5'-AMP NUCLEOTIDASE IS LOCALIZED IN THE AREA OF CELL-CELL CONTACT OF PRESPORE AND PRESTALK REGIONS DURING CULMINATION OF DICTYOSTELIUM-DISCOIDEUM

Microtechniques for enzymatic analysis were used to follow 5′-AMP nucleotidase activity during cell migrations leading to cellular aging in Dictyostelium discoideum. The activity was localized in the area of cell-cell contact of presumptive spore and stalk tissues. Activity converting ATP to adenosine demonstrated localization identical to that of 5′-AMP nucleotidase. The 5′-AMP nucleotidase could use p-nitrophenylphosphate as substrate when the reaction mixture was buffered at pH 9.0. The p-nitrophenylphosphate hydrolyzing activity (NHA) was not localized at the pseudoplasmodium stage although the prestalk contained about twice the activity of the prespore cells. At the culmination stage NHA was localized in the area between prespore and prestalk cells. Acid phosphatase was assayed with p-nitrophenylphosphate as substrate buffered at pH 4.8 and found to be localized in the stalk. This activity formed an increasing gradient as cells migrated into the stalk sheath and completed the process of stalk formation. Because the phosphatase activities at pH 4.8 and 9.0 resulted in opposing gradients, they were presumed to reflect two distinct enzyme proteins which serve different functions in differentiation. The possible presence of cryptic NHA in the prespore or mature stalk cells was investigated by altering the assay conditions to include: (1) addition of Mg2+ or inorganic phosphate (Pi) in the reaction mixture; (2) release of the enzyme protein from the membrane with Triton X-100; and (3) mixing and co-incubation of active and inactive cellular areas in the reaction mixture. It was found that Pi could mask 5′-AMP nucleotidase in the mature stalk because: (1) the levels of Pi known to be present in the stalk completely inhibited the enzyme; and (2) Pi inhibition was irreversible by dilution, so that the activity during the in vitro assay would remain masked even when Pi was diluted below inhibitory levels. The lack of immediate reversibility of Pi inhibition during the assay also indicates that any activity detected in vitro is not inhibited by Pi in vivo. © 1979.