Cell type specific inhibition of cAMP phosphodiesterase activity during terminal differentiation in Dictyostelium discoideum

Abstract

Cyclic AMP phosphodiesterase (PDE) activity reaches a peak during the aggregation stage of development where it functions to regulate extracellular levels of cAMP. During the subsequent differentiation of the two cell types at the culmination stage, the activity reappears but only in stalk cells. We found that extracts from the culmination stage contained PDE which could be activated by preincubation with Mg 2+ and dithiothreitol (DTT), a treatment which is known to release an endogenous inhibitor from the aggregation stage enzyme. When the culmination stage extracts were subjected to chromatography on Biogel P300, two peaks of activity were eluted, PDE-I ( M r > 260,000) and PDE-II ( M r 100,000). Treatment of the fractions with Mg-DTT did not affect the low-molecular-weight enzyme but caused activation of the high-molecular-weight enzyme and the appearance of a third, intermediate form. Kinetic analysis of the two peaks revealed K m values for cAMP of 2 m M and 10 μ M for PDE-I and PDE-II, respectively. We tested the possibility that these forms of the enzyme might be distributed differently in the two cell types by measuring the K m for cAMP and the effect of Mg-DTT treatment on isolated sections of stalk and spore cells. The spore sections contained a high K m form of the enzyme (0.3 m M) which was activated by preincubation with Mg · DTT whereas stalk sections contained a low K m form (3 μ M) which was not affected by the activation treatment. We conclude that both cell types contain enzyme protein and that the apparent localization of PDE activity in stalk cells is due to the inhibition of activity in spore cells.