Activation of respiratory burst oxidase is accompanied by desensitization of p47phox in nucleoside-triphosphate binding along with its translocation to cell membrane.
A cytosolic component of human neutrophils, p47phox, potentiates respiratory burst oxidase translocating from cytosol to membrane upon cell stimulation. In this study, the nucleotide-binding ability of p47phox was examined using [32P]GTP dialdehyde (oGTP), [32P]oATP, and [32P]oNADPH. p47phox showed affinities for both oGTP and oATP that were 14 times higher than that for oNADPH, suggesting that it is a nucleoside triphosphate (NTP)-binding protein rather than an NADPH-binding protein. Binding analysis of p47phox using either [32P]oGTP or [32P]oATP revealed an apparent binding constant for each individual NTP analogue and the same maximum binding value, which suggests that both NTPs share a common specific binding site. Stimulation of neutrophils with phorbol myristate acetate (PMA) resulted in enhancement of the oxidase activity to generate O2- anion and was accompanied by substantial translocation of p47phox to membrane. However, p47phox derived from the stimulated cell membrane had lost its NTP-binding ability, unlike that from the resting cytosol. These results suggest that the binding of NTP to p47phox may be involved in the process that activates the oxidase and is desensitized in translocated p47phox.
Mizunari
H
H
National Children's Medical Research Center, Tokyo, Japan.
Kuratsuji
T
T
Namiki
H
H
Tsunawaki
S
S
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000097:blood; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000097:blood; D006801:Humans; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D009249:NADP / Q000031:analogs & derivatives* / Q000097:blood; D009252:NADPH Dehydrogenase / Q000097:blood* / Q000302:isolation & purification; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000097:blood* / Q000302:isolation & purification; D012265:Ribonucleotides / Q000097:blood*; D013481:Superoxides / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010750:Phosphoproteins; D012265:Ribonucleotides; D013481:Superoxides; D009249:NADP; C017199:2',3'-dialdehyde ATP; C037756:2-(guanylformylmethoxy)-3-(triphospho)propanal; D006160:Guanosine Triphosphate; C038805:2',3'-dialdehyde NADP; D000255:Adenosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C022709:superoxide-forming enzyme; D009252:NADPH Dehydrogenase; D013755:Tetradecanoylphorbol Acetate