Characterization of the activity of purified recombinant human 5-lipoxygenase in the absence and presence of leukocyte factors.
Purified recombinant human 5-lipoxygenase was used to investigate the catalytic properties of the protein in the presence and absence of leukocyte stimulatory factors. Recombinant human 5-lipoxygenase was purified to apparent homogeneity (95-99%) from a high expression baculovirus system by chromatography on ATP-agarose with a yield of 0.6 mg of protein per 100 ml of culture (2 x 10(8) cells) and a specific activity of 3-6 mumol of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) per mg of protein in the presence of ATP, Ca2+, and phosphatidylcholine as the only factors. In the absence of leukocyte factors, the reaction catalyzed by the purified recombinant enzyme showed a half-time of maximal 5-HPETE formation of 0.5-0.7 min and was sensitive to the selective 5-lipoxygenase inhibitors BW755C (IC50 = 13 microM) and L-656,224 (IC50 = 0.8 microM). The reaction products of arachidonic acid oxidation were 5-HPETE and 6-trans- and 12-epi-6-trans-leukotriene B4, the nonenzymatic hydrolysis products of leukotriene A4 (LTA4), indicating that the purified protein expressed both the 5-oxygenase and leukotriene A4 synthase activities (ratio 6:1). The microsomal fraction and the 60-90% ammonium sulfate precipitate fraction from sonicated human leukocytes did not increase product formation by the isolated enzyme when assayed in the presence of ATP, Ca2+, and phosphatidylcholine. These factors were found to stabilize 5-lipoxygenase during preincubation of the enzyme at 37 degrees C with the assay mixture but they failed to stimulate enzymatic activity when added at the end of the preincubation period. The results demonstrate that human 5-lipoxygenase can be isolated in a catalytically active form and that protein factors from leukocytes protect against enzyme inactivation but are not essential for enzyme activity.
Denis
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D
Department of Biochemistry, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
Falgueyret
J P
JP
Riendeau
D
D
Abramovitz
M
M
D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D001798:Blood Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002384:Catalysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism*; D017572:Leukotriene A4; D015289:Leukotrienes / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D001798:Blood Proteins; D017572:Leukotriene A4; D015289:Leukotrienes; D011994:Recombinant Proteins; C025348:arachidonic acid 5-hydroperoxide; D001094:Arachidonate 5-Lipoxygenase