title: Activation of the leukocyte NADPH oxidase in a cell-free system: phosphorylation vs. amphiphiles.
abstract: We examined the ability of C-terminal deletion mutants of p47PHOX, a cytosolic subunit of the leukocyte NADPH oxidase, to support the activity of the oxidase in two different cell-free systems, one using protein kinase C and the other an anionic amphiphile (SDS or arachidonic acid) as the oxidase-activating agent. Two deletion mutants were studied: p47PHOXdelta330 and p47PHOXdelta348, each named according to the first residue of the deleted polypeptide. Wild-type (WT) p47PHOX and both mutants were phosphorylated by protein kinase C, but the WT protein was the most heavily phosphorylated, containing 6.0 +/- 0.5 mol phosphate/mol protein. Of the two deletion mutants, only p47PHOXdelta348 could support oxidase activity, and then only in the amphiphile-activated system; neither of the mutants supported oxidase activity in the system activated by protein kinase C. Translocation correlated with activity: WT p47PHOX translocated to the membrane in response to both protein kinase C and amphiphile, but p47PHOXdelta348 translocated only in the amphiphile-activated system. Comparison of these findings with the results of earlier studies suggests that the phosphorylation of p47PHOX is an important component of oxidase activation. The findings provide no information, however, about whether amphiphiles participate in the activation process in intact cells. Consequently, a mechanism of in vivo oxidase activation involving both phosphorylation and the generation of an amphiphile remains a distinct possibility.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Park
authors.forename: K E
authors.initials: KE
authors.lastname: Scott
authors.forename: B M
authors.initials: BM
authors.lastname: Babior
mesh_terms: D002474:Cell-Free System; D004789:Enzyme Activation / Q000235:genetics; D017353:Gene Deletion; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D019255:NADPH Oxidases