Regulation of the human neutrophil NADPH oxidase by rho-related G-proteins

Superoxide production by phagocytic white blood cells requires the assembly of an NADPH oxidase from membrane and cytosolic proteins. Recombinant cytosolic proteins p47phox and p67phox and neutrophil membranes were used to purify a third cytosolic component that is necessary and sufficient for cell-...

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Bibliographic Details
Published in:Biochemistry (Easton) 1993-06, Vol.32 (21), p.5711-5717
Main Authors: Kwong, Cheung H, Malech, Harry L, Rotrosen, Daniel, Leto, Thomas L
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Biological and medical sciences
cdc42 GTP-Binding Protein
Cell Membrane - enzymology
Cell-Free System
Chromatography, Ion Exchange
Cytosol - enzymology
Fundamental and applied biological sciences. Psychology
Fundamental immunology
GTP Phosphohydrolases - metabolism
GTP-Binding Proteins - analysis
GTP-Binding Proteins - genetics
GTP-Binding Proteins - isolation & purification
GTP-Binding Proteins - metabolism
Guanine Nucleotide Dissociation Inhibitors
Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology
Guanosine Diphosphate - analogs & derivatives
Guanosine Diphosphate - pharmacology
Guanosine Triphosphate - metabolism
Humans
Immunobiology
Kinetics
Molecular Sequence Data
Myeloid cells: ontogeny, maturation, markers, receptors
NADH, NADPH Oxidoreductases - blood
NADH, NADPH Oxidoreductases - isolation & purification
NADPH Oxidases
Neutrophils - enzymology
Polynuclears
Pregnancy Proteins - metabolism
rac GTP-Binding Proteins
Recombinant Proteins - metabolism
rho-Specific Guanine Nucleotide Dissociation Inhibitors
Sequence Homology, Amino Acid
Thionucleotides - pharmacology
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Summary:Superoxide production by phagocytic white blood cells requires the assembly of an NADPH oxidase from membrane and cytosolic proteins. Recombinant cytosolic proteins p47phox and p67phox and neutrophil membranes were used to purify a third cytosolic component that is necessary and sufficient for cell-free reconstitution of NADPH oxidase. The component was isolated as a complex of rho-GDP dissociation inhibitor (rho-GDI) and two members of the rho subfamily of ras-related guanine nucleotide binding proteins, rac2 and CDC42Hs. Oxidase reconstitution with these pure cytosolic proteins was unaffected by GTP gamma S but was inhibited by GDP beta S, suggesting that the active complex contained endogenous bound GTP. Direct binding of rho-GDI to the GTP gamma S-bound forms of these G-proteins was demonstrated by gel filtration following exchange with radiolabeled guanine nucleotide. rho-GDI was shown to be nonessential for cell-free oxidase reconstitution in experiments that compared the activities of pure recombinant forms of these G-proteins. Recombinant rac augmented superoxide production, while recombinant CDC42Hs, which shares 70% amino acid sequence identity with rac, did not. Three highly conserved regions of rac1 and rac2 were noted as markedly divergent in CDC42Hs. It is proposed that one or more of these regions of rac may be involved in the specific interaction of rac with the other NADPH oxidase protein(s).
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00072a029