Macrophage-derived superoxide-generating NADPH oxidase in an amphiphile-activated, cell-free system; partial purification of the cytosolic component and evidence that it may contain the NADPH binding site.

The cytosolic component of macrophage-derived superoxide generating NADPH oxidase was partially purified by affinity chromatography on 2',5'-ADP-agarose. Elution was nonspecific by elevated phosphate molarity. A single step attains at least 40-fold enrichment of specific activity, the recovery being over 20%. Elution with various ligands in the concentration range 2-3.5 mM was also tested. The most effective ligands were: ATP, dATP, GTP, NADPH and 2',5'-ADP. Ineffective were AMP, 2'-AMP, FMN, FAD and NADH. ADP was of medium potency. On the basis of the above and other results, we infer that the molecule (or complex) purified by us may contain the enzymatic NADPH binding site. This component is fully retained by a 100 kDa cutoff membrane and is labile at room temperature, the lability being cancelled by 2-mercaptoethanol.