A continuous fluorescence assay for lecithin cholesterol acyltransferase.
A continuous fluorescence assay was adapted to the measurement of the phospholipase reaction of lecithin cholesterol acyltransferase (LCAT). The fluorescent phospholipid 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl phosphatidylcholine (C6-NBD-PC) in micelle form reacted with LCAT to yield NBD-caproic acid, resulting in up to 5-fold increases in fluorescence in 30 min. The reaction rates were optimal in mixtures containing 0.1 M NaCl and 4 mM beta-mercaptoethanol at 37 degrees C. Apolipoprotein A-I did not activate the enzyme and bovine serum albumin bound monomeric substrate and interfered with the fluorescence assay. Under similar reaction conditions, bee venom phospholipase A2 was almost 100-fold more reactive than LCAT.
Bonelli
F S
FS
Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois 61801.
Kézdy
K E
KE
Jonas
A
A
D006801:Humans; D007700:Kinetics; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000032:analysis*; D010740:Phospholipases / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods
D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D010740:Phospholipases