scholarly journals Didecanoyl phosphatidylcholine is a superior substrate for assaying mammalian phospholipase D

1996 ◽  
Vol 319 (3) ◽  
pp. 861-864 ◽  
Author(s):  
Anne M VINGGAARD ◽  
Torben JENSEN ◽  
Clive P. MORGAN ◽  
Shamshad COCKCROFT ◽  
Harald S. HANSEN
Keyword(s):  
Detection Limit ◽  
Phospholipase D ◽  
Cultured Cells ◽  
Human Neutrophils ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Mammalian Tissues ◽  
Radioactive Concentration ◽  
Adp Ribosylation Factor ◽  
Cytosolic Factor ◽  
Assay Conditions

Phospholipase D (PLD) activity in crude or solubilized membranes from mammalian tissues is difficult to detect with the current assay techniques, unless a high radioactive concentration of substrate and/or long incubation times are employed. Generally, the enzyme has to be extracted and partially purified on one column before easy detection of activity. Furthermore, PLD activity in cultured cells can only be detected by the available assay techniques in the presence of guanosine 5´-[γ-thio]triphosphate (GTP[S]) and a cytosolic factor [usually ADP-ribosylation factor (Arf)]. In this paper we report that the use of didecanoyl phosphatidylcholine (C10-PC) in mammalian PLD assays considerably increases the detection limit. C10-PC was compared with the commonly used dipalmitoyl phosphatidylcholine (C16-PC) as a substrate for PLD activity from membranes of human neutrophils, human placenta and pig brain, and from placental cytosol. C10-PC was superior to C16-PC by a factor of 2–28 depending on assay conditions and tissue, and it allowed the detection of GTP[S]-and Arf-stimulated PLD activity without addition of phosphatidylinositol 4,5-bisphosphate.

scholarly journals ADP-ribosylation-factor-regulated phospholipase D activity localizes to secretory vesicles and mobilizes to the plasma membrane following N-formylmethionyl-leucyl-phenylalanine stimulation of human neutrophils

1997 ◽  
Vol 325 (3) ◽  
pp. 581-585 ◽  
Author(s):  
C. P. MORGAN ◽  
H. SENGELOV ◽  
J. WHATMORE ◽  
N. BORREGAARD ◽  
S. COCKCROFT
Keyword(s):  
Plasma Membrane ◽  
Phospholipase D ◽  
Small Gtpases ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Rho Proteins ◽  
Adp Ribosylation ◽  
Activated Neutrophils ◽  
Adp Ribosylation Factor ◽  
Stimulation Of

Phospholipase D (PLD) is responsible for the hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. Human neutrophils contain PLD activity which is regulated by the small GTPases, ADP-ribosylation factor (ARF) and Rho proteins. In this study we have examined the subcellular localization of the ARF-regulated PLD activity in non-activated neutrophils and cells ‘primed‘ with N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe). We report that PLD activity is localized at the secretory vesicles in control cells and is mobilized to the plasma membrane upon stimulation with fMetLeuPhe. We conclude that the ARF-regulated PLD activity is translocated to the plasma membrane by secretory vesicles upon stimulation of neutrophils with fMetLeuPhe in inflammatory/priming doses. We propose that this relocalization of PLD is important for the subsequent events occurring during neutrophil activation.

scholarly journals ADP-ribosylation Factor Functions Synergistically with a 50-kDa Cytosolic Factor in Cell-free Activation of Human Neutrophil Phospholipase D

1995 ◽  
Vol 270 (6) ◽  
pp. 2431-2434 ◽  
Author(s):  
J. David Lambeth ◽  
Jong-Young Kwak ◽  
Edward P. Bowman ◽  
David Perry ◽  
David J. Uhlinger ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Human Neutrophil ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor ◽  
Cytosolic Factor

scholarly journals ADP-ribosylation Factor and Rho Proteins Mediate fMLP-dependent Activation of Phospholipase D in Human Neutrophils

1998 ◽  
Vol 273 (21) ◽  
pp. 13157-13164 ◽  
Author(s):  
Amanda Fensome ◽  
Jacqueline Whatmore ◽  
Clive Morgan ◽  
David Jones ◽  
Shamshad Cockcroft
Keyword(s):  
Phospholipase D ◽  
Human Neutrophils ◽  
Rho Proteins ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Change in mutation frequency at a TP53 hotspot during culture of ENU-mutagenised human lymphoblastoid cells

Mutagenesis ◽  
2019 ◽  
Author(s):  
Masahiko Watanabe ◽  
Masae Toudou ◽  
Taeko Uchida ◽  
Misato Yoshikawa ◽  
Hiroaki Aso ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
Mutation Frequency ◽  
Cultured Cells ◽  
Tp53 Gene ◽  
Tumour Suppressor Genes ◽  
Restriction Sites ◽  
Mutation Spectra ◽  
Risk Of Cancer ◽  
Assay Conditions

