Arachidonate 5-lipoxygenase of porcine leukocytes studied by enzyme immunoassay using monoclonal antibodies.
The cytosol fraction of porcine leukocytes contained 5-lipoxygenase, the activity of which was masked by a predominant activity of 12-lipoxygenase. The 5-lipoxygenase was partially purified to a specific activity of about 10 nmol of arachidonic acid oxygenated/min/mg of protein and given to mice as an antigen to prepare monoclonal antibodies against the enzyme. Two species of antibodies recognized separate sites of the 5-lipoxygenase protein and did not cross-react with 12-lipoxygenase. They were utilized to develop a peroxidase-linked immunoassay of sandwich-type, which allowed a quantitative determination of the 5-lipoxygenase protein. The assay was applied to a screening of the 5-lipoxygenase content in various porcine tissues. By far the highest content of 5-lipoxygenase was found in leukocytes. About one-tenth the amount of the enzyme was found in lung, pancreas, ileum, and thymus, which could not be attributed to the contaminating leukocytes in these tissues.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D001094:Arachidonate 5-Lipoxygenase / Q000097:blood / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D007124:Immunoenzyme Techniques; D007962:Leukocytes / Q000201:enzymology*; D051379:Mice; D008809:Mice, Inbred C3H; D013552:Swine; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase