Analysis of activation factor for NADPH oxidase in neutrofhils

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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