Cytosolic Components to Activate Neutrophilic NADPH Oxidase in a Cell-Free System

• Published in: Journal of biochemistry (Tokyo) Vol. 108; no. 5; pp. 792 - 797
• Main Authors: Nisimoto, Yukio, Otsuka-Murakami, Hidetsugu
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.11.1990
• Subjects: Analytical, structural and metabolic biochemistry; Antibodies - immunology; arachidonic acid; Biological and medical sciences; Cell-Free System; Cytosol - enzymology; Detergents - pharmacology; Enzyme Activation - drug effects; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glucosides - pharmacology; Humans; Immunoglobulin G - immunology; Kinetics; magnesium; Molecular Weight; NADH, NADPH Oxidoreductases - immunology; NADH, NADPH Oxidoreductases - metabolism; NADPH oxidase; NADPH Oxidases; NADPH-Ferrihemoprotein Reductase - immunology; Neutrophils - drug effects; Neutrophils - enzymology; Neutrophils - immunology; Nitroblue Tetrazolium - pharmacology; Oxidoreductases; Superoxides - metabolism; NADPH; Purification; Enzymatic activity; Antibody; Gel electrophoresis; Enzyme; HPLC chromatography; Activation; Cytosol; Cell free system
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a123282

A soluble protein containing very weak NADPH-dependent nitroblue tetrazolium re-ductase 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, 46, and 40 kDa on non-denaturing gel electrophoresis in the presence of 36 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°C. The partially purified cytosolic protein(s) provided a concentration-dependent activation of NADPH oxidase in the cell-free system composed of the membrane, ara-chidonate 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.