Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase

Abstract

Human neutrophils and other phagocytes generate superoxide anion (O2-) as a means of destroying ingested microorganisms. O2- is produced by an NADPH-consuming oxidase composed of membrane and cytosolic components. Activation of the NADPH oxidase is absolutely dependent upon GTP, indicating the requirement for a GTP-binding protein in this process. We have utilized a five-step chromatographic procedure to isolate a GTP-binding protein from human neutrophil cytosol which can stimulate NADPH oxidase activity in a cell-free assay. Oxidase enhancing activity was shown to coisolate with this GTP-binding component, which was purified to apparent homogeneity. The GTP-binding protein was identified as Rac 2 by immunological reactivity and amino acid sequencing. Thus, Rac 2 appears to be a third cytosolic component required for human neutrophil NADPH oxidase activation. Recombinant Rac 2 was shown to bind guanine nucleotides in a Mg(2+)-dependent fashion. GDP dissociation rates were determined and shown to be regulated by the free Mg2+ concentration. Rac 2 was found to possess the highest rate of intrinsic GTP hydrolysis of any of the characterized members of the Ras superfamily. The biochemical properties of Rac 2 indicate it is likely to be subject to regulatory cofactors in vivo.