Open AccessEffect of Cyclic Nucleotide Analogs on Intrachain Site 1 of Protein Kinase Isozymes
Author(s) -
CORBIN Jackie D.,
RANNELS Stephen R.,
FLOCKHART David A.,
TIGANI Michael C.,
DØSKELAND Stein O.,
SUVA Robert H.,
SUVA Robert,
MILLER Jon P.
Publication year - 1982
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1982.tb06677.x
Subject(s) - isozyme , chemistry , binding site , selectivity , active site , protein subunit , stereochemistry , protein kinase a , nucleotide , dissociation constant , biochemistry , enzyme , gene , receptor , catalysis
The effects of numerous cAMP analogs present in the [ 3 H]cAMP binding reaction on subsequent dissociation of [ 3 H]cAMP from the regulatory subunit of CAMP‐dependent protein kinase I and I1 were analyzed. Certain analogs with modification at either C‐8 or C‐2 showed relative selectivity for one (site 1) of two intrachain cAMP binding sites of both isozymes. Modification at C‐6 caused selectivity for the second site (site 2). The combination of a site‐I‐directed and site‐Zdirected analog inhibited [ 3 H]cAMP binding much more than did either analog alone. In general, there was a correlation between the site 1 selectivity and the ability of the analog to stimulate the binding of [ 3 H]clMP, which selects site 2. The site‐1‐directed analogs stimulated the initial rate of [ 3 H]cIMP binding. The stimulatory effect was enhanced in the presence of a polycationic protein such as histone and was inhibited by high ionic strength. The type I and II isozymes exhibited large differences in analog specificity for this effect. For type I, of the analogs tested the most efficacious for stimulating [ 3 H]cIMP binding were those containing a nitrogen atom attached to C‐8, 8‐aminobutylamino‐CAMP being the most effective. Type I1 responded best to analogs con‐ taining a sulfur atom attached to C‐8, 8‐SH‐CAMP being the most effective of those tested. The stimulatory effect was accentuated in the presence of MgATP when using type I, but this nucleotide had no effect when using type II. It is proposed that in intact tissues cAMP binding to site 1 of either isozyme stimulates the binding to site 2.