1598 5 ' - Nucleotidase Activity of Mouse Peritoneal Macrophages

In the preceding paper (7), we reported on the activity and metabolism of 5'nucleotidase in resident and inflammatory mouse peritoneal macrophages. In this paper, we deal with the issue of the cellular distribution of the enzyme in these cells and with the distribution and metabolism of this enzyme in cells following two types of endocytic stimulus. In many cells, 5'-nucleotidase appears to be primarily, or even entirely, associated with the plasma membrane (for review, see 3). In mouse macrophages, the enzyme has also been identified in association with the membrane enclosing latex phagolysosomes, presumably as a result of its interiorization on portions of plasma membrane (14). Thus, quantitative information on the cellular distribution of the enzyme could shed light on the processes involved in the metabolism of plasma membrane. Endocytic stimulation, by altering the distribution and fate of plasma membrane, is another approach to an analysis of the cellular controls involved in the metabolism of the plasma membrane. We have used two different endocytic stimuli. Latex is a nondegradable particle which is ultimately included in secondary phagolysosomes. Concanavalin A, however, is a soluble protein which stimulates the formation ofpinocytic vesicles which do not readily fuse with lysosomes (5). Thus, although each agent ultimately leads to the formation of an intracellular pool of sequestered membrane, the mechanisms of stimulation and the fate of the interiorized membrane is quite different in the two cases. The diazonium salt of sulfanilic acid (DASA) ~ is a relatively membrane impermeable reagent, which was originally used by Berg to specifically label externally disposed components of the human red blood cell membrane (1). De Pierre and Karnovsky subsequently exploited it in their study of guinea pig neutrophil plasma membrane enzymes, including the neutrophil 5'-nucleotidase (4). In this study, DASA is used to assess the fraction of total 5'-nucleotidase activity which is externally accessible in the resident and inflammatory macrophage, and to examine the changes in this fraction related to endocytic stimulation.