Studies on porcine arachidonate 12-lipoxygenase using its monoclonal antibodies.
Monoclonal antibodies were raised against arachidonate 12-lipoxygenase using a partially purified enzyme from porcine leukocytes as an antigen. Immunohistochemical studies indicated a selective localization of 12-lipoxygenase in the cytosol of polymorphonuclear leukocytes. Two separate species of antibody (lox-1 and lox-2) recognizing different sites of the enzyme protein were utilized to develop a method to determine the amount of 12-lipoxygenase protein rather than the activity of enzyme. Fab fragment of lox-2 was conjugated to horseradish peroxidase, and the conjugate as a label was bound to 12-lipoxygenase. The complex was precipitated with the aid of the other antibody (lox-1) and protein A, and the peroxidase activity in the precipitate was correlated with the amount of 12-lipoxygenase. The immunoenzymometry of 12-lipoxygenase was more convenient and sensitive than the conventional assay to determine the conversion of [1-14C]arachidonic acid. Furthermore, in several porcine tissues this method allowed quantitation of the enzyme, the activity of which was masked due to the presence of certain endogenous inhibitors. A ubiquitous distribution of 12-lipoxygenase in porcine tissues was demonstrated by application of this method.
Shinjo
F
F
Yoshimoto
T
T
Yokoyama
C
C
Yamamoto
S
S
Izumi
S
S
Komatsu
N
N
Watanabe
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001091:Arachidonate Lipoxygenases; D006735:Horseradish Peroxidase / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments; D008084:Lipoxygenase / Q000276:immunology / Q000378:metabolism*; D008854:Microscopy, Electron; D009000:Monocytes / Q000201:enzymology; D013552:Swine; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D006735:Horseradish Peroxidase; D001091:Arachidonate Lipoxygenases; D008084:Lipoxygenase