Purification and partial characterization of lipoxygenase with dual catalytic activities from human term placenta.
Lipoxygenase possessing dual catalytic activities, i.e. dioxygenase and hydroperoxidase, was purified from the cytosols of term placentas from non-smoking women. Concanavalin A affinity chromatography followed by phenyl-Sepharose CL-4B chromatography resulted in the separation of one hydrophobic and one non-hydrophobic isoenzyme. The concanavalin A-purified enzyme was used in all subsequent experiments. The dioxygenase activity of the enzyme exhibited a Vmax. of 204.37 +/- 17.66 nmol/min per mg of protein and a Km of 0.79 mM for linoleic acid. The involvement of dioxygen in enzymic linoleic acid oxidation was confirmed by O2 uptake studies. Arachidonic acid and linolenic acid also served as substrates for the dioxygenase activity. The placental lipoxygenase co-oxidized benzidine in the presence of linoleic acid (hydroperoxidase activity). Both the dioxygenase and hydroperoxidase activities were significantly stimulated by Ca2+ (1-100 microM), ATP (10-400 nM) and H2O2 (1-10 nM). Similarly, these two activities were inhibited by nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid, gossypol, esculetin, butylated hydroxyanisole and butylated hydroxytoluene. Boiled enzyme was without significant dioxygenase and hydroperoxidase activities. Pyrogallol, 3,3'-dimethoxybenzidine, 3,3',5,5'-tetramethylbenzidine, tetramethylphenylenediamine and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) were also co-oxidized by the placental lipoxygenase. These results suggest that: (i) lipoxygenase from human term placenta exhibits both dioxygenase and hydroperoxidase activities, and (ii) this enzyme represents an important pathway for chemical oxidation in the placentas of non-smoking women.
Joseph
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P
Toxicology Program, College of Public Health, University of South Florida, Tampa 33612.
Srinivasan
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SN
Kulkarni
A P
AP
D002384:Catalysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008084:Lipoxygenase / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D015262:Xenobiotics / Q000737:chemistry
D015262:Xenobiotics; D008084:Lipoxygenase