Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane.
Human neutrophil respiratory burst oxidase (NADPH-oxidase) activity can be reconstituted in a cell-free system consisting of plasma membrane, cytosol and an anionic amphiphile [e.g., sodium dodecyl sulfate (SDS) or arachidonate]. Herein, we report reconstitution of oxidase activity using isolated neutrophil plasma membrane together with purified recombinant p47-phox and p67-phox which had been produced using a baculovirus expression system. Activity required an anionic amphiphile (SDS or arachidonate) and was potentiated by diacylglycerol and GTP gamma S. Serial washes of the plasma membrane failed to affect its ability to reconstitute activity, indicating that a dissociable membrane component was not present. The Km for NADPH, 43 microM, was the same as that determined using cytosol in place of recombinant factors. The EC50 values for p47-phox and p67-phox under optimal activation conditions were 220 nM and 80 nM, respectively, indicating a relatively high affinity of these components in an activation complex. Since neither cytosolic component contains a nucleotide binding consensus sequence, these data indicate that the NADPH binding component of the oxidase resides in the plasma membrane.
Uhlinger
D J
DJ
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.
Inge
K L
KL
Kreck
M L
ML
Tyagi
S R
SR
Neckelmann
N
N
Lambeth
J D
JD
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D006801:Humans; D007313:Insecta; D007700:Kinetics; D046911:Macromolecular Substances; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013481:Superoxides / Q000097:blood; D014162:Transfection
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme