Cell-bound Phosphodiesterase Research Articles

Correlations between prestalk-prespore tendencies and cAMP-related activities in Dictyostelium discoideum

Amoebae of the cellular slime mould Dictyostelium discoideum (strain Ax2) grown in axenic medium containing 86 mM glucose [G(+) cells] or no glucose [G(−) cells] were examined for the characteristics of aggregation, cAMP secretion rate, cAMP phosphodiesterase activity and cell surface cAMP-binding activity. (When G(+) and G(−) cells are mixed, G(+) cells preferentially differentiate into prespore cells and sort out to the posterior region of a slug.) Under the same conditions, G(−) cells aggregate later than G(+) cells and the formation of stable streams by G(−) cells was particularly delayed. The movements of G(−) cells during aggregation were less organized compared with the ordered behaviour of G(+) cells, yet G(−) cells seemed to be more sensitive to chemotactic signals. Furthermore, the cAMP-binding activity of G(−) cells was considerably higher than that of G(+) cells, a difference that is probably due to the difference in the number of the cAMP receptor sites. Also G(−) cells, compared to G(+) cells, secreted slightly more cAMP and showed higher activity of cell-bound phosphodiesterase activity at the aggregation stage, whereas the extracellular phosphodiesterase activity was lower, although these differences were minor.

The effects of cyclic AMP on disaggregation-induced changes in activities of developmentally regulated enzymes in [formula omitted

The addition of cyclic AMP to the shaking medium of cells disaggregated from pseudoplasmodia of Dictyostelium discoideum suppressed the accumulation of cell-bound phosphodiesterase which normally occurs (1) after disaggregation. The suppression was not secondarily brought about by its possible inhibitory effect of cyclic AMP on protein synthesis or by its stimulating effect on the release of the enzyme into the medium. The effect was reversible and specific to cyclic AMP. On the other hand, the inhibitory effect of cyclic AMP on the disaggregation-induced inactivation of UDP-galactose transferase was not apparent in the initial period, but thereafter it slowed down the decrease in the enzyme activity. These results indicate that exogenous cyclic AMP mimics at least in part the regulatory effects of cell-to-cell contact on certain enzymes.

Cyclic AMP, Cyclic AMP Phosphodiesterase, and the Duration of the Interphase in Dictyostelium discoideum

Summary After feeding has ended, amoebae of the cellular slime mold, Dictyostelium discoideum, enter a quiescent period (the interphase) before aggregation. Many characteristics required for aggregation develop in the amoebae during the interphase period, and the length of the interphase is affected by numerous factors including the density of the developing cell population. It was previously shown that the fewer the cells present, the longer the interphase [12, 13]. The present study is an investigation of the cell density dependence of the length of the interphase. It is shown that the interphase can be shortened for cell populations of low density by reducing the external cyclic AMP levels surrounding the amoebae. The same effect is obtained whether the cyclic AMP level is reduced by dilution into salt solution below the support medium, by absorption on to charcoal, or by destruction with the enzyme phosphodiesterase. Additional cyclic AMP in the support medium lengthens the interphase. Measurements of the cyclic AMP phophodiesterase levels produced by amoebae show that, under the condition of a reduced external cyclic AMP level, populations of low cell density not only aggregate earlier, but also aggregate at a lower level of cell-bound phosphodiesterase. At the time of aggregation, external cyclic AMP levels and cell-bound phosphodiesterase levels are seen to reach a specific ratio for lower cell densities. The relationship of cyclic AMP and cell-bound phosphodiesterase at aggregation is interpreted as important in the detection of a cyclic AMP gradient by the receptor system on the surface of the amoebae. It is suggested that low cell densities require a longer period of time to achieve the appropriate relationship between external cyclic AMP and cell-bound phosphodiesterase and therefore have a longer interphase.

Changes in activities of two developmentally regulated enzymes induced by disaggregation of the pseudoplasmodia of Dictyosteliumdiscoideum

Summary Changes in activities of two developmentally regulated enzymes were examined after disaggregation of the multicellular complex (pseudoplasmodium) of Dictyostelium discoideum . When disaggregated cells were incubated with shaking in a buffer containing EDTA, the activity of UDP-galactose transferase began to decrease almost immediately after disaggregation, with a half-life of 60–80 min (at 25°C). This decrease was induced even in the presence of cycloheximide and proceeded normally up to about 2 hrs of incubation. On the other hand, disaggregation caused an increase in the activity of cell-bound phosphodiesterase for cAMP. This process was sensitive to cycloheximide. These results indicate that at least two categories of changes are induced by loss of cell contact in the cellular slime mold.

Evidence for the existence of two types of cAMP binding sites in aggregating cells of dictyostelium discoideum

Inhibition by dithiothreitol of the cell-bound phosphodiesterase has allowed the measurement of cAMP binding to aggregation-competent Dictyostellum discoideum amebae. Two classes of binding sites were demonstrated: Type 1, of low affinity (Kd ≈ 160 nM) and high capacity (Ro ≈ 2 × 10 5 sites per cell); and Type 2, of high affinity (Kd ≈ 9 nM) but low capacity (Ro ≈ 1.5 × 10 4 sites per cell). Both sites are developmentally regulated and are expressed during aggregation. The specificities of both sites are consistent with the specificity observed in vivo for the chemotactic response.

The cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum. Binding characteristics of aggregation-competent cells and variation of binding levels during the life cycle.

