Uhlinger D J, Taylor K L, Lambeth J D
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322.
J Biol Chem. 1994 Sep 2;269(35):22095-8.
The neutrophil respiratory burst oxidase consists of both the plasma membrane-associated flavocytochrome b558 and cytosolic regulatory proteins including p47-phox, p67-phox, and a small GTP-binding protein (Rac1 and/or Rac2). Oxidase activation is thought to result from the assembly of the cytosolic components on the cytochrome. A model has been proposed in which p47-phox binds directly to the cytochrome, while p67-phox binding occurs indirectly by binding to p47-phox. We have carried out a steady state kinetic analysis using a cell-free semirecombinant activation system (isolated plasma membrane plus recombinant cytosolic factors) to analyze the effects of p47-phox and p67-phox on one another's association in the active oxidase complex. As predicted from the model, increasing p47-phox concentration markedly lowered the EC50 for p67-phox, indicating that p67-phox is dependent upon p47-phox for binding. Unexpectedly, increasing p67-phox concentration also produced a significant enhancement of p47-phox binding. Thus, a more complex binding model must be invoked in which p47-phox and p67-phox mutually enhance one another's binding.
中性粒细胞呼吸爆发氧化酶由与质膜相关的黄素细胞色素b558和胞质调节蛋白组成，后者包括p47-吞噬氧化蛋白、p67-吞噬氧化蛋白和一种小GTP结合蛋白（Rac1和/或Rac2）。氧化酶激活被认为是由于胞质成分在细胞色素上组装所致。有人提出了一个模型，其中p47-吞噬氧化蛋白直接与细胞色素结合，而p67-吞噬氧化蛋白通过与p47-吞噬氧化蛋白结合而间接结合。我们使用无细胞半重组激活系统（分离的质膜加重组胞质因子）进行了稳态动力学分析，以分析p47-吞噬氧化蛋白和p67-吞噬氧化蛋白对活性氧化酶复合物中彼此结合的影响。正如该模型所预测的，增加p47-吞噬氧化蛋白的浓度显著降低了p67-吞噬氧化蛋白的半数有效浓度（EC50），表明p67-吞噬氧化蛋白的结合依赖于p47-吞噬氧化蛋白。出乎意料的是，增加p67-吞噬氧化蛋白的浓度也显著增强了p47-吞噬氧化蛋白的结合。因此，必须引入一个更复杂的结合模型，其中p47-吞噬氧化蛋白和p67-吞噬氧化蛋白相互增强彼此的结合。
