Dialdehyde-GDP blocks activity of cytosolic components of neutrophil NADPH oxidase

Klinger, E; Aviram, Irit

doi:10.1016/0006-291x(91)92012-9

Cited by 2 publications

(5 citation statements)

References 18 publications

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“…These results corroborate our previous findings on the modification of cytolic oxidase component(s) by dialdehyde-guanine nucleotides [17]. In all cases, dialdehyde analogues exerted their activity in the absence of NaBH4, which is required for the stabilization of Schiff bases [22], implicating other types of stable linkage, e.g.…”

Section: Discussionsupporting

confidence: 91%

“…5) implicated p47-phox in covalent modification by ox-p[NH]ppG and suggested p47-phox as a possible target of modulation by ox-p[NH]ppG. This hypothesis is consistent with the previously reported binding of protein p47-phox to resinbound nucleotides [5,30] and with blockade of the oxidase activation-supporting activity of column fractions containing protein p47-phox by the dialdehyde analogue of GDP, oxidized GDP [17]. It is noteworthy that the exact role of protein p47phox in the activity/activation of the NADPH oxidase complex has not been elucidated [31][32][33][34].…”

Section: Discussionsupporting

confidence: 76%

“…dihydroxymorpholino compounds [27,28]. Oxidized GDP, however, inhibited activity [17], whereas ox-p[NH]ppG, used in the present study, prevented loss of basal activity. Moreover, the latter was effective at micromolar concentrations, implying high affinity and specificity of binding.…”

Section: Discussioncontrasting

confidence: 47%

“…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].…”

Section: Fractionation Of Neutrophilsmentioning

confidence: 99%

“…In a recent study we employed periodate-oxidized dialdehyde derivatives of GDP and GTP as affinity labels for the GTPbinding site involved in modulation of oxidase activity [17]. Our finding that dialdehyde-GDP blocked the capacity of cytosol to activate NADPH oxidase implicated cytosolic guaninenucleotide-binding site(s) in modulation of the enzyme.…”

Section: Introductionmentioning

confidence: 97%

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

Klinger

Aviram

1992

Biochemical Journal

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Activation of superoxide-producing NADPH oxidase of neutrophils requires the presence of cell membranes, cytosolic components and arachidonate and is markedly enhanced by non-hydrolysable analogues of guanine nucleotides, i.e. guanosine 5'-[gamma-thio]triphosphate and guanosine 5'[beta gamma-imido]triphosphate (p[NH]ppG). Gel filtration and ultrafiltration of the cytosol decreased the basal activity of NADPH oxidase. Activity could be restored by GTP, suggesting participation of the nucleotide in basal activation. Preincubation of neutrophil cytosol with periodate-oxidized p[NH]ppG (ox-p[NH]ppG) followed by gel filtration resulted in a time-dependent enhancement of basal oxidase activity. The presence of GDP or GTP, but not ATP, during the incubation with ox-p[NH]ppG abolished this enhancement. These data are consistent with a stable association of ox-p[NH]ppG with an oxidase-linked cytosolic protein. SDS/PAGE of neutrophil cytosol preincubated with [3H]ox-p[NH]ppG revealed radioactivity in bands migrating as 100, 70, 47, 34 and 22 kDa proteins. Evidence for covalent labelling of the cytosolic protein p47-phox with [3H]ox-p[NH]ppG is presented. Heterogeneity of cytosolic GTP-binding sites and possible participation of protein p47-phox in functional interaction with GTP analogues during cell-free activation are suggested.

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Section: Discussionsupporting

confidence: 91%