抄録
Tokyo Metropolitan Institute of Medical Science, 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 electrontransport 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 myeloperoxidase 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.
