Abstract
The two exocyclic oxygen atoms at phosphorus of cAMP have been replaced by a sulfur atom or by a dimethylamino group. These substitutions introduce chirality at the phosphorus atom; therefore, two diastereoisomers are known for each derivative: (SP)-cAMPS, (RP)-cAMPS, (SP)-cAMPN(CH3)2, and RP-cAMPN(CH3)2. We have investigated the agonistic and antagonistic activities of these compounds in four cAMP-dependent reactions: activation of the cellular slime mold Dictyostelium discoideum via its cell surface cAMP receptor, and phosphorylation by cAMP-dependent protein kinases type I, type II (both mammalian enzymes), and type D (derived from D. discoideum). The results show that 1) the compounds (SP)-cAMPS and (SP)-cAMPN(CH3)2 are (mostly full) agonists for the four proteins. Half-maximal activation is at micromolar concentrations (0.8-7 microM). 2) (RP)-cAMPS is a full antagonist for the cell surface receptor and protein kinases type I and II, with apparent inhibition constants between 0.8 and 8 microM. This compound is a partial agonist for protein kinase type D, where it induces maximally 50% activation of the enzyme if compared with cAMP. 3) (RP)-cAMPN(CH3)2 is a full antagonist for the cell surface receptor, and for protein kinase type II. This compound is a partial agonist for protein kinase type I (at least 50% activation if compared with cAMP), and inactive for protein kinase type D. This derivative is at least 25-fold less active as an antagonist than (RP)-cAMPS. 4) The activity of mixtures of different concentrations of the antagonist (RP)-cAMPS with different concentrations of cAMP reveals that the compound is a competitive antagonist of cAMP at micromolar concentrations.
Highlights
De Wit et al [4] were first to report on the differential activation of CAMP-dependent protein kinasetype I from rabbit muscle by(Sp)-CAMPS an(dRp)-CAMPS.They showed that binding of the S p isomer to the enzyme yields complete activation of the protein kinase
A Lineweaver-Burk plot of the data is shown in E; preincubation without (O),or with 2.5 p~ (0),or 8.33 p~ (A)(Rp)-cAMPS
CAMP, (Sp)-CAMPS, and (Sp)-cAMPN(CH3)2 concentrations ( 5 ) .To test the compounds for antagonistic stimulate protein kinase type 11, while both response in D. discoideum. (Rp) isomers are properties, the cells were mixed with the Rp stereoisomers inactive
Summary
A . c y c l ~ cnucleotide concentratlon antagonistic activities of the highly purified preparations of the S pand R p isomers of CAMPS and cAMPN(CH&for four. C y c l ~ cnucleotide concentratlon antagonistic activities of the highly purified preparations of the S pand R p isomers of CAMPS and cAMPN(CH&for four. CAMP-dependent proteins: the cell surfacecAMP receptor from D.discoideum, CAMP-dependent protein kinase type I z from beef heart, type I1 from rabbit muscle, and typeD from. The resultsshow that antagonists are present among these four derivatives
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