Cytosolic components to activate neutrophilic NADPH oxidase in a cell-free system.
A soluble protein containing very weak NADPH-dependent nitroblue tetrazolium reductase activity was partially purified from the cytosol of dormant human neutrophils by DEAE-5PW ion exchange chromatography. This preparation of cytosolic reductase exhibited three nitroblue tetrazolium-reducing bands with approximate molecular masses of 95, 45, and 40 kDa on non-denaturing gel electrophoresis in the presence of 35 mM n-octyl-glucoside, and two major bands with apparent masses of 45 and 40 kDa along with a few variable minor bands on SDS-polyacrylamide gel electrophoresis. The 45 kDa protein is susceptible to endogenous proteases and is rapidly converted to proteolysis products at 36 degrees C. The partially purified cytosolic protein(s) provided a concentration-dependent activation of NADPH oxidase in the cell-free system composed of the membrane, arachidonate and magnesium ion. In addition, polyclonal antibodies raised against rabbit hepatic NADPH:cytochrome P-450 reductase [EC 1.6.99.1] showed positive immunological reactivity toward cytosolic 45 kDa protein and also caused 30 to 40% inhibition of superoxide anion production in the cell-free system.
Nisimoto
Y
Y
Department of Biochemistry, Aichi Medical University.
Otsuka-Murakami
H
H
D000906:Antibodies / Q000276:immunology; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005960:Glucosides / Q000494:pharmacology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000276:immunology / Q000378:metabolism*; D019255:NADPH Oxidases; D009251:NADPH-Ferrihemoprotein Reductase / Q000276:immunology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D009580:Nitroblue Tetrazolium / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism
D000906:Antibodies; D003902:Detergents; D005960:Glucosides; D007074:Immunoglobulin G; D013481:Superoxides; D009580:Nitroblue Tetrazolium; C018619:octyl-beta-D-glucoside; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D019255:NADPH Oxidases