Characterization of leukotriene A4 and B4 biosynthesis.
We have studied LTA4 and LTB4 synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB4 from arachidonic acid are localized in the soluble fraction (100,000 x g supernatant) of these cells. The formation of LTA4 and LTB4 is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 microM, the synthesis of LTB4 leveled off at 30 microM and of LTA4 at 100 microM while 5-HETE had not reached a plateau at 300 microM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA4 and only a relatively small amount of LTB4. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA4 to LTB4. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA4 synthesis did not produce any additional LTA4 or LTB4. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.
D000818:Animals; D001091:Arachidonate Lipoxygenases; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000096:biosynthesis* / Q000378:metabolism; D002478:Cells, Cultured; D006893:Hydroxyeicosatetraenoic Acids; D007700:Kinetics; D007942:Leukemia, Experimental / Q000378:metabolism; D017572:Leukotriene A4; D007975:Leukotriene B4 / Q000096:biosynthesis*; D008084:Lipoxygenase / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors
D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D017572:Leukotriene A4; D007975:Leukotriene B4; D016718:Arachidonic Acid; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D001091:Arachidonate Lipoxygenases; D008084:Lipoxygenase