Grivennikov I A, Petukhov S P, Bulargina T V, Guliaev N N, Severin E S
Biokhimiia. 1984 Sep;49(9):1395-406.
The cAMP-dependent protein kinase from the soluble fraction of pigeon breast muscle is represented by two forms, PK I and PK II. The ratio of the phosphotransferase activity of the two forms is 35-40% and 60-65% for PK I and PK II, respectively. The regulatory subunit of PK I was isolated in a homogeneous state by affinity chromatography on 8-(2-oxoethylthio)-cAMP immobilized on epoxy-activated Sepharose 4B. The molecular weight of the regulatory subunit of PK I as determined by SDS polyacrylamide gel electrophoresis is 45 000. The specific cAMP-binding activity is equal to 16 nmol of [3H]cAMP per mg of protein. The apparent dissociation constant (Kd') for cAMP equals to 380 nM. The preparation of the regulatory subunit of PK II obtained by affinity chromatography on the same adsorbent is made up of polypeptides with Mr 56 000, 39 000, 29 000, 17 000 and 11 000. The preparation possesses a cAMP-binding activity of 22 nmol of [3H]cAMP per mg of protein. The interaction of several analogs of cAMP containing substituents at different positions of the nucleotide molecule with the regulatory subunit of PK I was studied. Practically all the analogs with substituents at positions 8 and 6 of the adenine ring in the cAMP molecule had the affinity which was 2-9 times less than that of cAMP. The only exceptions were 8-carboxymethylamino- and 8-(2-oxyethyl)-amino-cAMP whose binding to the regulatory subunit was 100 and 53 times lower than that of cAMP. The substitutions in position N-1 of the cAMP molecule leads to a 30-50-fold decrease of the analogs affinity. beta-Bromoethyl ester of cAMP does not reveal the ability to bind to the regulatory subunit. The carboxymethyl ester of cAMP possesses the affinity for the cAMP-binding site that is 35 times less than that of cAMP. Modification of the 2'-hydroxyl of ribose (as in the case of 2'-amino-2'-deoxy-8-hydroxy-cAMP, 2'-deoxy-cAMP and 2'-O-acrylyl-cAMP) decreases the affinity of these compounds 125-, 313- and 126-fold as compared with cAMP. It was assumed that the cAMP molecule is bound to the regulatory subunit in the syn-conformation. A structural model of the cAMP-binding site in the regulatory subunit of cAMP-dependent protein kinase I from pigeon breast muscle is proposed.
鸽胸肌可溶性部分的cAMP依赖性蛋白激酶有两种形式，即PK I和PK II。两种形式的磷酸转移酶活性之比，PK I为35 - 40%，PK II为60 - 65%。通过在固定于环氧活化的琼脂糖4B上的8 - (2 - 氧代乙基硫代)-cAMP上进行亲和层析，以均一状态分离出PK I的调节亚基。通过SDS聚丙烯酰胺凝胶电泳测定，PK I调节亚基的分子量为45000。其特异性cAMP结合活性等于每毫克蛋白质16 nmol的[³H]cAMP。cAMP的表观解离常数(Kd')等于380 nM。通过在相同吸附剂上进行亲和层析获得的PK II调节亚基制剂由分子量分别为56000、39000、29000、17000和11000的多肽组成。该制剂具有每毫克蛋白质22 nmol的[³H]cAMP的cAMP结合活性。研究了几种在核苷酸分子不同位置含有取代基的cAMP类似物与PK I调节亚基的相互作用。实际上，cAMP分子中腺嘌呤环8位和6位带有取代基的所有类似物的亲和力都比cAMP低2 - 9倍。唯一的例外是8 - 羧甲基氨基 - 和8 - (2 - 氧代乙基)-氨基 - cAMP，它们与调节亚基的结合比cAMP分别低100倍和53倍。cAMP分子N - 1位的取代导致类似物亲和力降低30 - 50倍。cAMP的β - 溴乙酯未显示出与调节亚基结合的能力。cAMP的羧甲基酯对cAMP结合位点的亲和力比cAMP低35倍。核糖2'-羟基的修饰（如2'-氨基 - 2'-脱氧 - 8 - 羟基 - cAMP、2'-脱氧 - cAMP和2'-O - 丙烯酰基 - cAMP的情况）使这些化合物的亲和力与cAMP相比分别降低125倍、313倍和126倍。据推测，cAMP分子以顺式构象与调节亚基结合。提出了鸽胸肌cAMP依赖性蛋白激酶I调节亚基中cAMP结合位点的结构模型。
