Friedman D L, Chambers D A
Proc Natl Acad Sci U S A. 1978 Nov;75(11):5286-90. doi: 10.1073/pnas.75.11.5286.
A photoaffinity labeling method was used to characterize and compare cyclic nucleotide-binding proteins of bovine liver cytosol with binding proteins of the nucleus. After photoaffinity labeling of cytosol with 8-azido cyclic [(32)P]AMP, autoradiographs of sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed two major labeled proteins of 47,000 and 52,000-55,000 daltons. DEAE-cellulose column-derived fractions suggested that the larger protein was the regulatory subunit of peak II cyclic AMP-dependent protein kinase and the smaller protein was the regulatory subunit of peak I kinase. The smaller protein was largely present as the free regulatory subunit. The two binding proteins differed in their ability to bind cyclic GMP. Binding to both proteins was abolished by excess unlabeled cyclic AMP but not by 5'-AMP. Photoaffinity labeling of a 0.14 M salt extract of nuclei and a nonhistone chromosomal protein preparation revealed two major binding proteins with the same molecular weight and competition profiles as those of the cytosol. Detergent-washed nuclei gave similar results. Several minor binding proteins were observed in both cytosol and nucleus. One protein (36,000 daltons) was unique to the nucleus and had low affinity for 8-azido cyclic AMP. Photoaffinity labeling with cyclic [(3)H]GMP revealed a cytosol protein, absent from the nucleus, of 31,000 daltons and the ligand was competed for by both cyclic GMP and 5'-GMP. These studies suggest that the major specific cyclic AMP-binding proteins of bovine liver are the type I and type II regulatory subunits of cyclic AMP-dependent protein kinase and are present in both nucleus and cytoplasm.
采用光亲和标记法对牛肝细胞溶质中的环核苷酸结合蛋白与细胞核中的结合蛋白进行表征和比较。用8-叠氮环[（32）P]AMP对细胞溶质进行光亲和标记后，十二烷基硫酸钠聚丙烯酰胺凝胶电泳的放射自显影片显示出两种主要的标记蛋白，分子量分别为47,000和52,000 - 55,000道尔顿。DEAE - 纤维素柱分离得到的组分表明，较大的蛋白是II型环磷酸腺苷依赖性蛋白激酶的调节亚基，较小的蛋白是I型激酶的调节亚基。较小的蛋白主要以游离调节亚基的形式存在。这两种结合蛋白结合环鸟苷酸的能力不同。过量未标记的环磷酸腺苷可消除与这两种蛋白的结合，但5'-AMP则不能。对细胞核的0.14M盐提取物和非组蛋白染色体蛋白制剂进行光亲和标记，结果显示出两种主要的结合蛋白，其分子量和竞争图谱与细胞溶质中的相同。用去污剂洗涤过的细胞核也得到了类似的结果。在细胞溶质和细胞核中均观察到几种次要的结合蛋白。一种蛋白（36,000道尔顿）是细胞核特有的，对8-叠氮环磷酸腺苷的亲和力较低。用环[（3）H]鸟苷酸进行光亲和标记，发现一种细胞核中不存在的31,000道尔顿的细胞溶质蛋白，环鸟苷酸和5'-鸟苷酸均可竞争该配体。这些研究表明，牛肝中主要的特异性环磷酸腺苷结合蛋白是环磷酸腺苷依赖性蛋白激酶的I型和II型调节亚基，它们同时存在于细胞核和细胞质中。
