CYTOSOLIC COMPONENTS TO ACTIVATE NEUTROPHILIC NADPH OXIDASE IN A CELL-FREE SYSTEM

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. © 1990 Copyright, 1990 by the Journal of Biochemistry.