Partial purification and characterization of 12-lipoxygenase in bullfrog erythrocytes.
12-Lipoxygenase (12-LO) in bullfrog (Rana catesbeiana) erythrocytes was purified partially by ion exchange chromatography and affinity chromatography. Bullfrog 12-LO was a single chain protein with a pI of 7.1-7.8 and MW of 7.77 kDa. This enzyme did not show typical Michaelis Menten type kinetics. At low substrate concentrations, it had a lag phase and at higher substrate concentrations, the activity was inhibited. The product of linoleic acid (LA), 13-hydroperoxy-9, 11-octadecadienoic acid (13-HpODE), was an activator for the enzyme. When arachidonic acid (AA) was used as substrate, 13-HpODE also affected the Km of bullfrog 12-LO towards AA. The affinity of LA towards bullfrog 12-LO was higher than the affinity of AA. Suicide inactivation was much more rapid than that of any mammalian 12-LO reported. Hemoglobin (Hb) inhibited the activity of 12-LO partially and removing Hb eliminated this inhibition. Both Hb and Met-Hb inhibited the 12-LO activity but did not denatured completely the Hb, suggesting that the inhibition was a direct interaction between 12-LO and Hb protein chain and was not due to competition between 12-LO and Hb for oxygen. This study characterizes bullfrog 12-LO with respect to stability, optimal pH, suicide inactivation and interaction with Hb and provides important evolutionary information about this enzyme.
Department of Biology, New Mexico State University, Las Cruces 88003, USA. keqiang.shen@dupontpharma.com
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D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000032:analysis* / Q000302:isolation & purification*; D016718:Arachidonic Acid / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D004912:Erythrocytes / Q000201:enzymology*; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008041:Linoleic Acids / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism; D008970:Molecular Weight; D011892:Rana catesbeiana / Q000097:blood*; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D006454:Hemoglobins; D008041:Linoleic Acids; D008054:Lipid Peroxides; C038649:13-hydroperoxy-9,11-octadecadienoic acid; D016718:Arachidonic Acid; D001092:Arachidonate 12-Lipoxygenase