Lam B K, Gagnon L, Austen K F, Soberman R J
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1990 Aug 15;265(23):13438-41.
Recently, we characterized the export of leukotriene (LT) C4 from human eosinophils as a carrier-mediated process (Lam, B. K., Owen, W. F., Jr., Austen, K. F., and Soberman, R. J. (1989) J. Biol. Chem. 264, 12885-12889). To determine whether a similar mechanism regulates the release of leukotriene B4 (LTB4), human polymorphonuclear leukocytes (PMN) were preloaded with LTB4 by incubation with 25 microM leukotriene A4 (LTA4) at 0 degrees C for 60 min. PMN converted LTA4 to LTB4 in a time-dependent manner as determined by resolution of products by reverse-phase high performance liquid chromatography and quantitation by integrated optical density. When PMN preloaded with LTB4 were resuspended in buffer at 37 degrees C for 0-90 s, there occurred a time-dependent release of LTB4 but little formation or release of 20-hydroxy-LTB4 and 20-carboxy-LTB4. When PMN were preloaded with increasing amounts of intracellular LTB4 by incubation with 3.1-50.0 microM LTA4 and were then resuspended in buffer at 37 degrees C for 20 s, there occurred a concentration-dependent and saturable release of LTB4 with a Km of 798 pmol/10(7) cells and a Vmax of 383 pmol/10(7) cells/20 s. The release of LTB4 was temperature-sensitive with a Q10 of 3.0 and an energy of activation of 19.9 kcal/mol. The rate of LTB4 release at 37 degrees C is about 50 times the rate of 20-carboxy-LTB4 release. PMN preloaded with LTB4 and resuspended at 0 degree C for 1-60 min in the presence of 30 microM LTA5 progressively converted LTA5 to LTB5. The rate of LTB4 release at 0 degree C was inhibited over the entire time period, peaking at about 50% at 30 min. These results indicate that the release of LTB4 from PMN is a carrier-mediated process that is distinct from its biosynthesis.
最近，我们将白三烯（LT）C4从人嗜酸性粒细胞的输出描述为一个载体介导的过程（林，B.K.，欧文，W.F.，小，奥斯汀，K.F.，和索伯曼，R.J.（1989年）《生物化学杂志》264，12885 - 12889）。为了确定类似的机制是否调节白三烯B4（LTB4）的释放，人多形核白细胞（PMN）在0℃下与25微摩尔白三烯A4（LTA4）孵育60分钟，从而预先加载LTB4。通过反相高效液相色谱法分离产物并通过积分光密度定量，PMN以时间依赖性方式将LTA4转化为LTB4。当预先加载LTB4的PMN在37℃的缓冲液中重悬0 - 90秒时，LTB4出现时间依赖性释放，但20 - 羟基 - LTB4和20 - 羧基 - LTB4的形成或释放很少。当PMN通过与3.1 - 50.0微摩尔LTA4孵育而预先加载增加量的细胞内LTB4，然后在37℃的缓冲液中重悬20秒时，LTB4出现浓度依赖性和饱和性释放，其米氏常数（Km）为798皮摩尔/10⁷个细胞，最大反应速度（Vmax）为383皮摩尔/10⁷个细胞/20秒。LTB4的释放对温度敏感，温度系数（Q10）为3.0，活化能为19.9千卡/摩尔。37℃时LTB4的释放速率约为20 - 羧基 - LTB4释放速率的50倍。预先加载LTB4并在30微摩尔LTA5存在下于0℃重悬1 - 60分钟的PMN逐渐将LTA5转化为LTB5。0℃时LTB4的释放速率在整个时间段内受到抑制，在30分钟时达到约50%的峰值。这些结果表明，PMN中LTB4的释放是一个载体介导的过程，与其生物合成不同。
