The cytosolic components of the respiratory burst oxidase exist as a M(r) approximately 240,000 complex that acquires a membrane-binding site during activation of the oxidase in a cell-free system.

Abstract

Sodium dodecyl sulfate (SDS) treatment of a mixture of cytosol and plasma membranes from resting neutrophils resulted in the activation of the respiratory burst oxidase, a complicated enzyme that catalyzes the production of O2- from NADPH and oxygen. Activation was accompanied by translocation to the plasma membranes of the oxidase components p47phox and p67phox, which in resting cytosol were found in a M(r) approximately 240,000 complex. This translocation, which appeared to take place without a major change in the size of the cytosolic complex, did not occur if the membranes lacked cytochrome b558, and was inhibited by the peptide PRGV-HFIFNK, a sequence found near the carboxyl terminus of cytochrome b558 that was known from earlier work to inhibit O2- production by the cell-free system (Rotrosen, D., Kleinberg, M. E., Nunoi, H., Leto T., Gallin, J. I., and Malech H. L. (1990) J. Biol. Chem. 265, 8745-8750). Cytosols pretreated with the cross-linking agents 3,3'-dithiobis(sulfosuccinimidyl) propionate (DTSSP) (cleavable by 2-mercaptoethanol) and bis-(sulfosuccinimidyl) suberate (not cleavable by 2-mercaptoethanol) lost most of their ability to support O2- production in the cell-free system, and oxidase components from DTSSP-treated cytosol failed to translocate to the plasma membrane. When DTSSP-treated cytosols were incubated with 2-mercaptoethanol, however, both O2- production and translocation were partly restored, indicating that the functional impairment in DTSSP-treated cytosols was probably due at least in part to a restriction in the conformational mobility of the cross-linked peptide chains in the approximately 240,000 complex. These findings provide further support for the idea that the cytosolic components of the respiratory burst oxidase exist in the form of a approximately 240,000 complex, and suggest that the exposure of this complex to SDS induces a structural change that may or may not be associated with the loss of an inhibitory subunit too small to cause a detectable change in the size of the complex. This SDS-induced change allows translocation to take place by creating a membrane-binding site on the surface of the complex.