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Immunochemical and electrophoretic analyses of phosphorylated native and
recombinant neutrophil oxidase component p47-phox
WM Nauseef, BD Volpp and RA Clark
Department of Medicine, VA Medical Center, Iowa City, IA.
Human polymorphonuclear neutrophils (PMNs) possess a potent oxygen-
dependent microbicidal system that depends on the activity of a
stimulus-activated multicomponent nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase. Patients with chronic granulomatous disease
(CGD) lack activity of this oxidase and consequently suffer severe and
frequent infections. Components of the oxidase include both membrane- bound
factors (most notably, cytochrome b559, which is absent in the X- linked
form of CGD) and at least two cytosolic factors, one or the other of which
is absent in autosomal CGD. Patients with CGD, particularly the autosomal
type, have defective phosphorylation of proteins in the 44 to 48 Kd range.
A polyclonal antiserum (B-1) that recognizes cytosolic oxidase components
of 47 and 67 Kd was used to identify phosphoproteins in a cell-free oxidase
system. Two-dimensional gel electrophoresis showed the identity of the
47-Kd cytosolic protein (p47-phox) recognized by B-1 and the cationic 47-Kd
protein that is phosphorylated in normal but not p47-phox-deficient CGD
cytosol during activation of the NADPH-dependent oxidase. All full-length
and C- terminal recombinant p47-phox proteins augmented the superoxide-
generating capacity of the cell-free system and were phosphorylated when
added to cytosol from normal subjects or from a patient with p47- deficient
autosomal CGD. These studies provide compelling evidence that the 47-Kd
cationic protein that is a substrate for phosphorylation during the
activation of PMNs is, in fact, p47-phox, a cytosolic protein previously
shown to be critical for normal activity of the NADPH-dependent oxidase of
PMNs.
Volume 76,
Issue 12,
pp. 2622-2629,
12/15/1990
Copyright © 1990 by The American Society of Hematology
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