Interfacial catalysis and production of a high ratio of leukotriene A4 to 5-HPETE by 5-lipoxygenase in a coupled assay with phospholipase A2.
The activity of purified 5-lipoxygenases (5-LO) shows a requirement for the presence of phosphatidylcholine or other lipids, in addition to Ca2+ and ATP. The enzymatic activity of purified human 5-lipoxygenase was dependent on the ratio of arachidonic acid to phospholipids rather than on the bulk arachidonic acid concentration, suggesting that the concentration of substrate at the lipid-water interface is important for the rate of the 5-LO reaction. Enzyme activity was also examined using vesicles of dimyristoylphosphatidylmethanol/1-palmitoyl-2-arachidonoylphosp hat idylcholine. Using PLA2 to release arachidonic acid from phospholipid, the ratio of leukotriene A4 (detected as trans-LTB4 isomers) to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) accumulated depended on the 5-LO concentration and was relatively independent of the amount of PLA2. The ratio of leukotriene A4 to 5-HPETE production increased with the amount of 5-LO (1-15 micrograms/ml) to reach values (> 10) similar to those observed with ionophore-challenged human leukocytes. The data are consistent with the catalysis of 5-LO occurring at the surface of phospholipid vesicles with the 5-HPETE product being re-utilized by the LTA4 synthase reaction of 5-lipoxygenase under conditions of limiting arachidonic acid availability.
Riendeau
D
D
Biochemistry Department, Merck Frosst Canada Inc., Pointe Claire-Dorval, Québec.
Falgueyret
J P
JP
Meisner
D
D
Sherman
M M
MM
Laliberté
F
F
Street
I P
IP
D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D016718:Arachidonic Acid / Q000494:pharmacology; D002384:Catalysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D017572:Leukotriene A4; D015289:Leukotrienes / Q000096:biosynthesis / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2
D017572:Leukotriene A4; D015289:Leukotrienes; D010713:Phosphatidylcholines; D016718:Arachidonic Acid; C025348:arachidonic acid 5-hydroperoxide; D001094:Arachidonate 5-Lipoxygenase; D010741:Phospholipases A; D054467:Phospholipases A2