergistic action of a small GTP-binding protein RhoA. 777

Purified budded virions of Autographa californica nuclear polyhedrosis virus (AcNPV) contain abundant amounts of free ubiquitin, which has an altered electrophoretic mobility on SDS gels as compared with standard ubiquitin. Phase extraction of virion proteins with Triton X-114 indicated that the modified form of ubiquitin behaved as an integral membrane protein. The membrane-bound form of ubiquitin was labeled with both phosphate and palmitate, and its electrophoretic mobility was altered by treatment with phospholipase A2 and a phosphatidylcholine-specific phospholipase D. Mild trypsin digestion indicated that the acyl group was not linked to the C-terminus of the protein. Acylated ubiquitin could not be radiolabeled with a membrane-impermeable Bolton-Hunter reagent unless virus was pretreated with detergent. Together, these experiments suggest that ubiquitin is attached to the inner face of the viral membrane by a novel type of phospholipid anchor.
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PMID:Ubiquitin is attached to membranes of baculovirus particles by a novel type of phospholipid anchor. 783 50

The activation of phospholipase D (PLD) is a receptor-mediated event that has been implicated in signal transduction and membrane traffic in eukaryotic cells. Little is known about the biochemical and molecular properties of signal-activated PLDs, and none has been isolated. Here we report that phosphatidylinositol 4,5-bisphosphate (PIP2) potently stimulates brain membrane PLD activity in vitro in a highly specific manner. PIP2 increases 10-fold the maximal activity of a partially purified PLD with an EC50 of < 0.5 mol %. Other acidic phospholipids, including phosphatidylinositol 4-phosphate, phosphatidylinositol, phosphatidylserine, and phosphatidic acid, are completely or nearly ineffective. Neomycin, a high affinity ligand of PIP2, inhibits membrane-bound PLD but has no effect on the activity of a detergent-solubilized or partially purified enzyme. The addition of PIP2 restores the sensitivity of partially purified PLD to neomycin inhibition, indicating that neomycin blocks membrane PLD activity by binding to endogenous PIP2. These results define a novel function of PIP2 as a cofactor for brain membrane PLD and suggest that PIP2 synthesis and hydrolysis could be important determinants in regulating PLD action in signal transduction and membrane transport.
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PMID:Novel function of phosphatidylinositol 4,5-bisphosphate as a cofactor for brain membrane phospholipase D. 806 70

Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP-binding protein that activates PLD can be functionally coupled. However, assay of membrane preparations at increasing dilutions showed that stimulation of PLD by the compounds studied, with the partial exception of ATP[S], involved diffusible rather than protein-bound intermediates.
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PMID:Stimulation of phospholipase D in rabbit platelet membranes by nucleoside triphosphates and by phosphocreatine: roles of membrane-bound GDP, nucleoside diphosphate kinase and creatine kinase. 819 58

Photoreactive lipid analogs, namely, 1-acyl-2-(12-azidooleoyl)glycero-3-phosphocholine (N3-PC) and 1-acyl-2-(12-azidooleoyl)glycero-3-phosphoethanolamine (N3-PE) have been synthesized as previously described [R. Rajasekharan and J. D. Kemp (1994) J. Lipid Res. 35, 45-51]. Azidophosphatidic acid was produced by hydrolyzing N3-PC with phospholipase D. All of the lysophospholipid analogs, 2-(12-azidooleoyl)glycero-3-phosphate (N3-LPA), 2-(12-azidooleoyl)glycero-3-phosphocholine (N3-LPC), and 2-(12-azidooleoyl)glycero-3-phosphoethanolamine (N3-LPE), were produced from appropriate azidophospholipids by lipase treatment. The photoactive lysophospholipid analogs were recognized as substrates by acyltransferases in the dark and as irreversible inhibitors after photolysis with uv light. The photoinactivation of acyltransferases by azidolysophospholipids was protected by the addition of natural lysophospholipids. Incubation of developing soybean microsomal membranes with N3-LPA followed by photolysis resulted in 69% inhibition of lysophosphatidic acid (LPA) acyltransferase and also had significant inhibitory effects on lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) acyltransferases, indicating that the LPA analog interacts with all the lysophospholipid acyltransferases. When the membranes were photolyzed with N3-LPC or N3-LPE and assayed, the membranes showed approximately 50% inactivation of LPC and LPE acyltransferase activities, whereas LPA acyltransferase was unaffected, suggesting that a single enzyme might acylate both LPC and LPE. The recognition of these photoreactive lipid analogs by acyltransferases will facilitate the identification and purification of these membrane-bound enzymes.
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PMID:Use of photoreactive substrates for characterization of lysophosphatidate acyltransferases from developing soybean cotyledons. 820 2

