ABSTRACT
A genetic selection procedure has been developed which makes the growth of E. coli dependent on expression of a cGMP phosphodiesterase cDNA. E. coli does not contain a cGMP phosphodiesterase, and guanine auxotrophs cannot extract the guanine from cGMP. If a functional cGMP phosphodiesterase is introduced, then guaA auxotrophs will grow on cGMP as a guanine source. The method also selects GMP synthetase cDNAs, which complement the guanine auxotrophy directly. Expression of a Dictyostelium discoideum or human heart cyclic nucleotide phosphodiesterase cDNA permits growth of the E. coli guaA auxotroph in the presence of cGMP. Several commercial cDNA libraries were corrupt and contained phosphodiesterase and/or GMP synthetase sequences that were from a contaminating DNA source.
Subject(s)
3',5'-Cyclic-GMP Phosphodiesterases/metabolism , DNA/genetics , Escherichia coli/enzymology , Gene Expression Regulation, Enzymologic , Guanosine Monophosphate/metabolism , 3',5'-Cyclic-GMP Phosphodiesterases/genetics , Animals , Cyclic GMP/metabolism , Dictyostelium/enzymology , Escherichia coli/genetics , Humans , Hydrolysis , Myocardium/enzymology , Plasmids , Selection, GeneticABSTRACT
8-Chloroadenosine 3',5'-monophosphate has been reported to inhibit growth of various mammalian cell lines at micromolar concentrations. We have used Chinese hamster ovary cell lines with mutated cyclic AMP-dependent protein kinase or altered cyclic nucleotide metabolism to show that a metabolite, 8-chloroadenosine, is formed in the medium and is the active inhibitor of cell growth in Chinese hamster ovary cells. Adding adenosine deaminase to the Chinese hamster ovary cell growth media removes the inhibition of cell growth attributed to 8-chloroadenosine 3',5'-monophosphate. Adenosine deaminase or dipyridamole also protects Molt-4 lymphoblasts from the growth-inhibitory effects of 8-chloroadenosine 3',5'-monophosphate.
Subject(s)
8-Bromo Cyclic Adenosine Monophosphate/analogs & derivatives , Adenosine/metabolism , Antineoplastic Agents/pharmacology , 8-Bromo Cyclic Adenosine Monophosphate/metabolism , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , 8-Bromo Cyclic Adenosine Monophosphate/therapeutic use , Adenosine Deaminase/pharmacology , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/therapeutic use , Cell Division/drug effects , Cells, Cultured , Cricetinae , Protein Kinases/metabolismABSTRACT
(Rp)-Adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) is a highly specific antagonist of the cAMP-dependent protein kinase from eukaryotic cells and is a very poor substrate for phosphodiesterases. It is therefore a useful tool for investigating the role of cAMP as a second messenger in a variety of biological systems. Taking advantage of stereospecific inversion of configuration around the alpha-phosphate during the adenylate cyclase reaction, we have developed a method for the preparative enzymatic synthesis of the Rp diastereomer of adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) from the Sp diastereomer of adenosine 5'-O-(1-thiotriphosphate) ((Sp)-ATP alpha S). The adenylate cyclase from Bordetella pertussis, partially purified by calmodulin affinity chromatography, cyclizes (Sp)-ATP alpha S approximately 40-fold more slowly than ATP, but binds (Sp)-ATP alpha S with about 10-fold higher affinity than ATP. The triethylammonium salt of the reaction product can be purified by elution from a gravity flow reversed-phase C18 column with a linear gradient of increasing concentrations of methanol. Yields of the pure (Rp)-cAMPS product of a synthesis with 2 mg of substrate are about 75%.
