Kinetic studies on arachidonate 5-lipoxygenase from rat basophilic leukemia cells.
Arachidonate 5-lipoxygenase of a 10,000 x g supernatant from RBL-1 cell homogenate was studied by a continuous assay measuring enzyme-catalysed oxygen consumption. Parallel HPLC and TLC analysis of arachidonic acid metabolites revealed that the oxygen consumption measured is solely due to 5-lipoxygenation of arachidonic acid. Oxygen consumption by this lipoxygenase was strictly dependent upon Ca2+, ATP and 5-HPETE. Removal of any of these three cofactors caused a complete inhibition of enzyme activity. Addition of the missing cofactor instantly restored the 5-lipoxygenase-dependent consumption of oxygen which remained linear for 10-20 s. Later on the velocity of the reaction decreased and after 2-3 min the enzyme became inactivated. Kinetic data were obtained from the initial velocity of the reaction using constant and saturating concentrations of CaCl2 and ATP. From Lineweaver-Burk plots substrate inhibition is evident for arachidonic acid concentrations greater than 45-50 microM. Km(app) for arachidonic acid is 182 +/- 16 microM (mean +/- SD, n = 5) and Vmax(app) is 425 +/- 140 nmol O2/(min x mg protein) (mean +/- SD, n = 5).
Haurand
M
M
Grünenthal GmbH, Center of Research, Aachen.
Flohé
L
L
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000378:metabolism*; D001095:Arachidonic Acids / Q000302:isolation & purification; D001491:Basophils / Q000201:enzymology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D007942:Leukemia, Experimental / Q000201:enzymology*; D010101:Oxygen Consumption; D051381:Rats
D001095:Arachidonic Acids; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase