Analysis of activation factor for NADPH oxidase in neutrofhils

中性粒细胞中的NADPH氧化酶在杀死入侵细菌方面起着重要作用。在由质膜、胞浆和花生四烯酸组成的无细胞体系中，我们在豚鼠中性粒细胞中发现了一种对NADPH氧化酶的低分子胞浆激活因子。用Sephadex G-25凝胶过滤层析将胞质组分分离成大于10KDA的高分子量组分和低分子量组分。在无细胞体系中，用高相对分子质量组分和每个低相对分子质量组分代替胞浆来激活NADPH氧化酶。含有约1 kDa物质的低分子基团不具有激活NADPH酶的能力，而高分子量组分则增强了NADPH酶的活性。低分子量官能团经加热和蛋白酶处理后不影响其活性的提高。在FAD和GTPsigmaS存在下，众所周知的低分子量激活…因子越多，低分子量组分越能增强NADPH氧化酶的活性。另一方面，我们还研究了豚鼠中性粒细胞中46 kDa蛋白的磷酸化和去磷酸化，以阐明该蛋白的磷酸化在NADPH氧化酶激活中的重要性。在激活的中性粒细胞制备的质膜中，我们发现46 kDa蛋白的脱磷与NADPH氧化酶的失活有关。此外，蛋白磷酸裂解酶抑制剂可抑制NADPH氧化酶的失活。中性粒细胞经抑制剂处理后，趋化肽fMLP对中性粒细胞的激活由微弱的、短暂的转变为持续的、持续的激活。这些结果表明，46 kDa蛋白的去磷酸化导致NADPH氧化酶失活，而且不仅46 kDa蛋白的磷酸化增加，而且蛋白的去磷酸化降低激活了NADPH氧化酶。较少

NADPH oxidase in neutrophils plays an important role in killing invaded bacteria. We found a low molecular cytosolic activation factor in guinea pig neutrophils for NADPH oxidase in a cell-free system, which consisting plasma membranes, cytosol and arachidonic acid. The cytosolic fraction was separated into a high molecular weight fraction larger than 10 kDA (a through fraction) and low molecular weight fractions by sephadex G-25 gel filtration chromatography. Inthe cell-free system, NADPH oxidase was activated using the high molecular weight fraction and each of the low molecular weight fractions instead of cytosol. The low molecular weight faction, which contained about 1kDa materials and had no ability to activate NADPH oxidase, enhanced NADPH oxidase activation by the high molecular weight fraction. Treatment of the low molecular weight faction with heat and proteinase did not affect the enhancement. In the presence of FAD and GTPsigmaS, well-known low molecular weight activation f … More actors, the low molecular weight fraction enhanced NADPH oxidase activation. These results indicate the low molecular weight fraction contains a new cytosolic activation factor for NADPH oxidase.Onthe other hand, we also investigated phosphorylation and dephosphorylation of 46 kDa protein in guinea pig neutrophils to clarify the importance of phosphorytion of this protein to NADPH oxidase actvation. In the plasma membranes prepared from activaed neutrophils, we found the correlation between dephosphrylaion of the 46 kDa protein and deactivation of NADPH oxidase. Furthermore, the deactivation of NADPH oxidase was suppressed by a protein phoshatase inhibitor. By the treatment of neutrophils with the inhibitor, a weak and temporal activation by fMLP, a chemotactic peptide, changed to substantial and prolonged activation. These reslts shows that the dephosphorylation of 46 kDa protein cause the deactivation of NADPH oxidase, and not only the increase in the phosphorylation of 46 kDa protein but also the decrease in the dephosohorylation of the protein activate NADPH oxidase. Less