Effects of Diacylglycerol on the Activation and Kinetics of the Respiratory Burst Oxidase in a Cell-Free System from Human Neutrophils: Evidence That Diacylglycerol May Regulate Nucleotide Uptake by a GTP-Binding Protein

The respiratory burst oxidase from human neutrophils is a membrane-associated enzyme that catalyzes the reduction of oxygen to O−2 at the expense of NADPH. The oxidase is dormant in resting neutrophils, but comes to life when the cells are exposed to certain activating agents. Activation requires GTP or a GTP analog and is associated with the transfer of cytosolic oxidase polypeptides to the plasma membrane. Treatment of resting neutrophil membranes with phospholipase C caused an eightfold rise in their diacylglycerol content, increased the sodium dodecyl sulfate-mediated transfer of cytosolic polypeptides to the membrane, and enhanced O−2 production by the membranes after treatment with cytosol and sodium dodecyl sulfate. Use of phospholipase C-treated membranes in the cell-free system caused only a minor change in the Km and Vm for NADPH as compared with the same system containing untreated membranes, but caused the Km for the nonhydrolyzable GTP analog GTPγS to fall from 200 to 34 nM. Similar kinetic changes were observed with membranes treated with dioctanoylglycerol. These findings are consistent with the idea that the activity of a G protein can be regulated by diacylglycerol.