| Abstract: | We demonstrate that 5'-nucleotidase (5'NT), an ectoenzyme of guinea pig polymorphonuclear leukocytes, is largely excluded from phagosomal membrane, rather than internalized randomly during phagocytosis of heat-killed bacteria, latex microbeads, or zymosan particles. Cells were fixed in 0.25% glutaraldehyde (pH 6.3) at 4 degrees C for 10 min and incubated in a cytochemical medium for the demonstration of 5'NT. In the nonphagocytosing cells, 5'NT was evenly distributed on the external side of the plasma membrane. In cells phagocytosing bacteria, 5'NT appeared to be cleared from the nascent phagosomal membrane; after 5 min of phagocytosis, most of the phagocytic vacuoles containing bacteria, latex, or zymosan particles were devoid of reaction product. When phagosomes containing latex particles were isolated and biochemically assayed, they contained less than 3% of the total cellular 5'NT activity even after 60 min of phagocytosis, and at that time the total cellular 5'NT activity had not declined. When the diazonium salt of sulfanilic acid (DASA), a nonpermeable ectoenzyme inhibitor, was used to determine the distribution of extracellular and intracellular 5'NT activity, no increase in DASA-insensitive intracellular 5'NT was found after phagocytosis of latex or opsonized zymosan. Cytochemical and biochemical evidence led us to conclude that 5'NT is excluded from phagosomal membrane, and that the exclusion is due to redistribution rather than to inactivation by granule enzymes. |
