A chemiluminescence method to analyze phosphatidylcholine-phospholipase activity in plasma membrane preparations and in intact cells.
We present a new method to analyze the hydrolysis of phosphatidylcholine by either phospholipase C (PLC) or phospholipase D. The method is nonradioactive, rapid, and very sensitive and is based on chemiluminescence. It relies on the peroxidase-catalyzed chemiluminescent oxidation of luminol by the H2O2 derived from choline oxidation. The enzyme activity can be quantified by calculation of produced choline in a standard curve. Data are accurate and reproducible in a large range of choline concentration. In fact, the response of PLC to GTP in plasma membranes was similar to the activity measured with radioactive methods of diacylglycerol or phosphatidylinositol triphosphate determination. The method can be applied to study the hydrolysis of phosphatidylcholine in plasma membrane preparations and in intact cells.
Lucas
M
M
Department of Medical Biochemistry and Molecular Biology, Virgen Macarena Hospital, Medical School of Sevilla, Spain.
Sänchez-Margalet
V
V
Pedrera
C
C
Bellido
M L
ML
D000818:Animals; D001782:Blood Donors; D000001:Calcimycin / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D016014:Linear Models; D008163:Luminescent Measurements; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000819:agonists / Q000097:blood*; D051381:Rats; D012680:Sensitivity and Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000819:agonists / Q000097:blood*
D010713:Phosphatidylcholines; D000001:Calcimycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010738:Type C Phospholipases; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate