A novel and sensitive assay for phospholipase D in intact cells

A novel and sensitive assay for phospholipase D (PLD) that measures the incorporation of high specific activity [ 3H]butan-1-ol into [ 3H]phosphatidylbutanol has been developed. The assay has been used to measure PLD activation in human neutrophils and platelets. Both the chemotactic peptide fMet-Le...

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Veröffentlicht in:	FEBS letters 1990-05, Vol.264 (1), p.87-90
Hauptverfasser:	Randall, Roger W., Bonser, Robert W., Thompson, Neil T., Garland, Lawrence G.
Format:	Artikel
Sprache:	eng
Schlagworte:	1-Butanol Analytical, structural and metabolic biochemistry Biological and medical sciences Blood Platelets - enzymology Butanols Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Humans Hydrolases Kinetics leukocytes (neutrophilic) N-Formylmethionine Leucyl-Phenylalanine - pharmacology Neutrophil Neutrophils - enzymology Opsonised-zymosan PAF, platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycerophosphorylcholine) PBut, phosphatidylbutanol Phospholipase D Phospholipase D - analysis Phospholipase D - blood Phospholipases - blood Platelet platelets PLD, phospholipase D Radioisotope Dilution Technique TLC, thin-layer chromatography Transphosphatidylation Tritium Zymosan - pharmacology
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Beschreibung
Zusammenfassung:	A novel and sensitive assay for phospholipase D (PLD) that measures the incorporation of high specific activity [ 3H]butan-1-ol into [ 3H]phosphatidylbutanol has been developed. The assay has been used to measure PLD activation in human neutrophils and platelets. Both the chemotactic peptide fMet-Leu-Phe and opsonised-zymosan stimulated PLD in the human neutrophil. In the platelet, PLD was stimulated by thrombin and collagen but responses were small and only occurred at high agonist concentrations. This assay has a number of advantages over existing techniques and should be valuable for investigating PLD activation in a variety of isolated cells and possibly intact tissues.
ISSN:	0014-5793 1873-3468
DOI:	10.1016/0014-5793(90)80772-B