Activation of O2(-)-generating oxidase in an heterologous cell-free system derived from Epstein-Barr-virus-transformed human B lymphocytes and bovine neutrophils. Application to the study of defects in cytosolic factors in chronic granulomatous disease.
Epstein-Barr-virus-transformed human B lymphocytes (EBV B lymphocytes) stimulated by 4 beta-phorbol 12-myristate 13-acetate exhibit an NADPH-dependent oxidase activity capable of generating the superoxide anion O2-, similar to, but less efficient than that of activated neutrophils. A cell-free system of oxidase activation consisting of a membrane fraction and cytosol from EBV B lymphocyte homogenate supplemented with guanosine 5'-[gamma-thio]triphosphate (GTP[S]), arachidonic acid and Mg2+ was found to be competent in the production of O2-, assessed by the superoxide-dismutase-sensitive reduction of cytochrome c in the presence of NADPH. However, cytochrome c reduction was slow and largely insensitive both to superoxide dismutase, and to iodonium biphenyl, a powerful inhibitor of the oxidase activity in neutrophils. A markedly faster reduction of cytochrome c in the presence of NADPH was obtained with a heterologous system consisting of cytosol from EBV B lymphocytes and bovine neutrophil membranes, GTP[S], arachidonic acid and Mg2+; in this system, reduction of cytochrome c was totally inhibited by superoxide dismutase and iodonium biphenyl. These results show that EBV B lymphocytes contain a substantial amount of cytosolic factors of oxidase activation, and that the limiting factors for O2- production in B lymphocytes are the membrane components of the oxidase complex. The heterologous system of EBV B lymphocyte cytosol and bovine neutrophil membranes provided a rapid and convenient method to diagnose cytosolic defects in autosomal forms of chronic granulomatous disease. In addition, it might be a useful tool to explore the mechanism of action of the cytosolic factors in oxidase activation.
Cohen-Tanugi
L
L
Laboratoire d'Enzymologie, Centre Hospitalier Universitaire, Grenoble, France.
Morel
F
F
Pilloud-Dagher
M C
MC
Seigneurin
J M
JM
Francois
P
P
Bost
M
M
Vignais
P V
PV
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001402:B-Lymphocytes / Q000201:enzymology*; D015153:Blotting, Western; D002417:Cattle; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006105:Granulomatous Disease, Chronic / Q000378:metabolism*; D004854:Herpesvirus 4, Human; D006801:Humans; D009504:Neutrophils / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
D013481:Superoxides; D016718:Arachidonic Acid; D010088:Oxidoreductases