Membrane‐Bound Cyclic AMP Phosphodiesterase in Chemotactically Responding Cells of Dictyostelium discoideum

Abstract

I n cells of the cellular slime mold, Dictyostelium discoideum, that respond chemotactically to cyclic AMP, cell-bound cyclic AMP phosphodiesterase was studied under two conditions : in suspensions of living cells with external cyclic AMP as a substrate, and in the 27000 x g sediment of cell homogenates. Under both conditions a drastic increase of cell-bound phosphodiesterase activity in the developmental phase preceding cell aggregation was found. I n non-aggregating mutants this increase is smaller or absent. The particle-bound cyclic AMP phosphodiesterase has an optimum a t pH 7.0-8.0. I n vivo it is resistant to inhibition by external glutathione and to a macromolecular phosphodiesterase inhibitor from Dictyostelium discoideum ; in particle fractions enzyme activity is partially sensitive to these inhibitors. A possible function of this enzyme in the chemotactic receptor system is discussed.Development of the cellular slime mold, Dictyostelium discoideum, proceeds from single amoeboid cells to the formation of a multicellular organism by cell aggregation. Cells remain single during growth and differentiate later on within a preaggregation phase of several hours to aggregation-competent ones. These aggregation-competent cells are more sensitive to the chemotactic action of cyclic AMP than growth phase cells [I] ; they are expected, therefore, to possess either a greater number of, or more sensitive cyclic-AMP receptors on their surface. Precise chemotactic orientation over a period of time requires continuous re-evaluation of the concentration gradient of the chemotactic agent by the responding cells. The following receptor mechanisms would fulfil such a function. (1) The receptor is a cyclic-AMPbinding compound without catalytic activity. Continuous functioning of the receptor system requires in this case either (a) high turnover of receptor molecules, or (b) rapid readjustment of the equilibrium of free to receptor-bound cyclic AMP, or (c) it requires enzymatic conversion of the bound cyclic AMP into a nonbinding product, e.g. by phosphodiesterase action. (2) The receptor itself acts as a phosphodiesterase. I n this case the free energy change associated with hydrolysis of the phosphodiester ring would enable the cell to couple with hydrolysis of cyclic AMP a t the membrane an endergonic reaction, the product of which could mediate chemotactic motion. The AGO of cyclic AMP hydrolysis is -11.9 kcal/mol (pH 7.3; 25 "C; Mg2+ equimoles to nucleotide) [2]. If possibility 1 c or 2 is the case then a phosphodiesterase activity should be detectable on the cell surface, and this activity should be correlated with the responsiveness of the cells to cyclic AMP. I n this paper we study the properties of such an enzyme with regard to its possible function as part of the chemotactic receptor system. METHODS Cell CulturesAll Dictyostelium discoideum strains were cultivated in suspension on a gyratory shaker a t 23 "C. The ax-2 strain was grown, if not stated otherwise, in peptone-yeast extract medium supplemented with 1....

Unified mechanism for relay and oscillation of cyclic AMP in Dictyostelium discoideum.

“…Here v denotes a constant ATP input rate, divided by Ks; a denotes the maximum adenylate cyclase activity, divided by Ks; q = KS/Kp; kt measures the assumed proportionality of intracellular cAMP transport rate to intracellular cAMP concentration; the dilution factor h is the ratio of extracellular fluid volume to cell volume in the experiments with cell suspensions; k measures the assumed linear rate at which extracellular cAMP is destroyed by the membrane-bound (20) We can view the situation somewhat differently, by concentrating our attention on the interior of the cell. It has been shown (15) that the activation of adenylate cyclase by 5'-AMP, which is one of the regulatory loops found experimentally by Rossomando and Sussman in D. discoideum (16) (23).…”

Short-Term Binding and Hydrolysis of Cyclic 3′:5′-Adenosine Monophosphate by Aggregating Dictyostelium Cells

“…Aggregating cells of D. discoideum which rpspond chemotactically to cAMP contain cell-bound phosphodiesterase active enough to hydrolyze 108 extracellular cAMP molecules per cell per minute (5). The question was asked whether the catalytic site of the phosphodiesterase is itself the receptor site so that the cells count cAMP molecules strictly by hydrolyzing them; or if the two sites are different (5).…”

“…Only 4% of the label in the cell sediment had been released. Membrane-bound phosphodiesterase activity was assayed in 17 mM phosphate buffer, pH 6.0, with 2 X 107 ilving cells per ml, 50 pM cAMP, and 900 units of inhibitor of extracellular phosphodiesterase per ml (5). The inhibitor was purified from the mutant ga 93 by DEAE-cellulose and Sephadex G-75 chromatography.…”

“…Wild-type strain vi2IMS, the UV-mutant aggr 60-2, and the spontaneous revertant aggr 60-3 (rev) were cultivated in shaker suspensions of 1 X 1010 cells per ml of either Escherichia coli B/r or SalmoneUa minnesota R595 and washed three times in 17 mM phosphate buffer, pH 6.0, at the time of exhaustion of bacteria (to) (5 (7).…”

“…Optical recording of the cellular response to a single cAMP pulse has shown that the response reaches a maximum at half a minute after cAMP addition and declines thereafter with a time constant of not more than 10 see (3). Extracellular cAMP is hydrolyzed in this organism both by extracellular and cell-bound phosphodiesterases, the latter exhibiting its highest activity at the stage of aggregation-competence (4,5).…”