Nucleoside-triphosphate binding of the two cytosolic components of the respiratory burst oxidase system: evidence for its inhibition by the 2',3'-dialdehyde derivative of NADPH and desensitization in their translocated states.
Affinity labeling of the two cytosolic components of the respiratory burst oxidase system, p49-phox and p63-phox, from resting porcine neutrophils was carried out with [32P]NADPH dialdehyde (oNADPH), [32P]oGTP and [32P]oATP. p49-phox and p63-phox showed 10-times higher affinities for both oGTP and oATP than for oNADPH, suggesting that they are nucleoside triphosphate (NTP)-binding proteins, rather than the NADPH-binding site of the oxidase. In addition, oNADPH markedly inhibited the affinity labeling of p49-phox with [32P]oGTP and [32P]oATP, well reflecting its inhibitory effect on the oxidase activity in the cell-free system, which was previously reported to propose the NADPH-binding site in a cytosolic component. Stimulation of porcine neutrophils with either myristic acid or phorbol myristate acetate resulted in great enhancement of the oxidase activity, and in considerable translocation of p49-phox and p63-phox. Nevertheless, the affinity labeling of the stimulated cell membranes in both cases revealed no labeled bands corresponding to molecular masses of 49 kDa and 63 kDa. p49-phox derived from the stimulated membranes had lost its [32P]oGTP binding ability in contrast with that from resting cytosol, suggesting that the NTP-binding sites of the two cytosolic components may be desensitized on NTP binding in their translocated states.
Mizunari
H
H
National Children's Medical Research Center, Tokyo, Japan.
Kakinuma
K
K
Suzuki
K
K
Namiki
H
H
Kuratsuji
T
T
Tsunawaki
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D003600:Cytosol / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D013045:Species Specificity; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D009227:Myristic Acids; D010750:Phosphoproteins; D019814:Myristic Acid; D009249:NADP; D006160:Guanosine Triphosphate; 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