Both cyclic guanosine 3':5'-monophosphate and dithiothreitol stimulate binding of cyclic adenosine 3':5'-monophosphate (cAMP) to aggregation-competent amoebae. Both compounds appear to function solely by preventing the hydrolysis of cAMP by the cell-bound phosphodiesterase. The dissociation constant for binding of cAMP is 36 nM. Both cAMP binding and membrane-bound phosphodiesterase activities increase dramatically as cells develop aggregation competence, reach a maximum at about 11 hours, and remain at high levels for up to 48 hours if cells are maintained in shaken suspension. When amoebae are allowed to aggregate and develop naturally, binding of cAMP increases during aggregation, decreases during tip formation, and disappears during culmination. Phosphodiesterase activity parallels binding activity except that the decreased level after tip formation is retained throughout culmination. Two N-6-modified cAMP derivatives compete with cAMP for binding sites. One derivative is fluorescent (1,N-6-etheno-cAMP); the other is photolyzable [N-6(ethyl-2-diazomalonyl)cAMP]. This result opens the possibilities of using fluorescence quenching for assay of in vitro binding and of affinity labeling of binding sites. Competition by the derivatives is only partial, indicating possible heterogeneity of binding sites. Both compounds inhibit hydrolysis of cAMP by the membrane-bound phosphodiesterase.

Induction of stalk cell differentiation by cyclic-AMP in a susceptible variant of Dictyostelium discoideum

The P-4 variant of Dictyostelium discoideum (DdH) was found to produce a great excess of stalk cells compared to the wild type DdH. If the vegetative cells of P-4 were repeatedly washed, the variant changed back to the wild type phenotype, and if cyclic-AMP was added to the washed P-4 cells, the variant character was restored. Furthermore, if the concentration of added cyclic-AMP was increased, it was possible to induce 100% stalk cells in P-4. Phosphodiesterase would cause the variant to change to the wild type, while 5-AMP and cyclic-nucleotides other than cyclic-AMP have no effect at all. Therefore it was concluded that cyclic-AMP plays a key role in stalk cell differentiation. A comparison between wild type DdH and the variant P-4 showed that DdH is ten times less sensitive to cyclic-AMP induction. They both produce the same amount of cyclic-AMP and extracellular phosphodiesterase, but the specific activity of P-4 cell-bound phosphodiesterase during development is significantly less than that in the DdH. One hypothesis that accounts for the P-4-DdH difference is that because of the lack of cell-bound phosphodiesterase, more cyclic-AMP enters the variant cells and hence more stalk cell differentiation.

A plausible role for a membrane-bound cyclic AMP phosphodiesterase in cellular slime mold chemotaxis

1. 1. Kinetics of membrane-bound cyclic AMP phosphodiesterase of the cellular slime mold, Dictyostelium discoideum, were studied under two conditions: in the 27 000 × g sediment of cell homogenates (particle-bound phosphodiesterase) and in cell suspensions using external cyclic AMP as a substrate (cell-bound phosphodiesterase). Both methods revealed non-Michaelian kinetics with interaction coefficients less than 1. 2. 2. The membrane-bound phosphodiesterase has a specificity different from that of the cyclic AMP receptor, also present at the cell surface. 3. 3. The membrane-bound enzyme was solubilized by lithium 3,5-diiodosalicylate and partially purified. In this state the non-linear kinetics were still retained; however, the enzyme was now inhibited by the D. discoideum inhibitor, unlike the cell-bound phosphodiesterase in vivo. This indicates that both enzymes share an inhibitor binding site and that this site is cryptic in the cell-bound state. 4. 4. Production of periodic cyclic AMP pulses by centers, and their relay by other cells, is believed to occur during aggregation. It is suggested that the cell-bound enzyme determines a “time window” significantly smaller than the period of pulsing, and optimizes stimulation of the cyclic AMP receptors in chemotaxis and signal relaying.

Short-term binding and hydrolysis of cyclic 3':5'-adenosine monophosphate by aggregating Dictyostelium cells.

Transient binding of cyclic AMP to aggregating cells of Dictyostelium discoideum was measured under cyclic GMP excess in order to inhibit cyclic AMP hydrolysis by cell-bound phosphodiesterase. Cyclic GMP extended the period of half-maximal cyclic AMP binding from less than 5 sec to 1-2 min. With the same time course as bound cyclic AMP was released from the cells, labeled 5'-AMP appeared in the medium. Specificity, kinetics, and developmental regulation suggest that the cyclic AMP-binding sites exposed in living cells are identical with receptor sites for the chemotactic response. The functioning of the cyclic AMP-receptor/phosphodiesterase system and its formal similarity with the synaptic acetylcholine-receptor/esterase system are discussed.

Cyclic AMP binding to living cells of Dictyostelium discoideum in presence of excess cyclic GMP

Summary [ 3 H]-cyclic AMP binding to living, aggregating cells of Dictyostelium discoideum can be detected if, simultaneously, excess cyclic GMP is added. The binding reaches a peak within less than half a minute after the cAMP pulse and the label is subsequently released into the intercellular space. It is argued that cGMP blocks cAMP-hydrolysis by cell-bound phosphodiesterase and interferes only to a minor extent with binding. Sensitivity, selectivity and developmental regulation are in accord with the function of the binding sites in the receptor system for the chemotactic response to cAMP.

Membrane-bound cyclic AMP phosphodiesterase in chemotactically responding cells of Dictyostelium discoideum.

In 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 ×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. In non‐aggregating mutants this increase is smaller or absent. The particle‐bound cyclic AMP phosphodiesterase has an optimum at pH 7.0–8.0. In vivo it is resistant to inhibition by external glutathione and to a macro‐molecular phosphodiesterase inhibitor from Dictyostelium discoideum; in particle fractions enzyme activity is partially sensitive to these inhibitors. A possible function of this enzyme in the chemo‐tactic receptor system is discussed.