Structural Studies on the Leukocyte NADPH-Oxidase Generating Superoxide Oxyanion

Abstract

The membrane bound neutrophil NADPH-oxidase which catalyzes the one electron transfer from NADPH to molecular oxygen generating superoxide is a complex enzyme system made up of components whose structure and distribution in the membrane vary according to the system's state of activation. Cytochrome b558 is an important constituent of the NADPH-oxidase in neutrophils and B lymphocytes. Four components of the NADPH-oxidase have been identified: two membrane bound components, gp9 1 phox and p22phox, both subunits of cytochrome b558 ; and two cytosolic components, p47phox and p67phox (Chancock et al, 1994). The aim ofthis study was to gain further knowledge of the tertiary structure and arrangement of the large membrane bound component of cytochrome b558 (gp9 1 phox) in the bovine neutrophil plasma membranes during the resting state using proteolytic digestion with trypsin and papain to possibly identify the extra membranous components of cytochrome b558; i.e. In this neutrophil membrane preparation, the NADPH and FAD binding sites are postulated to be external to the membrane bilayer and should be exposed to proteolyticaction. Papaindigestion was also performed on octylglucoside solubilized and partially purified neutrophil membrane preparations. We will report on the superoxide-generating activity, NADPH oxidation and Iodonitrotetrazolium violet (INT) dye reduction profiles of membrane preparations and the correlation of such activities with proteolyticdigestiontime. Iodonitrotetrazolium violet (INT) is a dye which can be directly reduced by reduced flavins (FADH2). In addition to activity profiles, we will report on an analysis of the components of the membrane released by proteolysis.