concanavalin-a and benzidine

1. Human lung lipoxygenase (HLLO) was partially purified by concanavalin-A (Con-A) affinity chromatography that provided an easy and rapid one-step procedure for the removal (> or = 96%) of haemoglobin from cytosol. 2. HLLO exhibited dioxygenase activity towards arachidonic acid (AA) and linoleic acid (LA). The dioxygenase activity towards LA varied approximately 12-fold (48-591 nmol/min/mg protein) among different human lung samples examined. 3. Reverse-phase HPLC analysis of AA metabolites indicated the predominance of 15-lipoxygenase in human lung cytosol. 4. HLLO exhibited co-oxidase activity towards benzidine (BZD) and several other model compounds. The co-oxidase activity towards BZD was significantly inhibited by several lipoxygenase inhibitors. 5. HLLO and soybean lipoxygenase (SLO), used as a model enzyme, metabolized acrylonitrile (ACN) to 2-cyanoethylene oxide (CEO) and ultimately to cyanide. 6. HLLO was a approximately 6-fold better catalyst than SLO in converting ACN to cyanide. The generation of cyanide by HLLO was dependent on the concentration of enzyme and the reaction was inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the anti-oxidant butylated hydroxytoluene (BHT). 7. Under optimal assay conditions, the covalent binding of HLLO-generated reactive intermediate(s) from [14C]ACN to protein and DNA (nmol equivalent bound/15 min/mg HLLO/mg bovine serum albumin or calf thymus DNA) was observed at approximately 1.20+/-0.13 and 2.20+/-0.50 respectively. Both protein and DNA binding were inhibited by NDGA, butylated hydroxyanisole (BHA) and BHT.

Topics: Acrylonitrile; Animals; Antioxidants; Benzidines; Butylated Hydroxytoluene; Cattle; Chromatography, Affinity; Concanavalin A; Cyanides; Cytosol; DNA; Glycine max; Humans; Lipoxygenase; Lipoxygenase Inhibitors; Lung; Masoprocol; Oxidation-Reduction; Plant Lectins; Radiometry; Serum Albumin, Bovine

The evidence presented here constitutes the first report on the occurrence of lipoxygenase (LO) activity in the adult human liver. LO activity was isolated free of hemoglobin from the whole liver cytosol by affinity chromatography using a concanavalin-A sepharose 4B column, and some properties of its dioxygenase and co-oxidase activities were examined. High-pressure liquid chromatography (HPLC) analyses of arachidonic acid metabolites suggested the presence of 5-, 12-, and 15-LO activities in the human liver. Linoleic acid was converted into 13-hydroperoxyoctadecadienoic acid. The dioxygenase activity with a Vmax value of 1.74 mumoles/min/mg protein and a Km value of 0.48 mM was noted in the presence of different concentrations of linoleic acid at pH 10. The activity was markedly stimulated by the presence of calcium, ATP, hydrogen peroxide, and KCl in the assay medium. Under optimum conditions, all the xenobiotics tested were cooxidized by the enzyme preparations in the presence of linoleic acid. Kinetic data obtained for benzidine oxidation yielded a Km value of 0.53 mM and a Vmax value of 90.9 nmoles/min/mg protein. At present, the significance of these findings in in vivo toxicity of benzidine is unknown. The linoleic acid-dependent dioxygenase and co-oxidase activities were thermolabile and inhibited by micromolar concentrations of several classical LO inhibitors, further confirming the involvement of LO in these reactions.

Topics: Adenosine Triphosphate; Adult; Arachidonic Acid; Benzidines; Calcium; Chromatography, Affinity; Chromatography, High Pressure Liquid; Concanavalin A; Cytosol; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Female; Hemoglobins; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Linoleic Acid; Linoleic Acids; Lipoxygenase; Liver; Male; Middle Aged; Oxidation-Reduction; Potassium Chloride; Xenobiotics