Abstract Mutations in oncogenes or tumour suppressor genes cause increases in cell growth capacity. In some cases, fully malignant cancer cells develop after additional mutations occur in initially mutated cells. In such instances, the risk of cancer would increase in response to growth of these initially mutated cells. To ascertain whether such a situation might occur in cultured cells, three independent cultures of human lymphoblastoid GM00130 cells were treated with N-ethyl-N-nitrosourea to induce mutations, and the cells were maintained for 12 weeks. Mutant frequencies and spectra of the cells at the MspI and HaeIII restriction sites located at codons 247–250 of the TP53 gene were examined. Mutant frequencies at both sites in the gene exhibited a declining trend during cell culture and reached background levels after 12 weeks; this was also supported by mutation spectra findings. These results indicate that the mutations detected under our assay conditions are disadvantageous to cell growth.

scholarly journals MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

2018 ◽  
Vol 116 (1) ◽  
pp. 303-312 ◽  
Author(s):  
Erol C. Bayraktar ◽  
Lou Baudrier ◽  
Ceren Özerdem ◽  
Caroline A. Lewis ◽  
Sze Ham Chan ◽  
...  
Keyword(s):  
Metabolite Profiling ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Cell Types ◽  
Tissue Level ◽  
Specific Cell ◽  
Mammalian Tissues ◽  
Cell Type Specific ◽  
Untargeted Metabolite Profiling

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

scholarly journals MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

2018 ◽  
Author(s):  
Erol Can Bayraktar ◽  
Lou Baudrier ◽  
Ceren Özerdem ◽  
Caroline A. Lewis ◽  
Sze Ham Chan ◽  
...  
Keyword(s):  
Metabolite Profiling ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Cell Types ◽  
Tissue Level ◽  
Specific Cell ◽  
Mammalian Tissues ◽  
Cell Type Specific ◽  
Untargeted Metabolite Profiling

ABSTRACTMitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell-types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag (“MITO-Tag”) for the fast isolation of mitochondria from cultured cells to now generate “MITO-Tag Mice.” Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

scholarly journals Monosodium urate-crystal-stimulated phospholipase D in human neutrophils

1999 ◽  
Vol 337 (2) ◽  
pp. 185 ◽  
Author(s):  
Josée MARCIL ◽  
Danielle HARBOUR ◽  
Martin G. HOULE ◽  
Paul H. NACCACHE ◽  
Sylvain BOURGOIN
Keyword(s):  
Phospholipase D ◽  
Human Neutrophils ◽  
Monosodium Urate ◽  
Urate Crystal

Simple Thermodynamically-Derived Model for Predicting the Hydrolase and Transferase Activity of Phospholipase D in the Synthesis of Phosphatidylglycerol

2012 ◽  
Vol 5 ◽  
pp. LPI.S8376
Author(s):  
Andrew Nair ◽  
Shahidan Radiman ◽  
Mamot Said
Keyword(s):  
Phospholipase D ◽  
Lung Surfactant ◽  
Entropy Change ◽  
Transferase Activity ◽  
Surface Response Methodology ◽  
Streptomyces Species ◽  
Dipalmitoyl Phosphatidylcholine ◽  
The Difference ◽  
Layer Chromatography ◽  
Enzyme Source

Preparation of a single and pure phospholipid via transphosphatidylation has been a much sought after endeavor in the pharmaceutical and nonmedical industries. For this reason, phosphatidylglycerol, a lung surfactant, was produced from phosphatidylcholine with defined fatty acids, ie, dipalmitoyl phosphatidylcholine. Substrate type and concentration, enzyme source, and reaction temperature were investigated. Phospholipase D from two sources, ie, savoy cabbage, was purified in the authors’ laboratory and a commercially available Streptomyces species was used for this study. The substrates used were glycerol, a polyhydric alcohol, and solketal, a monohydric form of glycerol. The progress of the reaction was monitored using thin layer chromatography, and synthesis with solketal, an unusual form of glycerol, was confirmed by liquid chromatography mass spectrometry. Surface response methodology used on four combinations of enzyme and substrate at various temperatures (30 °C–60 °C) and concentration (0.25–1 mM) revealed that yield and selectivity was temperature-driven and predictable. To validate further the thermodynamic attributes, a modified version of the Eyring equation was derived from selectivity and the Arhenius equation. These equations provide some useful insights into the difference in activation of enthalpy change(ΔΔH++) and difference in activation of entropy change(ΔΔS++). Plots of ln[PG]/[PA] versus 1/T gave good linear fits for these four combinations. In addition, a new thermodynamic parameter known as T PG = PA has emerged as a theoretical temperature for equivalent transferase and hydrolase activity.

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