Fluorimetric assay of phospholipase A acting on biomembrane phospholipids.
A sensitive phospholipase A assay suitable for organelle activities is described. Activation of the enzyme produces an increase in membrane lysophospholipids. The amino groups of extracted lipids were derivatized with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and the amounts of phosphatidylethanolamine and its lysoform were quantitated using HPLC equipped with a SiO2 column and a fluorescence detector. The procedure was tested with porcine enzyme acting on liposomes and mitochondria, and with endogenous mitochondrial enzyme.
Saris
N E
NE
Department of Medical Chemistry, University of Helsinki, Finland.
Somerharju
P
P
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004530:Egg Yolk / Q000032:analysis; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010741:Phospholipases A / Q000032:analysis* / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013552:Swine
D005456:Fluorescent Dyes; D008244:Lysophosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D010741:Phospholipases A; D009327:4-Chloro-7-nitrobenzofurazan