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

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 O2.- generating oxidase activation of bovine neutrophils. Evidence for synergism of multiple cytosolic factors in a cell-free system.

Activation of the O2.- generating oxidase in neutrophils can be achieved with a cell-free oxidase-activating system, which consists of a high speed supernatant (cytosol), a particulate fraction enriched in plasma membrane, GTP-gamma-S, arachidonic acid and Mg ions. Cytosolic proteins from bovine neutrophils were fractionated by chromatography on Mono Q and Mono S columns into two fractions, neither of which was able to activate efficiently the membrane-bound oxidase. However, when combined and added to the cell-free system under optimized conditions, they acted synergistically, activating the oxidase to virtually the same extent as crude cytosol. This synergism demonstrates that more than one cytosolic factor is required for oxidase activation, and can be used to trace the cytosolic factors during the course of their purification.

Parameters of activation of the membrane-bound O2- generating oxidase from bovine neutrophils in a cell-free system.

Parameters governing the extent of activation of the O2- generating oxidase in a cell-free system derived from bovine neutrophils were examined. The reconstituted system consisted of the following: a particulate fraction enriched in plasma membrane and containing the oxidase, a soluble fraction containing cytosolic factor(s) required for oxidase a soluble fraction containing cytosolic factor(s) required for oxidase activation, a non hydrolyzable analog of GTP, and either arachidonic acid or sodium dodecyl sulfate. When the amount of arachidonic acid or sodium dodecyl sulfate was maintained at a fixed value with respect to the amount of membrane used, a sigmoidal response of oxidase activity to increasing amounts of cytosol added was observed. In contrast, when the concentration of arachidonic acid or sodium dodecyl sulfate was properly adjusted with respect to that of membrane and cytosol, the curve relating oxidase activity to cytosol was hyperbolic, pointing to a simple michaelian relationship for the dependence of oxidase activation on the activating factor(s) of cytosol. Another parameter affecting oxidase activation was the ionic strength of the reconstitution medium, the extent of activation being lower at high ionic strength.

Activation of bovine neutrophil oxidase in a cell free system. GTP-dependent formation of a complex between a cytosolic factor and a membrane protein.

The effect of guanine nucleotides on activation of the O2-. generating oxidase in a cell free system consisting of bovine neutrophils membranes, cytosol and arachidonic acid has been studied. In a complete system, GTP-gamma-S was stimulatory and GDP-beta-S inhibitory. When cytosol was omitted, both nucleotides acted as inhibitors. Activation parameters have been explored in a preincubation step prior to the oxidase assay. Stimulation was found to be maximal at 7 to 100 microM GTP-gamma-S. Whereas the time course of activation was monophasic when activation was performed at room temperature, it became biphasic at 2 degrees C, with a first plateau of activation attained after 1 min, followed by a slow rise lasting for more than 30 min. The following lines of evidence demonstrated that oxidase activation resulted from the formation of a complex between cytosolic factor(s) and a target protein in the plasma membrane. 1/ When activated membranes, in a suspension containing cytosol, arachidonic acid and GTP-gamma-S, were separated from soluble components by centrifugation and washed, their oxidase remained fully active. 2/ The activity of the washed membranes was lost upon addition of GDP-beta-S, urea and deoxycholate, but was preserved by addition of glutaraldehyde, a cross-linking reagent. The results of experiments in which cytosol and membrane fractions were incubated separately with GTP-gamma-S, suggested that GTP-gamma-S first interacts with a factor present in the cytosol, before reacting with a target protein in the plasma membrane.