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.
Shen
K
K
Department of Biology, New Mexico State University, Las Cruces 88003, USA. keqiang.shen@dupontpharma.com
Herman
C A
CA
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