In a wide range of Cell types, stimulus-response coupling is accompanied by a rise in cytoplasmic pH (pHi). It is shown that stimulation of developing Dictyostelium discoideum cells with the chemoattractant cAMP also results in a rise in pHi. About 1.5 rain after stimulation, pHi starts increasing from pHi~7.45 to pHi ~ 7.60, as is revealed independently by two different pH null-point methods. The rise in pHi is transient, also with a persistent stimulus, and effectively inhibited by diethylstilbestrol (DES), strongly suggesting that the rise in phi is accomplished by the DES-sensitive plasma membrane proton pump which has been demonstrated in D. discoideum.

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A series CAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for ;the two types of CAMP-binding sites in mammalian protein kinase type I. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that CAMP is bound to both sites in a comparable manner : the adenine appears to have no hydrogen-bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2', 3' and 5' positions of CAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation.The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocyclic oxygen atoms may result in a reduced maximal activation level and in one case, (RJ-adenosine 3', 5'-monophosphorothioate [R,-CAMPS], in total absence of activation even at concentrations at which the analog saturates both binding sites. Since occupancy of the CAMPbinding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize: the activation by CAMP. Indeed (RJ-CAMPS was found to inhibit CAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type I1 (R,)-CAMPS showed antagonistic activity, while with a CAMP-dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed.Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et al. (1982) Eur. J. Biochem. 122, 95 -991. This was based on measurements with impure preparations of (R,)-CAMPS and the R, and S, isomers adenosine 3', 5'-monophosphodimethylamidate, cAMPN(CH,), . The present work using highly purified compounds suggests the absence of a charge interaction, since the uncharged analog (S,)-cAMPN(CH,), activates the kinase effectively. The data seem compatible with an activation model involving the formation of a covalent bond with phosphorus in both CAMP binding sites.

Studies of the binding of four folate derivatives to the cell surface of Dictyostelium discoideum indicate the existence of five types of sites. About 99% of the total number of binding sites (160000 per cell) belongs to the 'non-selective' type, which recognizes folate, 2-deaminofolate and metbotrexate with equal affinity. As judged by the kinetics of association and dissociation this class consists of two distinct subtypes; a high-affinity site, designated by A n, and a low-affinity site A L. Upon addition of ligand a number of the low-affinity sites is converted to the high-affinity state. Prolonged dissociation revealed the presence of extremely slowly dissociating sites. While the A-sites released bound ligand within 5 s, the slow (B) type yielded a half-time of about 6 rain. This class (550 sites per cell) showed a clear selectivity for the four folates, with Nt0-methylfolate being the best ligand. From ~the kinetics of association and dissociation it is concluded that the B-sites are interconvertible with another binding type. In addition a class of sites was detected, which binds Nl0-methyifolate and folate with high affinity but 2-deaminofolate and methotrexate with approx. 100-fold lower affinity. Kinetic studies reveal that this C-class is also composed of two subtypes; a fast equilibrating site (within 1 s) designated as C r, and a slower site C s. It is proposed that before binding of ligand only C F exists, while after binding this binding type is converted into C s. At equilibrium more than 90% of the C-sites have attained the C s state.