Overexpression, purification and characterization of human recombinant 15-lipoxygenase.
Human 15-lipoxygenase was expressed to high levels (approx. 20% of cellular protein) in a baculovirus/insect cell expression system. Catalytically active enzyme was readily purified (90-95% pure) from cytosolic fractions by anion-exchange chromatography on a Mono Q column with approx. 95% recovery of enzymatic activity. Routinely, a yield of 25-50 mg of pure enzyme per L of culture and a specific activity of 7.1-21 mumol 13-hydroxyoctadecadienoic acid (13-HODE)/mg.min (turnover rate of 8.4-25 s-1) were obtained. Both the specific activity and the enzyme's iron content was significantly increased by the addition of ferrous ions to either the purified enzyme or to the insect cell culture medium during production. An isoelectric point of 5.85 was determined and the N-terminal amino acid sequence was found to be identical to that predicted from the cDNA. The purified recombinant enzyme exhibits a dual positional specificity with arachidonic acid (formation of 15S- and 12S-hydroxyeicosatetraenoic acid (12S-HETE) in a ratio of 12:1). Double oxygenation products 14R,15S- and various 8,15-DiHETE isomers were also identified. With linoleic acid as substrate, a pH-optimum of 7.0 and a KM of 3 microM were determined. The enzyme undergoes suicidal inactivation during fatty acid oxygenation, is sensitive to standard lipoxygenase inhibitors, and oxygenates phospholipids, cholesterol esters, biomembranes and human low-density lipoprotein. Contrary to prior studies on the rabbit enzyme, no glycosylation was detected.
Kühn
H
H
Cardiovascular Research Institute, University of California, San Francisco.
Barnett
J
J
Grunberger
D
D
Baecker
P
P
Chow
J
J
Nguyen
B
B
Bursztyn-Pettegrew
H
H
Chan
H
H
Sigal
E
E
D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D016367:Baculoviridae / Q000201:enzymology; D002417:Cattle; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta / Q000382:microbiology; D015289:Leukotrienes / Q000378:metabolism; D019787:Linoleic Acid; D008041:Linoleic Acids / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D015289:Leukotrienes; D008041:Linoleic Acids; D008054:Lipid Peroxides; D008077:Lipoproteins, LDL; D011994:Recombinant Proteins; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D019787:Linoleic Acid; D001093:Arachidonate 15-Lipoxygenase