Characterization of two unusual guanylyl cyclases from dictyostelium.
Guanylyl cyclase A (GCA) and soluble guanylyl cyclase (sGC) encode GCs in Dictyostelium and have a topology similar to 12-transmembrane and soluble adenylyl cyclase, respectively. We demonstrate that all detectable GC activity is lost in a cell line in which both genes have been inactivated. Cell lines with one gene inactivated were used to characterize the other guanylyl cyclase (i.e. GCA in sgc(minus sign) null cells and sGC in gca(minus sign) null cells). Despite the different topologies, the enzymes have many properties in common. In vivo, extracellular cAMP activates both enzymes via a G-protein-coupled receptor. In vitro, both enzymes are activated by GTPgammaS (K(a) = 11 and 8 microm for GCA and sGC, respectively). The addition of GTPgammaS leads to a 1.5-fold increase of V(max) and a 3.5-fold increase of the affinity for GTP. Ca(2+) inhibits both GCA and sGC with K(i) of about 50 and 200 nm, respectively. Other biochemical properties are very different; GCA is expressed mainly during growth and multicellular development, whereas sGC is expressed mainly during cell aggregation. Folic acid and cAMP activate GCA maximally about 2.5-fold, whereas sGC is activated about 8-fold. Osmotic stress strongly stimulates sGC but has no effect on GCA activity. Finally, GCA is exclusively membrane-bound and is active mainly with Mg(2+), whereas sGC is predominantly soluble and more active with Mn(2+).
Roelofs
Jeroen
J
Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Van Haastert
Peter J M
PJ
D000818:Animals; D002384:Catalysis; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000502:physiology; D016837:Dictyosteliida / Q000201:enzymology*; D005492:Folic Acid / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009997:Osmotic Pressure
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008345:Manganese; D005492:Folic Acid; D000242:Cyclic AMP; C437313:SgcA protein, Dictyostelium discoideum; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D008274:Magnesium