Citation

Shen K, Herman CA. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2000; 127(4): 563-573.

Copyright

(Copyright © 2000, Elsevier Publishing)

DOI

10.1016/s0305-0491(00)00288-1

PMID

11281273

Abstract

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.

Language: en

Keywords

Animals; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Chromatography, Thin Layer; Erythrocytes; Hemoglobins; Hydrogen-Ion Concentration; Isoelectric Focusing; Linoleic Acids; Lipid Peroxides; Molecular Weight; Rana catesbeiana; Subcellular Fractions; Substrate Specificity; Temperature