Cytosolic factors in bovine neutrophil oxidase activation. Partial purification and demonstration of translocation to a membrane fraction

Abstract

The O2(.-)-generating oxidase of bovine neutrophils is activated in a cell-free system consisting of a particulate fraction enriched in plasma membrane and containing the dormant oxidase, a high-speed supernatant from neutrophil homogenate (cytosol), Mg ions, GTP gamma S, and arachidonic acid [Ligeti, E., Doussiere, J., & Vignais, P.V. (1988) Biochemistry 27, 193-200]. The cytosolic components participating in the activation of the membrane-bound oxidase have been investigated. These components were resolved into several active peaks by Q Sepharose chromatography. The oxidase-activating potency of these peaks was synergistically enhanced by combining samples from separate peaks, or by supplying them with a threshold amount of crude cytosol. Partial purification of two active fractions containing a limited number of proteins of 65, 56, 53, and 45 kDa was achieved by gel filtration of cytosol on Ultrogel AcA44, followed by chromatography on hydroxylapatite and Mono Q. The specific oxidase-activating potency of these partially purified fractions (activating potency per milligram of soluble protein) was 6-8-fold higher than that of crude cytosol; it was enhanced up to 75-fold by complementation with a minute amount of crude cytosol, which per se had a limited efficiency. These data indicate that oxidase activation requires more than one cytosolic component to be activated. To check whether translocation of cytosolic proteins to the membrane occurred concomitantly with oxidase activation, use was made of radiolabeled cytosolic proteins. Cytosol was treated with phenyl[14C]isothiocyanate ([14C]PITC), such that 60% of its activation potency was still present.(ABSTRACT TRUNCATED AT 250 WORDS)

The superoxide‐generating oxidase of phagocytic cells

“…In doing so, it mimics a cytosolic factor. Another pitfall which may lead to erroneous interpretation originates from the presence of SOD in some of the fractions utilized in complementation studies (Doussiere et al, 1990). A number of attempts aimed at the purification of cytosolic factors apparently failed to appreciate these requirements, which explains some of the contradictory results in the early literature.…”

NADPH-binding protein of the neutrophil superoxide-generating oxidase of guinea pigs.

“…Stimulation of neutrophils under in vivo conditions such as bacterial infection or under in vitro conditions by the addition to a neutrophil cell suspension of compounds activating neutrophils (41)(42)(43) results in the activation of the superoxide-generating system. A number of experimental observations suggest that activation of the oxidase involves the translocation of cytosolic factors to the plasma membranes (19)(20)(21). Activation, which mimics in a number of aspects activation of the intact cell system, can be achieved by using a neutrophil cell-free system.…”

“…The superoxide-generating oxidase has been postulated as being a multicomponent electron-transport chain that consists of a cytochrome b558 (5-8), a flavin-adenine dinucleotide (FAD)-containing flavoprotein (9-12), and certain cytosolic factors (13-16) that catalyzes a one-electron reduction of oxygen at the expense of NADPH (17,18). Activation of the oxidase system is thought to be associated with the translocation of cytosolic components to the plasma membranes (19)(20)(21).The availability of a cell-free system demonstrating NADPH-dependent superoxide formation (22-26) opened up experimental avenues for investigation previously not available and eliminates the complications presented by the presence of an intact neutrophil plasma membrane. In the cellfree system, superoxide formation requires the simultaneous presence of plasma membranes and the cytosol fraction obtained from unstimulated neutrophils.…”

“…The superoxide-generating oxidase has been postulated as being a multicomponent electron-transport chain that consists of a cytochrome b558 (5)(6)(7)(8), a flavin-adenine dinucleotide (FAD)-containing flavoprotein (9)(10)(11)(12), and certain cytosolic factors (13)(14)(15)(16) that catalyzes a one-electron reduction of oxygen at the expense of NADPH (17,18). Activation of the oxidase system is thought to be associated with the translocation of cytosolic components to the plasma membranes (19)(20)(21).…”

Phosphorylation of the leucocyte NADPH oxidase subunit p47phox by casein kinase 2: conformation-dependent phosphorylation and modulation of oxidase activity

“…, a heterodimer of p22 phox and gp91 phox , to assemble the functioning oxidase [7][8][9][10][11][12].…”