Studies on the NADPH oxidase of phagocytes. Production of a monoclonal antibody which blocks the enzymatic activity of pig neutrophil NADPH oxidase.
We describe in this paper a monoclonal antibody to pig NADPH oxidase which inhibits enzymatic activity. This antibody, designated 1H8.2, was selected from a group of monoclonal antibodies produced against active preparations of purified NADPH oxidase and which showed selectivity of binding. 1H8.2 is an IgM restricted in binding to pig NADPH oxidase and showing higher binding to NADPH oxidase purified from phorbol myristate acetate-stimulated than from resting neutrophils. The antibody inhibits by about 90% the oxidase activity at 20-50 micrograms/ml. Inhibition is due to a decrease of the Vmax of the oxidase, and the Km is not affected. Incubation of the NADPH oxidase with 1H8.2 in the presence of concentrations of NADPH up to 25-fold the Km does not prevent the inhibition. Together with the evidence that the antibody does not inhibit the neutrophil superoxide dismutase-insensitive NADPH cytochrome c reductase and the liver NADPH-cytochrome c reductase this observation indicates that the 1H8.2 does not bind to an epitope belonging to the NADPH-binding site. Experiments of immunoprecipitation of iodinated membrane proteins and of immunoaffinity purification showed that 1H8.2 recognizes a heterodimer of apparent molecular mass of 16/18 and 14 kDa. These polypeptides can be involved in the NADPH oxidase activity or represent still unrecognized molecules able to modulate its function.
Berton
G
G
Institute of General Pathology, University of Verona, Italy.
Dusi
S
S
Serra
M C
MC
Bellavite
P
P
Rossi
F
F
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000276:immunology; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D013552:Swine
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases