Analysis of leukotriene B4 metabolism in human promyelocytic HL-60 cells.
We previously reported that human alveolar macrophages rapidly metabolize the chemotactic active lipid mediator leukotriene B4 (LTB4) into the dihydro-LTB4 by reduction of one of the conjugated double bonds. We herein report that human HL-60 cells (a myeloid precursor which can be differentiated into granulocyte- as well as monocyte-like cells by dimethyl sulphoxide or phorbol myristate acetate) express a highly active LTB4 reductase in the undifferentiated state. Differentiation by dimethyl sulphoxide (1.3%) along the granulocyte lineage, as confirmed by light microscopy, conversion of NitroBlue Tetrazolium into formazan, failed to induce a substantial capacity for omega-oxidation of LTB4; this reaction is exclusively found in mature granulocytes. Studies with the cell homogenate of undifferentiated HL-60 cells indicated that the activity of the enzyme depends on the presence of NADPH, Ca2+ and Mg2+, with a pH optimum of 7.5 at 37 degrees C. The enzyme was not released into the supernatant after stimulation of HL-60 cells with phorbol myristate acetate (100 ng) or Ca2+ ionophore (7.5 microM). Subcellular fractionation revealed evidence that the LTB4 reductase is located within the membrane fraction. Purification of the enzyme by gel filtration and gel electrophoresis suggests an apparent molecular mass of 40 kDa.
Kasimir
S
S
Ag Infektabwehrmechanismen, Med. Mikrobiologie und Immunologie, Ruhr Universität Bochum, Federal Republic of Germany.
Schönfeld
W
W
Hilger
R A
RA
König
W
W
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000201:enzymology / Q000378:metabolism*; D007975:Leukotriene B4 / Q000378:metabolism*; D008163:Luminescent Measurements; D009580:Nitroblue Tetrazolium; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007975:Leukotriene B4; D009580:Nitroblue Tetrazolium; D010088:Oxidoreductases; C071224:leukotriene B4 reductase; D013755:Tetradecanoylphorbol Acetate