Kinetics of cell-free activation of neutrophil NADPH oxidase. Effects of neomycin and guanine nucleotides

Abstract

The effects of neomycin, fluoride and the non-hydrolysable guanine nucleotide analogue GTP gamma S on the kinetics of cell-free activation of NADPH oxidase in membranes of resting human neutrophils were investigated. Arachidonate-mediated activation of the oxidase followed a first-order reaction course (kobs. = 0.39 min-1 at 26 degrees C). In the presence of NaF during the activation process, activity was enhanced while the activation rate was slightly reduced (kobs. = 0.25 min-1 at 26 degrees C). Neomycin blocked activation (half-maximal effect at 25 microM) without affecting rates of superoxide release by preactivated enzyme in vitro or in vivo. In spite of reduced specific activity neither the first-order rate constant of the activation nor the Km of the oxidase were altered by neomycin. Oxidase activated in the presence of GTP gamma S exhibited increased specific activity and unchanged Km; the course of the reaction deviated from first-order kinetics. Kinetic evidence is presented for two separate activation reactions: a GTP gamma S-independent, basal, first-order process and a GTP gamma S-dependent sigmoid activation process. The results are compatible with the existence in neutrophil membranes of two separate pools of dormant oxidase. An alternative scheme of the formation of two active forms of NADPH oxidase is also presented.

In vitro Activation of the NADPH Oxidase by Fluoride

“…Similar results were obtained when activation of the NADPH oxidase was investigated in an in vitro system consisting of a washed membrane fraction from bovine neutrophils and recombinant p47,,,,, p67,,,,,,, and Rac proteins (data not shown). All these findings are in agreement with previously published data (Seifert et al, 1986;Gabig et al, 1987; Ligeti et a]., 1988; Aviram and Sharabani, 1989) and support that the activatory effect of fluoride is not a The effect of fluoride is inhibited by GDP and its analog but it is potentiated by GTP. In addition to its activatory effect on heterotrimeric G proteins, fluoride is also known to be an inhibitor of glycolytic enzymes and various phosphatases.…”

“…Sporadic results obtained both in intact (Della Bianca et al, 1988) and in electropermeabilized cells (Hartfield and Robinson, 1990) suggested that fluoride might have additional effects, too. Early reports characterizing the properties of the activation of NADPH oxidase under cell-free condition also demonstrated the stimulatory effect of fluoride ions (Seifert et al, 1986;Gabig et al, 1987;Ligeti et al, 1988;Aviram and Sharabani, 1989).…”

Involvement of GTP in cell-free activation of neutrophil NADPH oxidase. Studies with GTP analogues

“…Human neutrophils were isolated from fresh buffy coats by standard procedures. Cells (108/ml) in 10 mM-potassium phosphate-buffered saline (pH 7.0) (PBS) containing 1 mM-EGTA and proteinase inhibitors were disrupted by sonication and fractionated into soluble cytosolic and light membrane fractions as described [17,21].…”

“…Arachidonate-dependent activation was performed in two successive steps [21]. The optimal arachidonate concentration was determined for each experiment.…”

Human neutrophil cytosolic phospholipase C: partial characterization

“…The cells were broken and fractionated as described elsewhere [14]. 7.4) 1 mM EGTA/250 mM sucrose/0.5% CHAPS.…”