Subcellular localization of O2- generating enzyme in guinea pig polymorphonuclear leukocytes; fractionation of subcellular particles by using a Percoll density gradient.
An iso-osmotic Percoll density gradient was applied to determine the subcellular localization of the O2- generating enzyme, NADPH oxidase, in guinea pig polymorphonuclear leukocytes (PMN). [14C]Myristate (MA) was employed as a metabolic stimulant in order to clarify whether the myristate binding site on PMN membrane was identical with the O2- generating site. The distribution pattern of the O2- generating activity of MA-activated PMN was compared with that of unactivated PMN in parallel experiments to find fractions showing an enhanced O2- generating activity (i.e., above the background values due to O2-generation by other electron-transport systems). We observed very high O2- generating activity concentrated in a single peak with MA-activated PMN but little activity was seen with unactivated PMN in the Percoll density gradient. The O2- generating activity of MA-activated PMN was consistently associated with 5'-nucleotidase activity as a membrane marker enzyme, but was not associated with lysosomal marker enzymes such as myeloper-oxidase and lysozyme. O2- generating and 5'-nucleotidase activities in the peak fraction of MA-activated PMN were increased to about six and four times those of whole cells in terms of specific activity, respectively. These results indicate that the O2- generating enzyme is located on PMN plasma membrane. Furthermore, [14C]myristate-binding activity was mainly found in the peak fraction containing O2 - generating enzyme. This suggests that [14C]myristate binds to plasma membrane, and the O2 - generating enzyme may thus be activated.
D015720:5'-Nucleotidase; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D004789:Enzyme Activation / Q000187:drug effects; D006168:Guinea Pigs; D008854:Microscopy, Electron; D009226:Myristates / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D009708:Nucleotidases / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013481:Superoxides / Q000378:metabolism*
D009226:Myristates; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D009708:Nucleotidases; D015720:5'-Nucleotidase; D010100:Oxygen