Effects of DNA and synthetic oligodeoxyribonucleotides on the binding properties of a cGMP-binding protein from Dictyostelium discoideum.
Previously, we identified a cGMP-binding protein (cGBP) in Dictyostelium discoideum that can exist in two forms: a fast-dissociating (F-type) activity and a slow-dissociating (S-type) activity. Moreover, the F-type activity was converted effectively to S-type by the addition of nucleic acids, especially DNA (Parissenti, A.M. and Coukell, M. B. (1989) J. Cell Sci. 92, 291-301). In this study, we examined the effects of heterologous DNA and various synthetic homo-oligodeoxyribonucleotides on the cGMP-binding properties of partially purified F-type activity. Equilibrium and kinetic binding experiments revealed that DNA increased the affinity of the protein for cGMP without altering the number of binding sites. However, the presence of DNA decreased only slightly the apparent Kd of the protein for cGMP because the nucleic acid also reduced the rate of cGMP association. Addition of oligo(dGMP)8 or oligo(dCMP)8 to the protein increased both total cGMP binding and the conversion of F-type activity to S-type; in contrast, oligo(dAMP)8 or oligo(dTMP)8, at the same concentration, had no effect. Oligodeoxycytidylic acids with chain lengths less than about eight nucleotides were also ineffective or inhibitory. Analysis of cGMP binding to intact, filipin-permeabilized cells revealed a binding activity with association and dissociation rates comparable to isolated S-type activity. This observation suggests that in vivo the cGBP might exist in its S-form.
Parissenti
A M
AM
Department of Biology, York University, North York, Ontario, Canada.
Coukell
M B
MB
D002352:Carrier Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D006152:Cyclic GMP / Q000378:metabolism*; D004247:DNA / Q000494:pharmacology*; D004023:Dictyostelium / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009838:Oligodeoxyribonucleotides; C022994:cyclic GMP-binding protein; D004247:DNA; D006152:Cyclic GMP