INACTIVATION OF NADPH OXIDASE FROM HUMAN NEUTROPHILS BY AFFINITY LABELING WITH PYRIDOXAL 5'-DIPHOSPHO-5'-ADENOSINE

When a particulate NADPH oxidase prepared from phorbol ester-activated human neutrophils was treated with pyridoxal 5′-diphospho-5′-adenosine (PLP-AMP), the superoxide anion-producing activity was inhibited according to affinity labeling kinetics. NADPH afforded a protection against inactivation which was competitive with respect to PLP-AMP; 2′,5′-ADP and 2′-phospho-5′ diphosphoadenosine (ATP ribose) appeared to be as potent as NADPH as protecting agents. NADP+ and ATP were less effective, while ADP and GTP-γ-S did not protect significantly. These results suggest that PLP-AMP can be used, in conjunction with tritiated cyanoborohydride, to identify the elusive NADPH-dependent flavoprotein which is part of the electron transfer chain of NADPH oxidase. © 1991.