Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer.
Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer.
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DOI：
10.1016/s0021-9258(17)38401-6
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发表时间：
1986-06
期刊：
影响因子：
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通讯作者：
L. Schwartz;T. Bradford
中科院分区：
文献类型：
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作者：
L. Schwartz;T. Bradford
在37℃生理缓冲液和血浆中，类胰蛋白酶通过与肝素结合而稳定为具有酶活性的四聚体，并在无肝素的情况下解离为非活性单体。在37℃下，生理缓冲液和血浆中的类胰蛋白酶活性在6-8分钟内都下降了50%。当肝素糖胺多聚糖存在时，类胰蛋白酶在缓冲液和血浆中几乎完全保持活性2小时。在合成酯和多肽底物存在的情况下，类胰蛋白酶活性在标准的检测条件下也会衰退，除非与肝素结合。肝素-琼脂糖层析、凝胶过滤和速度沉淀表明，在所使用的pH和生理盐分浓度下，类胰蛋白酶与肝素结合。在0.8M的氯化钠溶液中，类胰蛋白酶从肝素-琼脂糖中得到洗脱。当肝素对类胰蛋白酶的重量比等于或大于1时，肝素对类胰蛋白酶的稳定性最大。在0.15M氯化钠和37℃条件下，对甲苯磺酰-L-甘氨酸-Pro-赖氨酸-对硝基苯胺和对-对甲苯磺酰-L-精氨酸甲酯的Kcat/Km比值分别为0.9X10(6)S-1M-1和1.7X10(6)S-1M-1，是已报道的最高值之一。用Superose 12高效液相色谱对类胰蛋白酶的稳定性进行了分析，发现类胰蛋白酶对肝素的稳定机制不是由于肝素的间接离子结合作用。活性酶洗脱的表观MR为132,000+/-10,000(n=3，+/-S.D.)，而无肝素孵育失活的类胰蛋白酶洗脱的表观MR为34,000。在37℃与肝素孵育后，二异丙基氟磷酸抑制类胰蛋白酶的四聚体结构也保持不变，但在没有肝素的情况下孵育后，四聚体结构被还原为单体亚基。在十二烷基硫酸钠-聚丙烯酰胺凝胶中的电泳法直接表明，在引起亚基解离的条件下，类胰蛋白酶没有明显的降解。完整的酶(计算为134,000 mR)中存在的两个不同的亚基34,000和33,000 mR(还原后)也被检测到在通过解离其亚基使类胰蛋白酶失活后保持不变。因此，肝素和类胰蛋白酶在人肥大细胞分泌颗粒中的选择性定位和结合很可能在类胰蛋白酶分泌后的调节中起主要作用。
Tryptase was shown to be stabilized as an enzymatically active tetramer by association with heparin and dissociated to inactive monomers in the absence of heparin at 37 degrees C in physiologic buffer and in plasma. There was a 50% loss of tryptase activity at 37 degrees C by 6-8 min in both physiologic buffer and plasma. When heparin glycosaminoglycan was present, tryptase retained nearly full activity for 2 h in buffer and in plasma. Tryptase activity also decayed under standard assay conditions in the presence of synthetic ester and peptide substrates unless bound to heparin. That tryptase is bound to heparin at the pH and physiologic NaCl concentrations employed was shown by chromatography of tryptase on heparin-agarose, gel filtration, and velocity sedimentation. Elution of tryptase from heparin-agarose occurred at 0.8 M NaCl. Maximal stabilization of tryptase by heparin occurred at a weight ratio to tryptase that was equal to or greater than unity. Kcat/Km ratios for tryptase-heparin at 0.15 M NaCl and 37 degrees C were 0.9 X 10(6) s-1 M-1 for tosyl-L-Gly-Pro-Lys-p-nitroanilide and 1.7 X 10(6) s-1 M-1 for p-tosyl-L-arginine methyl ester and are among the highest reported for tryptic enzymes. The mechanism of heparin-dependent stabilization of tryptase was not due to indirect ion binding properties of heparin and was analyzed by Superose 12 high performance liquid chromatography. Active enzyme eluted with an apparent Mr of 132,000 +/- 10,000 (n = 3, +/- S.D.), whereas tryptase inactivated by incubation without heparin eluted with an apparent Mr of 34,000. The tetrameric structure of diisopropyl fluorophosphate-inhibited tryptase was also preserved after incubation with heparin at 37 degrees C but was reduced to monomeric subunits after incubation without heparin. That no appreciable degradation of tryptase occurs under conditions that cause dissociation of subunits was directly shown by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Two different subunits of 34,000 and 33,000 Mr (after reduction) present in the intact enzyme (calculated to be 134,000 Mr) were also detected unchanged after inactivation of tryptase by dissociation of its subunits. Thus, the selective localization and association of heparin and tryptase in the human mast cell secretory granule most likely plays a major role in the regulation of tryptase after secretion.