GTP‐dependent and ‐independent activation of superoxide producing NADPH oxidase in a neutrophil cell‐free system

Abstract

GTP and GTP-~,-S enhanced several-fold the NADPH-depcndent supcroxide production induced by sodium dodeeyl sulfate in a eell-free system of pig neutrophils consisting of the mebrane fraction and two cytosolic fractions separated by gel filtration. The enhanced activity was decreased by the addition of GDP in a dose-dgl~endcnt manner, but 70~ .of the activity in the absenee of GTP remained even at 1 mM GDP. Only one cytosol fraction besides the membrane fraction was required for the activation in the presence of GTP. The cytosol fraction was analyzed by chromatography on 2%5'-ADP agarose and two components responsible for the (3TP-depcndent and independent activation were separated. These results suggest that at least two pathways are available for the activation of supcroxide production in the cell-free system of pig neutrophils.

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

“…The basal activity of NADPH oxidase activated in a mixture of membranes, cytosol and arachidonate can be augmented by the addition of poorly hydrolysable analogues of GTP, guanosine 5'-[fl,y-imido]triphosphate (p[NH]ppG) or guanosine 5'-[y-thio]triphosphate (GTP[S]) [11,12], implicating membrane and/or cytosolic GTP-binding proteins in the process. The question of the location and identity of GTP-modulated component(s) of the oxidase has been addressed by several investigators [7,[13][14][15].…”

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

“…The search for the isolation and identification of this factor has been intensive. So far, attempts to purify this cytosolic factor by size-exclusion chromatography (Gabig et al, 1987; Curnutte et al, 1987;Fujita et al, 1987;Ishida et al, 1989) and affinity chromatography on 2',5'-ADP-agarose (Sha'ag & Pick, 1988) or GTP-agarose (Volpp et al, 1988) have resulted in the recovery of fractions only partially enriched in cytosolic factor activity. It was recently recognized that neutrophils of patients suffering from an autosomal recessive form of chronic granulomatous disease (CGD)1 are deficient in cytosolic proteins (Curnutte et al, that the cytosolic factor consisted of different components (Volpp et al, 1988;Nunoi et al, 1988;Caldwell et al, 1988;Curnutte et al, 1989;Bolscher et al, 1989).…”