Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187.

C A Rouzer,S Kargman

doi:10.1016/s0021-9258(18)38066-9

Abstract

Challenge of human peripheral blood leukocytes with ionophore A23187 resulted in leukotriene (LT) synthesis, a decrease in total cellular 5-lipoxygenase activity, and a change in the subcellular localization of the enzyme. In homogenates from control cells, greater than 90% of the 5-lipoxygenase activity and protein was localized in the cytosol (100,000 X g supernatant). Ionophore challenge (2 microM) resulted in a loss of approximately 55% of the enzymatic activity and 35% of the enzyme protein from the cytosol. Concomitantly, there was an accumulation of inactive 5-lipoxygenase in the membrane (100,000 X g pellets) which accounted for at least 45% of the lost cytosolic protein. There was a good correlation between the quantities of LT synthesized and 5-lipoxygenase recovered in the membrane over an ionophore concentration range of 0.1-6 microM. The time course of the membrane association was similar to that of LT synthesis. Furthermore, although the pellet-associated enzyme recovered from ionophore-treated leukocytes was inactive, an irreversible, Ca2+-dependent membrane association of active 5-lipoxygenase could be demonstrated in cell-free systems. To determine whether ionophore treatment induced proteolytic degradation of 5-lipoxygenase, the total activity and protein content of 10,000 X g supernatants from control and ionophore-treated cells were examined. These supernatants, which included both cytosolic and membrane-associated enzyme, showed a 35% loss of 5-lipoxygenase activity but only an 8% loss of enzyme protein as a result of ionophore challenge (2 microM). Therefore, the majority of the loss of 5-lipoxygenase activity was most likely due to suicide inactivation during the LT synthesis, rather than to proteolytic degradation. Together these results are consistent with the hypothesis that ionophore treatment results in a Ca2+-dependent translocation of 5-lipoxygenase from the cytosol to a membrane-bound site, that the membrane-associated enzyme is preferentially utilized for LT synthesis, and that it is consequently inactivated. Thus, membrane translocation of 5-lipoxygenase may be an important initial step in the chain of events leading to full activation of this enzyme in the intact leukocyte.

Highlights

The time course of the membrane association was sim- sophilic leukemia cells [2,5,6]
Effect of Ionophore Stimulation on LT Synthesis, and the Subcellular Localization of 5-Lipoxygenase Activity-Leukocyte suspensions were challenged for 10 min at 37 "C with 2 ~ L Mionophore
In the present studies we have attempted to ascertain the fate of 5-lipoxygenase in leukocytes challenged with ionophore A23187

Summary

Introduction

The time course of the membrane association was sim- sophilic leukemia cells [2,5,6]. Pellet-associated enzyme recovered from ionophore- the human enzyme was stimulated by threedistinct treated leukocytes was inactive,an irreversible, Cas+- protein fractionsfrom the leukocytes, one of which was memdependent membrane association of active 5-lipoxy- brane-associated [5, 7] These findings suggested a genase could be demonstrated in cell-free systems. Solic and membrane-associated enzyme, showed a 35% When subcellular fractions of human leukocyte homogeloss of 5-lipoxygenase activity but only an 8%loss of enzyme protein as a result of ionophore challenge (2 p ~ )T.herefore, the majority of the lossof B-lipoxygenase activity wasmost likely due to suicide inactivation during the LT synthesis, rather than to proteolytic degradation. Membrane translocation of 5-lipoxygenase may homology with the lipid interfacial binding sites of pancreatic be animportantinitialstep in the chain of events lipase and lipoprotein lipase [9].Third, 5-lipoxygenase activleading to full activation of this enzyme in the intact ity in cell-free systems is stimulated by phosphatidylcholine leukocyte

Methods

Results

Conclusion