No abstract

The cellular slime mold Dictyostelium discoideum has an intracellular phosphodiesterase which specifically hydrolyzes cGMP. The enzyme is activated by low cGMP concentrations, and is involved in the reduction of chemoattractant-mediated elevations of cGMP levels. The interaction of 20 cGMP derivatives with the activator site and with the catalytic site of the enzyme has been investigated. Binding of cGMP to the activator site is strongly reduced (more than 80-fold) if cGMP is no longer able to form a hydrogen bond at N2Hz or OZ'H. Modifications at N7, C8, 03' and 0'' induce only a small reduction of binding affinity. A cyclic phosphate structure, as well as a negatively charged oxygen atom at phosphorus, are essential to obtain activation of the enzyme. Substitution of the axial exocyclic oxygen atom by sulphur is tolerated; modification of the equatorial oxygen atom reduces the binding activity of cGMP to the activator site by 90-fold.Binding of cGMP to the catalytic site is strongly reduced if cGMP is modified at NIH, C'O, Cs and 03', while modifications at NZHz, N3, N7, 02'H, and 05' have minor effects. Both exocyclic oxygen atoms are important to obtain binding of cGMP to the catalytic site. The results indicate that activation of the enzyme by cGMP and hydrolysis of cGMP occur at different sites of the enzyme. cGMP is recognized at these sites by different types of molecular interaction between cGMP and the protein.cGMP derivatives at concentrations which saturate the activator site do not induce the same degree of activation of the enzyme (activation 2.3 -6.6-fold). The binding affinities of the analogues for the activator site and their maximal activation are not correlated. Our results suggest that the enzyme is activated because cGMP bound to the activator site stabilizes a state of the enzyme which has a higher affinity for cGMP at the catalytic site.Cyclic nucleotides have important functions in the cellular slime mold Dict-yostelium discoideum. This organism lives in the soil where it feeds on bacteria. When the food supply is exhausted the single cells aggregate to form a multicellular slug, which finally differentiates into a fruiting body. Cell aggregation is mediated by chemotaxis to CAMP, which is detected by cell surface receptors. Extracellular cAMP induces a rapid transient accumulation of intracellular cGMP levels, which reach a maximal concentration after about 10 s (for reviews see [l -31).D. discoidewn cells contain two classes of cyclic nucleotide phosphodiesterase activity. One class of enzymes hydrolyzes cAMP and cGMP with similar rates; these enzymes are located extracellularly, intracellularly and on the cell surface [4-61. A second class of enzymes hydrolyzes only cGMP and is localized only intracellularly [7 -91. Non-specific phosphodiesterase is present in large excess over the cCMP-specific enzyme. These enzyme activities can be easily separated by concanavalin-A -Sepharose column chromatography, since all CAMP-hydrolyzing activity binds to the column, while the cGMP-specific enz...

No abstract

No abstract

Dictyostelium discoideutn cells show 2 distinct classes of cell surface binding sites for folates. One type is non-specific, i.e., binds folic acid (FA), 2-deaminofolic acid (DAFA), and methotrexate (MTX) with similar affinity (K,, N 140 nM). Scatchard analysis of this non-specific binding type suggests either heterogeneity or negative cooperativity. Isolated D. discoideum membranes show similar binding characteristics. Guanine nucleotides changed the binding levels of [3H]MTX. In the presence of 0.1 mM GTP, the number of binding sites remains unchanged, while the affinity decreases. GDP and guanylyl imidodiphosphate (GPPNP) are required at about 20-fold higher concentration than GTP, which elicits a half-maximal effect at 15 FM. Other guanine and adenine nucleotides are ineffective up to 1 mM. These results suggests that the nonspecific cell surface receptor for folic acid interacts with a guanine nucleotide regulatory (G-) protein. Folate receptor Guanine nucleotide