Guanylate cyclase activity in permeabilized Dictyostelium discoideum cells.

Dictyostelium discoideum cells respond to chemoattractants by transient activation of guanylate cyclase. Cyclic GMP is a second messenger that transduces the chemotactic signal. We used an electropermeabilized cell system to investigate the regulation of guanylate cyclase. Enzyme activity in permeabilized cells was dependent on the presence of a nonhydrolysable GTP analogue (e.g., GTP gamma S), which could not be replaced by GTP, GDP, or GMP. After the initiation of the guanylate cyclase reaction in permeabilized cells only a short burst of activity is observed, because the enzyme is inactivated with a t1/2 of about 15 s. We show that inactivation is not due to lack of substrate, resealing of the pores in the cell membrane, product inhibition by cGMP, or intrinsic instability of the enzyme. Physiological concentrations of Ca2+ ions inhibited the enzyme (half-maximal effect at 0.3 microM), whereas InsP3 had no effect. Once inactivated, the enzyme could only be reactivated after homogenization of the permeabilized cells and removal of the soluble cell fraction. This suggests that a soluble factor is involved in an autonomous process that inactivates guanylate cyclase and is triggered only after the enzyme is activated. The initial rate of guanylate cyclase activity in permeabilized cells is similar to that in intact, chemotactically activated cells. Moreover, the rate of inactivation of the enzyme in permeabilized cells and that due to adaptation in vivo are about equal. This suggests that the activation and inactivation of guanylate cyclase observed in this permeabilized cell system is related to that of chemotactic activation and adaptation in intact cells.

Transformation with vectors harboring the NEOR selection marker induces germination-specific adenylylcyclase activity in Dictyostelium cells.

Dictyostelium cells express an aggregative adenylylcyclase (ACA), responsible for oscillatory cAMP signaling, and a spore germination-specific adenylylcyclase (ACG). Overexpression of PKA regulatory (R) subunits blocks oscillatory cAMP signaling but increases basal cAMP levels, while neither ACA nor ACG mRNA could be detected. To test whether a novel type of adenylylcyclase (AC) was responsible for cAMP synthesis, dominant-negative PKA-R subunits (PKA-RM) and control R-subunits (PKA-RC) were overexpressed in ACA null mutants. Both transformations as well as transformation with an unrelated vector, carrying the same NEOR selection marker, induced an AC activity in growing cells with the biochemical characteristics of ACG. Similar vectors with a different URA selection marker did not increase AC activity. We conclude that the amino-glycoside phosphotransferase encoded by the very commonly used NEOR selection marker induces ectopic ACG activity in Dictyostelium cells.

A soluble factor and GTP gamma S are required for Dictyostelium discoideum guanylate cyclase activity.

Amoeba of Dictyostelium discoideum show a rapid, transient cGMP synthesis in response to chemotactic stimulation. Using Mg(2+)-GTP as a substrate, guanylate cyclase (E.C. 4.6.1.2.) activity is found exclusively in the particulate fraction of Dictyostelium cells. Here we show that the activity is dependent on the presence of the non-hydrolysable GTP-analogue GTP gamma S, which itself is only a poor substrate for the enzyme under the prevailing conditions. Evidence is presented that a transient exposure of the enzyme to GTP gamma S is sufficient to constitutively activate the enzyme. GTP gamma S-dependent activity is found to require a factor that can be separated from the enzyme by washing the particulate fraction with low salt buffer. Addition of the soluble cell fraction to these washed membranes restores enzyme activity.