Loss of cytosolic 15-lipoxygenase activity in A23187-stimulated human leukocytes: involvement of a translocation process?
We focused our study on the subcellular redistribution of 15-lipoxygenase in human leukocytes challenged with A23187 and arachidonic acid (AA). We found that in cytosolic fractions of stimulated cells the 15-lipoxygenase (15-LO) activity, measured as 15-HETE, was 65% less than in controls. However, no activity was found in cell membranes. This effect was complete within 2 min of incubation and was correlated in a dose dependent manner to exogenously added AA. No significant difference in cytosolic distribution of 15-LO activity was observed when cells were stimulated in presence of various concentrations of Ca++. Immunoblot analysis showed that the loss of cytosolic 15-LO activity registered after challenging with A23187 was associated with a concomitant loss of the enzyme content in the cytosol, suggesting the possibility of a translocation process. Neither the 15-LO activity nor the enzyme was, however, found in the cell membrane under our present experimental conditions. But, addition of protease inhibitors showed a slight increase of 15-LO activity in the membrane fraction. Despite the small effect, this may indicate a translocation of 15-LO following challenge of human leukocytes with A23187.
Bongrazio
M
M
Department of Gynecology, Free University Berlin, Federal Republic of Germany.
Roscher
A
A
Ludwig
P
P
Nigam
S
S
D000328:Adult; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D001692:Biological Transport; D000001:Calcimycin / Q000494:pharmacology*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology*; D006801:Humans; D015151:Immunoblotting; D007962:Leukocytes / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D016859:Lipoxygenase Inhibitors
D016859:Lipoxygenase Inhibitors; D016718:Arachidonic Acid; D000001:Calcimycin; D001093:Arachidonate 15-Lipoxygenase