Immunoprecipitation Of A NadPH Oxidase System GTPase, Rac2, From The Human Neutrophil And The GTP-Dependent Activity Of Rac2 In A Phosphorylation-Dependent Cell-Free System

Abstract

The neutrophil is an integral part of the body’s line of defense against infectious microbial agents. Its microbicidal activity is carried out by NADPH oxidase, an enzyme that uses electron transfer to create superoxide (O2"), which is then transformed into various toxins that are responsible for the demise of the foreign microorganism. The oxidase system is composed of membrane and cytosolic components. One of the cytosolic components is a low molecular-weight member of the Rho family of GTPases, Rac2. A genetic link has recently been established between Rac2 and Chronic Granulomatomous Disease (CGD), a disease in which the body’s immune system is compromised as a result of a defect in the NADPH oxidase system. To further elucidate the role ofRac2, cytosolic subcellular fractions were immunoprecipitated and reconstituted in two different cell-free systems, SDS and McPhail, to determine the specific amount of Rac2 present, as well as how much of this protein can be depleted before immunity is compromised. Results indicated that immunoprecipitation (IP) of Rac2 was incomplete with concurrent IP of two other essential proteins, p47phox and p67phox, noted as well. The best results were obtained using 10% bovine serum albumin to block the sepharose beads, which were incubated with the cytosol separately from the antibody. It is possible that some Rac2 escaped IP or was present in the membrane prior to activation. IP of both Racl and Rac2 was completed to test a hypothesis that Racl was capable of substituting for Rac2 when its levels are diminished in the cell. Addition of purified Rac2 to IP cytosol demonstrated an optimal level of 1 pg, after which the increase in superoxide production began to level off. Finally, the GTP-dependency ofthis protein was studied using GTP-depleted cytosol in the McPhail cell-free assay. Control cytosol reached peak performance levels with the addition of GTP alone; GTP-depleted cytosol was shown to be dependent on the addition ofboth ATP and GTP.