title: Regulation of Dictyostelium discoideum adenylate cyclase by manganese and adenosine analogs.
abstract: Adenylate cyclase is the critical enzyme in the chemotactic signal relay mechanism of the slime mold amoeba, Dictyostelium discoideum. However, few studies examining the regulation of this enzyme have been performed in vitro due to the instability of enzyme activity in crude lysates. For studies presented in this communication, a membrane preparation has been isolated that exhibits a high specific activity adenylate cyclase that is stable during storage at -70 degrees C and under assay conditions at 27 degrees C. The enzyme was activated by micromolar concentrations of MnCl2. GTP and its non-hydrolyzable analog, guanosine 5'-(beta, gamma-imino)triphosphate, inhibited the enzyme non-competitively in the presence of either Mg2+ or Mn2+. However, this inhibition was more pronounced in the presence of Mn2+. Since guanylate cyclase activity in the D. discoideum membranes was less than 10% of the adenylate cyclase activity, there could not be a significant contribution by guanylate cyclase toward the production of cyclic AMP. Experiments indicate that D. discoideum adenylate cyclase was also regulated by adenosine analogs. The enzyme was inhibited by 2',5'-dideoxyadenosine and 2'-deoxyadenosine and inhibition was augmented by the presence of Mn2+. However, the inhibition was not entirely consistent with that which would be expected for the P-site of eukaryotic systems because some purine-modified adenosine analogs also inhibited the enzyme. Guanine nucleotides had no effect on the inhibition by either purine-modified or ribose-modified adenosine analogs. The binding of cyclic AMP to its receptor on the D. discoideum membranes was not affected by either MnCl2 or adenosine analogs.
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000494:Allosteric Regulation; D002712:Chlorides; D000242:Cyclic AMP / Q000096:biosynthesis; D004023:Dictyostelium / Q000201:enzymology*; D005656:Fungal Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008345:Manganese / Q000494:pharmacology*; D017895:Manganese Compounds; D008565:Membrane Proteins / Q000378:metabolism; D013696:Temperature
chemical_list: D002712:Chlorides; D005656:Fungal Proteins; D017895:Manganese Compounds; D008565:Membrane Proteins; D015636:Magnesium Chloride; D008345:Manganese; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; D008274:Magnesium; D000241:Adenosine; C025340:manganese chloride