Diradylglycerol synergizes with an anionic amphiphile to activate superoxide generation and phosphorylation of p47phox in a cell-free system from human neutrophils.
The superoxide-generating respiratory burst oxidase (NADPH oxidase) from human neutrophils can be activated in a cell-free system consisting of plasma membranes, cytosol, and an anionic amphiphile such as sodium dodecyl sulfate (SDS) or arachidonate, and guanosine 5'-(3-O-thio)triphosphate (GTP(gamma)S) augments activation. We report herein that short-chain diacylglycerols (e.g. dioctanoylglycerol (diC8)) synergize with SDS in the activation of superoxide generation in a dose- and time-dependent manner, resulting in rates up to 1400 nmol/min/mg plasma membrane protein, or 250-700% higher than the rate seen with SDS alone. diC8 did not affect significantly the dose response for either cytosol or SDS, indicating that the activation was not due to increased sensitivity of the oxidase toward either of these components. At optimal concentrations of SDS and diC8, additional activation was observed in the presence of GTP(gamma)S, indicating that diC8 and GTP activate by separate mechanisms. In contrast to diC8, other known activators of protein kinase C (phorbol myristate acetate and mezerein) augmented SDS activation only minimally (typically 20-30%), and neither diacylglycerols nor tumor promoters activated in the absence of SDS. Activation by diC8 was calcium and phosphatidylserine independent, and the specificity for neutral lipids was atypical for protein kinase C. Inhibitors of protein kinase C (staurosporine and a peptide substrate analog) also failed to inhibit the response. Nevertheless, phosphorylation of several neutrophil proteins including p47phox was seen with both SDS and diC8, and synergistic phosphorylation of p47phox was seen when both activating factors were present. Thus, diacylglycerol synergizes with SDS in activating both superoxide generation and p47phox phosphorylation in the cell-free activation system, but the activation is atypical of a protein kinase C mechanism.
Burnham
D N
DN
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322.
Uhlinger
D J
DJ
Lambeth
J D
JD
D000328:Adult; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D013481:Superoxides / Q000097:blood*
D004075:Diglycerides; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme