Lipoxin synthase activity of human platelet 12-lipoxygenase.
Human platelets and megacaryocytes generate lipoxins from exogenous leukotriene A4 (LTA4). We examined the role of human 12-lipoxygenase (12-LO) in lipoxin generation with recombinant histidine-tagged human platelet enzyme (6His-12-LO), partially purified 12-LO from human platelets (HPL 12-LO) and, for the purposes of direct comparison, permeabilized platelets. Recombinant and HPL 12-LO catalysed the conversion of intact LTA4 into both lipoxin A4 (LXA4) and lipoxin B4 (LXB4). In contrast, only negligible quantities of LXA4 were generated when recombinant 12-LO was incubated with the non-enzymic hydrolysis products of LTA4.6His-12-LO also converted a non-allylic epoxide, 5(6)-epoxy-(8Z,11Z,14Z)-eicosatrienoic acid. The apparent Km and Vmax. for lipoxin synthase activity of 6His-12-LO were estimated to be 7.9 +/- 0.8 microM and 24.5 +/- 2.5 nmol/min per mg respectively, and the LXB4 synthase activity of this enzyme was selectively regulated by suicide inactivation. Aspirin gave a 2-fold increase in lipoxin formation by platelets but did not enhance the conversion of LTA4 by the recombinant 12-LO. These results provide direct evidence for LXA4 and LXB4 synthase activity of human platelet 12-LO. Moreover, they suggest that 12-LO is a dual-function enzyme that carries both oxygenase and lipoxin synthase activity.
Romano
M
M
Department of Medicine, Brigham and Women's Hospital, Boston, MA.
Chen
X S
XS
Takahashi
Y
Y
Yamamoto
S
S
Funk
C D
CD
Serhan
C N
CN
D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000096:biosynthesis / Q000097:blood* / Q000302:isolation & purification; D001792:Blood Platelets / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D006639:Histidine; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology; D017572:Leukotriene A4 / Q000378:metabolism; D044045:Lipoxins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
D018076:DNA, Complementary; D006893:Hydroxyeicosatetraenoic Acids; D017572:Leukotriene A4; D044045:Lipoxins; D011994:Recombinant Proteins; C040527:lipoxin A4; D006639:Histidine; C042651:lipoxin B4; C084486:lipoxin A4 synthase; C084488:lipoxin B4 synthase; D001092:Arachidonate 12-Lipoxygenase