PURIFICATION, CHARACTERIZATION, AND STRUCTURAL-PROPERTIES OF A SINGLE PROTEIN FROM RAT BASOPHILIC LEUKEMIA (RBL-1) CELLS POSSESSING 5-LIPOXYGENASE AND LEUKOTRIENE-A4 SYNTHETASE ACTIVITIES

  • 1 December 1986
    • journal article
    • research article
    • Vol. 30  (6) , 510-519
Abstract
Arachidonate 5-lipoxygenase of rat basophilic leukemia (RBL-1) cells was purified more than 1000-fold by gel filtration and anion exchange protein-high performance liquid chromatography (HPLC). Physical properties of the purified 5-lipoxygenase such as molecular weight (74,000-76,000), N-terminal sequence (30 amino acids), and amino acid composition were determined. The purified enzyme converted [14C]arachidonic acid at 20.degree.C to [14C]5-hydroperoxyeicosatetraenoic acid (5-HPETE) and to [14C]dihydroxyeicosatetraenoic acids (diHETEs). Utilizing [14C]5(S)HPETE as substrate, the purified enzyme also converted the hydroperoxy acid to [14C]diHETES. The [14C]diHETE reaction products were identified primarily (> 80% of recovered radioactivity) as the nonenzymatic hydrolysis products of leukotriene A4 (i.e., 6-trans-leukotriene B4 and 12-epi-6-trans-leukotriene B4) by reverse phase HPLC, scanning spectrophotometry, and gas chromatography-mass spectrometry. The bioconversion of [14C]arachidonate and [14C]5(S)HPETE to reaction products by the purified enzyme was dependent on the presence of both Ca2+ and ATP. The enzymatic activities were inhibited in a similar manner by the lipoxygenase inhibitors nordihydroguairetic acid, diphenyldisulfide, and SK and F 86002. The data provide evidence that RBL-1 cells 5-lipoxygenase and leukotriene A4 synthetase activities reside on a single monomeric protein with a free N-terminus and that they possess similar biochemical characteristics.