Seville M, Holbrook J J
Anal Biochem. 1984 Mar;137(2):330-4. doi: 10.1016/0003-2697(84)90094-0.
A nondenaturing method for the preparation of R subunits from type II cyclic AMP-dependent protein kinase is described. The procedure is based on the exchange of cyclic AMP, which is tightly bound to the R subunit, for more weakly bound cyclic GMP, which can be removed by washing and dialysis. Less than 5% of the available cyclic nucleotide-binding sites of R subunit prepared by this method contained cyclic AMP and less than 3% contained cyclic GMP. The C-subunit contamination (mol of C/mol of R monomer) was approximately 0.2%. These levels of contamination did not affect the properties of the R subunit as judged by (a) the ability of the R subunit to inhibit the activity of the C subunit and (b) the rate of exchange of cAMP into R2 . etheno-cAMP. The advantages of our method are that the protein is not subjected to denaturing conditions and that large amounts of material can be processed relatively rapidly.
本文描述了一种用于从II型环磷酸腺苷依赖性蛋白激酶制备R亚基的非变性方法。该方法基于将紧密结合在R亚基上的环磷酸腺苷与结合较弱的环磷酸鸟苷进行交换，环磷酸鸟苷可通过洗涤和透析去除。用这种方法制备的R亚基中，不到5%的可用环核苷酸结合位点含有环磷酸腺苷，不到3%含有环磷酸鸟苷。C亚基污染（C亚基摩尔数/R单体摩尔数）约为0.2%。根据以下方面判断，这些污染水平不影响R亚基的性质：(a)R亚基抑制C亚基活性的能力；(b)环磷酸腺苷与R2·乙烯基环磷酸腺苷的交换速率。我们方法的优点是蛋白质不经过变性条件，并且可以相对快速地处理大量材料。
