Open AccessOn the nature of the 5-lipoxygenase reaction in human leukocytes: enzyme purification and requirement for multiple stimulatory factors.
Author(s) -
Carol A. Rouzer,
Bengt Samuelsson
Publication year - 1985
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.82.18.6040
Subject(s) - enzyme , size exclusion chromatography , arachidonate 5 lipoxygenase , chemistry , lipoxygenase , biochemistry , chromatography , polyacrylamide gel electrophoresis , coomassie brilliant blue , gel electrophoresis , specific activity , ammonium sulfate , hydroxylapatite , leukotriene , high performance liquid chromatography , enzyme assay , ion chromatography , staining , biology , arachidonic acid , genetics , asthma , immunology
Arachidonate 5-lipoxygenase was purified 400-fold from homogenates of human peripheral blood leukocytes by a combination of ammonium sulfate fractionation, gel filtration chromatography, and HPLC on anion exchange and hydroxylapatite columns. NaDodSO4/polyacrylamide gel electrophoresis of the purified protein revealed the presence of a single major band (apparent Mr, 80,000). Densitometric analysis of the Coomassie blue staining pattern of the gels revealed that a 90-97% purity had been achieved. As has been reported for the 5-lipoxygenase from other mammalian sources, the human leukocyte enzyme required Ca2+ and ATP for maximal activity. In addition, a number of factors were isolated during the course of the purification, which possessed significant 5-lipoxygenase stimulatory activities. These were obtained in a high-speed pellet of leukocyte homogenate, a 60-90% ammonium sulfate precipitate fraction, and the unabsorbed protein from the first anion exchange HPLC step. In the absence of stimulatory factors, little activity was detected in the purified enzyme, even in the presence of Ca2+ and ATP. The specific function of these various factors is unknown, but their existence suggests that the human leukocyte 5-lipoxygenase is regulated by a complex mechanism that is likely to play an important role in the control of leukotriene and lipoxin biosynthesis.