Sasaki J, Hiura M, Yamaguchi M, Sakai M, Aoki K, Abe H, Okamura N, Ishibashi S
Department of Physiological Chemistry, Hiroshima University School of Medicine, Japan.
Arch Biochem Biophys. 1994 Nov 15;315(1):16-23. doi: 10.1006/abbi.1994.1465.
It is well known that sodium dodecyl sulfate (SDS) activates NADPH oxidase in a cell-free system independently of protein kinase C (PKC). However, in intact neutrophils, direct evidence has never been presented to show that O2- production by SDS is actually due to the NADPH oxidase activation observed in the cell-free system. So, in this paper, we investigated the activation mechanism by SDS in intact guinea pig neutrophils. We previously reported that hypotonic treatment reversibly enhanced O2- production stimulated by PKC activators in intact neutrophils (M. Hiura et al., 1991, Arch. Biochem. Biophys. 291, 31-37). In this paper, SDS also significantly stimulated O2- production in the intact cells under the hypotonic condition. This enhancement was gradual and was PKC inhibitor resistant. Furthermore, phosphorylation of the 46-kDa protein, one of cytosolic activation factors, was not detected by autoradiography of two-dimensional electrophoresis. Translocation of cytosolic activation factors was demonstrated by a decrease in the activity of the factors remained in the cytosol. In the presence of SDS, addition of 1-oleoyl-2-acetylglycerol, a PKC activator, further enhanced O2- production and translocation of the cytosolic activation factors. On the other hand, SDS remarkably increased membrane fluidity in intact neutrophils as well as in the cell-free system. These results indicate that activation of NADPH oxidase by SDS in intact neutrophils seems to be partly due to the same mechanism observed in cell-free activation, and that SDS alone slightly activates the oxidase and other stimulation, such as hypotonic and/or PKC activator treatments, is required for significant activation. The increase in the membrane fluidity may be one of the activation mechanisms of NADPH oxidase by SDS.
众所周知，十二烷基硫酸钠（SDS）在无细胞体系中可独立于蛋白激酶C（PKC）激活NADPH氧化酶。然而，在完整的中性粒细胞中，从未有直接证据表明SDS产生的O₂⁻实际上是由于在无细胞体系中观察到的NADPH氧化酶激活所致。因此，在本文中，我们研究了SDS在完整豚鼠中性粒细胞中的激活机制。我们之前报道过，低渗处理可可逆地增强完整中性粒细胞中PKC激活剂刺激产生的O₂⁻（M. Hiura等人，1991年，《生物化学与生物物理学报》291卷，31 - 37页）。在本文中，SDS在低渗条件下也显著刺激了完整细胞中O₂⁻的产生。这种增强是渐进的，并且对PKC抑制剂有抗性。此外，通过二维电泳放射自显影未检测到46 kDa蛋白（一种胞质激活因子）的磷酸化。通过胞质中残留因子活性的降低证明了胞质激活因子的易位。在SDS存在的情况下，添加PKC激活剂1 - 油酰基 - 2 - 乙酰甘油可进一步增强O₂⁻的产生以及胞质激活因子的易位。另一方面，SDS显著增加了完整中性粒细胞以及无细胞体系中的膜流动性。这些结果表明，SDS在完整中性粒细胞中激活NADPH氧化酶似乎部分归因于在无细胞激活中观察到的相同机制，并且单独的SDS可轻微激活氧化酶，而显著激活则需要其他刺激，如低渗和/或PKC激活剂处理。膜流动性的增加可能是SDS激活NADPH氧化酶的机制之一。
