Optimization of cofactors which regulate RBL-1 arachidonate 5-lipoxygenase.
The arachidonate lipoxygenase from rat basophilic leukemia cells (RBL-1) is widely utilized as a model to dissect the primary enzymatic reactions leading to leukotriene formation. The purpose of the present study was to optimize the specific activity of 5-lipoxygenase prepared from a high speed supernatant of RBL-1 cell homogenates. Activation of 5-lipoxygenase was observed in the presence of micromolar levels of calcium. A synergistic enhancement of 5-lipoxygenase was observed upon addition of equally low levels of ATP; maximal activation was induced by 5 microM CaCl2 plus 5 microM ATP. Addition of a microsomal-membrane preparation and NADPH further augmented 5-HETE biosynthesis. High concentrations (330 microM) of NADPH reversed the microsomal-induced stimulation of RBL-1 5-lipoxygenase, resulting in enzyme inhibition.
Cochran
F R
FR
Department of Allergy and Inflammation, Hoffmann-LaRoche, Nutley, NJ 07110.
Finch-Arietta
M B
MB
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D002460:Cell Line; D006706:Homeostasis; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D051381:Rats
D006893:Hydroxyeicosatetraenoic Acids; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D009249:NADP; D000255:Adenosine Triphosphate; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase; D002122:Calcium Chloride