| Autor: |
Van Lookeren Campagne MM; Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032., Villalba Díaz F, Chason KW, Kessin RH |
| Jazyk: |
angličtina |
| Zdroj: |
Analytical biochemistry [Anal Biochem] 1990 Jul; Vol. 188 (1), pp. 86-90. |
| DOI: |
10.1016/0003-2697(90)90531-d |
| Abstrakt: |
(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%. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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