The activation of phospholipase D (PLD) by platelet-derived growth factor (PDGF), prostaglandin F2 alpha and 12-O-tetradecanoylphorbol 13-acetate (TPA) was studied in NIH-3T3 fibroblasts. PLD activation was determined by measuring the production of both [3H]phosphatidic acid and [3H]phosphatidylpropanol (products of the PLD-catalyzed hydrolysis and transphosphatidylation reactions, respectively), in cells that were metabolically pre-labeled with [3H]oleic acid. All mitogens caused a rapid (within 2 min) activation of PLD. Activation of PLD by prostaglandin F2 alpha and PDGF was transient and declined to near basal levels by 15 min and 55 min, respectively. In contrast, TPA-induced activation of PLD was sustained for at least 60 min of incubation. A combination of maximally effective concentrations of PDGF and TPA stimulated PLD activity in a non-additive manner, while the effect of prostaglandin F2 alpha was additional to that of either PDGF or TPA. The protein kinase inhibitor staurosporine inhibited PLD activation by PDGF or TPA with almost identical dose/response curves. In contrast, staurosporine potentiated prostaglandin-F2 alpha-induced PLD activation. The specific protein kinase C inhibitor GF109203X (a bisindolylmaleimide) inhibited PLD activation by prostaglandin F2 alpha and PDGF at concentrations higher than those required for inhibition of PLD activation induced by TPA. Depletion of cellular protein kinase C abolished PLD activation by all three mitogens without affecting in vitro activity of membrane-bound PLD. The distinct kinetics of PLD activation and its differential susceptibility to protein kinase inhibitors suggest the existence of agonist-specific activation and/or inactivation mechanisms. The results indicate also that protein kinase C participates in the mechanism of PLD activation via PDGF, while the effect of prostaglandin F2 alpha involves a pathway independent of protein kinase C.
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PMID:Distinct mechanisms of phospholipase D activation and attenuation utilized by different mitogens in NIH-3T3 fibroblasts. 834 13

The specific activity of cGMP-inhibited cAMP phosphodiesterase (PDE III) of rat liver microsomal membranes is increased in a concentration-dependent way by adding phospholipase C from Clostridium perfringens or phospholipase D from Streptomyces chromofuscus. The effect depends on an increase in Vmax of the enzyme. Treatment of microsomal membranes with phospholipase C causes a marked increase in the relative amounts of phosphatidylserine and phosphatidylinositol, and mild stimulation of PDE III activity. Treatment with phospholipase D increases phosphatidic acid and strongly increases PDE III activity. These data suggest that phosphatidic acid is the most important regulator of membrane-bound PDE III activity in liver.
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PMID:Stimulation of rat liver microsomal cGMP-inhibited cAMP phosphodiesterase (PDE III) by phospholipase C and D. 838 Dec 82

Activation of the membrane-bound phospholipase D (PLD) requires cytosolic factor(s), and ADP-ribosylation factor (ARF) has been identified as a cytosolic PLD activator. In the present study, we demonstrate that calmodulin (CaM) and ARF are both involved in PLD activation in rabbit peritoneal neutrophils. The PLD activity of streptolysin O-permeabilized, cytosol-depleted rabbit neutrophils was significantly enhanced when the permeabilized cells were reconstituted with bovine brain cytosol in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), whereas there was little activation of the enzyme in the absence of cytosol. The GTP gamma S-stimulated PLD activity in the presence of cytosol was augmented on increasing the concentration of free Ca2+. The PLD activity stimulated by GTP gamma S and Ca2+ in this system was inhibited by the calmodulin inhibitor W-7. These findings suggest that CaM plays a role as a cytosolic PLD activator. Moreover, highly purified CaM alone, as well as partially purified ARF alone, promoted a slight stimulation of the PLD activity in permeabilized neutrophils. Interestingly, ARF-stimulated PLD activity was augmented by CaM in the presence of GTP gamma S and Ca2+. This augmentation was again inhibited by W-7, as well as by the structurally unrelated CaM inhibitor trifluoperazine. These data imply that CaM stimulates the PLD activity of rabbit neutrophils in concert with ARF.
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PMID:Augmentation by calmodulin of ADP-ribosylation factor-stimulated phospholipase D activity in permeabilized rabbit peritoneal neutrophils. 855 2

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