PMID- 8334154
OWN - NLM
STAT- MEDLINE
DCOM- 19930824
LR  - 20230120
IS  - 0006-3002 (Print)
IS  - 0006-3002 (Linking)
VI  - 1169
IP  - 1
DP  - 1993 Jul 21
TI  - Overexpression, purification and characterization of human recombinant 
      15-lipoxygenase.
PG  - 80-9
AB  - 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.
FAU - Kuhn, H
AU  - Kuhn H
AD  - Cardiovascular Research Institute, University of California, San Francisco.
FAU - Barnett, J
AU  - Barnett J
FAU - Grunberger, D
AU  - Grunberger D
FAU - Baecker, P
AU  - Baecker P
FAU - Chow, J
AU  - Chow J
FAU - Nguyen, B
AU  - Nguyen B
FAU - Bursztyn-Pettegrew, H
AU  - Bursztyn-Pettegrew H
FAU - Chan, H
AU  - Chan H
FAU - Sigal, E
AU  - Sigal E
LA  - eng
GR  - HL-24136/HL/NHLBI NIH HHS/United States
GR  - R01 HL 48591/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - Netherlands
TA  - Biochim Biophys Acta
JT  - Biochimica et biophysica acta
JID - 0217513
RN  - 0 (Leukotrienes)
RN  - 0 (Linoleic Acids)
RN  - 0 (Lipid Peroxides)
RN  - 0 (Lipoproteins, LDL)
RN  - 0 (Recombinant Proteins)
RN  - 67675-14-3 (15-hydroperoxy-5,8,11,13-eicosatetraenoic acid)
RN  - 9KJL21T0QJ (Linoleic Acid)
RN  - EC 1.13.11.33 (Arachidonate 15-Lipoxygenase)
SB  - IM
MH  - Animals
MH  - Arachidonate 15-Lipoxygenase/*biosynthesis/chemistry/isolation & purification
MH  - Baculoviridae/enzymology
MH  - Cattle
MH  - Cell Line
MH  - Humans
MH  - Hydrogen-Ion Concentration
MH  - Insecta/microbiology
MH  - Leukotrienes/metabolism
MH  - Linoleic Acid
MH  - Linoleic Acids/metabolism
MH  - Lipid Peroxides/metabolism
MH  - Lipoproteins, LDL/metabolism
MH  - Rats
MH  - Recombinant Proteins/*biosynthesis/chemistry/isolation & purification
EDAT- 1993/07/21 00:00
MHDA- 1993/07/21 00:01
CRDT- 1993/07/21 00:00
PHST- 1993/07/21 00:00 [pubmed]
PHST- 1993/07/21 00:01 [medline]
PHST- 1993/07/21 00:00 [entrez]
AID - 0005-2760(93)90085-N [pii]
AID - 10.1016/0005-2760(93)90085-n [doi]
PST - ppublish
SO  - Biochim Biophys Acta. 1993 Jul 21;1169(1):80-9. doi: 
      10.1016/0005-2760(93)90085-n.