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

Abstract：

: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.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Doussiere J,Pilloud MC,Vignais PV

doi

10.1016/s0006-291x(88)80382-6

subject

Has Abstract

pub_date

1988-05-16 00:00:00

pages

993-1001

issue

3

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(88)80382-6

journal_volume

152

pub_type

杂志文章

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