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 O2- 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 O2- 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 GTP gamma S 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.
Park
J W
JW
Scripps Research Institute, La Jolla, California 92037.
Babior
B M
BM
D002474:Cell-Free System; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D012265:Ribonucleotides / Q000378:metabolism*; D013481:Superoxides / Q000097:blood; D010738:Type C Phospholipases / Q000494:pharmacology
D004075:Diglycerides; D012265:Ribonucleotides; C044295:1,2-dioctanoylglycerol; D013481:Superoxides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins