Botterell S H, Jans D A, Hemmings B A
Eur J Biochem. 1987 Apr 1;164(1):39-44. doi: 10.1111/j.1432-1033.1987.tb10989.x.
The catalytic (C) subunit activity of the cAMP-dependent protein kinase (cAMP-PK) from the mutant cell lines, FIB4 and FIB6, is only 10% compared with the parent cell line, LLC-PK1 [Jans and Hemmings (1986) FEBS Lett. 205, 127-131]. In order to understand the nature of the mutant phenotypes the cAMP-PK from parent and mutant cell lines was studied in more detail. Analysis of mutant cAMP-PK activity by ion-exchange chromatography revealed that kinase activity associated with type I holoenzyme of both FIB4 and FIB6 was only 5% parental, and the activity of the type II holoenzyme was about 20% parental. The type I regulatory (RI) subunits associated with the type I were also found to be reduced by 70-80% in both mutants, whereas the type II R subunit levels were similar to that of the parent. The residual kinase activity associated with the type I holoenzyme from FIB4 and FIB6 could not be activated by cAMP whereas the type II holoenzyme was activated by cAMP (Ka of 5.5 X 10(-8) M), and showed normal affinities for Kemptamide and ATP. A polyclonal antibody to the catalytic subunit was used to quantify the level of this protein in wild-type and mutant cells. This analysis showed that FIB4 and FIB6 had nearly normal levels of C subunit, suggesting that the C subunit synthesized by the mutants was mostly inactive. As both type I and type II cAMP-PK holoenzymes were abnormal, the most likely explanation of the mutant phenotype is a defect either in the structural gene for the C subunit or in an enzyme involved in its posttranslational processing. However, a second lesion affecting the RI subunit cannot be ruled out at this moment.
与亲本细胞系LLC-PK1相比，突变细胞系FIB4和FIB6中的环磷酸腺苷依赖性蛋白激酶（cAMP-PK）的催化（C）亚基活性仅为10%[扬斯和赫明斯（1986年）《欧洲生物化学学会联合会快报》205，127 - 131]。为了了解突变表型的本质，对亲本细胞系和突变细胞系中的cAMP-PK进行了更详细的研究。通过离子交换色谱分析突变型cAMP-PK活性发现，FIB4和FIB6的I型全酶相关的激酶活性仅为亲本的5%，II型全酶的活性约为亲本的20%。还发现与I型相关的I型调节（RI）亚基在两个突变体中也减少了70 - 80%，而II型R亚基水平与亲本相似。FIB4和FIB6的I型全酶相关的残余激酶活性不能被环磷酸腺苷激活，而II型全酶可被环磷酸腺苷激活（解离常数为5.5×10⁻⁸M），并且对肯普酰胺和三磷酸腺苷表现出正常亲和力。使用针对催化亚基的多克隆抗体来定量野生型和突变细胞中该蛋白的水平。该分析表明，FIB4和FIB6的C亚基水平几乎正常，这表明突变体合成的C亚基大多无活性。由于I型和II型cAMP-PK全酶均异常，突变表型最可能的解释是C亚基的结构基因或参与其翻译后加工的一种酶存在缺陷。然而，此时不能排除影响RI亚基的第二个损伤。
