Subcellular localization of O2- generating enzyme in guinea pig polymorphonuclear leukocytes; fractionation of...

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.