Suter S, Lew P D, Waldvogel F A
Pediatr Res. 1986 Sep;20(9):848-52. doi: 10.1203/00006450-198609000-00008.
It was previously shown that inhibitors such as tosylamido-phenylethyl-chloromethylketone (TPCK) inhibit superoxide production by human neutrophils. These studies suggested that a chymotrypsin-like protease inhibited by TPCK was involved in the activation of the neutrophils oxidative system. In this study, we attempted to define the step in cellular activation and/or cell function inhibited by TPCK. TPCK 10(-5) M did not inhibit the following early events thought to be involved in the activation of oxidase. 1) f-met-leu-phe-induced activation of phospholipase C assessed by the production of inositol-tris-phosphate (IP3), 2) f-met-leu-phe-induced membrane potential changes, 3) f-met-leu-phe-induced increase in free cytosolic calcium, and 4) phorbol-myristate acetate-induced protein phosphorylation in 32P labeled neutrophils. We also showed that TPCK 10(-5) M inhibited bactericidal activity of neutrophils on Staphylococcus aureus, whereas it did not inhibit the ingestion rate of endotoxin-coated Oil red O particles. We conclude that 1) TPCK at the concentration of 10(-5) M inhibits superoxide production but not ingestion of Oil red O particles and 2) TPCK inhibits superoxide production at a step distal from calcium mobilization and protein phosphorylation. Radiolabeled TPCK may therefore be a useful tool to study, whether a protease is involved in the activation of the oxidative system distal to second messenger generation.
先前的研究表明，诸如甲苯磺酰胺基苯乙基氯甲基酮（TPCK）之类的抑制剂可抑制人中性粒细胞产生超氧化物。这些研究表明，一种受TPCK抑制的胰凝乳蛋白酶样蛋白酶参与了中性粒细胞氧化系统的激活。在本研究中，我们试图确定受TPCK抑制的细胞激活和/或细胞功能步骤。10⁻⁵ M的TPCK并未抑制以下被认为与氧化酶激活有关的早期事件。1）通过肌醇三磷酸（IP3）的产生评估的f-甲硫-亮-苯丙氨酸诱导的磷脂酶C激活；2）f-甲硫-亮-苯丙氨酸诱导的膜电位变化；3）f-甲硫-亮-苯丙氨酸诱导的游离胞质钙增加；4）佛波醇肉豆蔻酸酯乙酸酯诱导的32P标记的中性粒细胞中的蛋白质磷酸化。我们还表明，10⁻⁵ M的TPCK抑制中性粒细胞对金黄色葡萄球菌的杀菌活性，而不抑制内毒素包被的油红O颗粒的摄取率。我们得出结论：1）浓度为10⁻⁵ M的TPCK抑制超氧化物的产生，但不抑制油红O颗粒的摄取；2）TPCK在远离钙动员和蛋白质磷酸化的步骤抑制超氧化物的产生。因此，放射性标记的TPCK可能是一种有用的工具，用于研究蛋白酶是否参与第二信使产生远端的氧化系统激活。
