J. Biochem, 1982, Vol. 91, No. 1 31-40
© 1982 Japanese Biochemical Society
research-article |
Subcellular Localization of O2– Generating Enzyme in Guinea Pig Polymorphonuclear Leukocytes; Fractionation of Subcellular Particles by Using a Percoll Density Gradient
Tokyo Metropolitan Institute of Medical Science Honkomagome, Bunkyo-ku, Tokyo 113
An iso-osmotic Percoll density gradient was applied to determine the subcellular localization of the O2– generating enzyme, NADPH oxidase, in guinea pig polymor-phonuclear 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.