Wilk H E, Werr H, Friedrich D, Kiltz H H, Schäfer K P
Eur J Biochem. 1985 Jan 2;146(1):71-81. doi: 10.1111/j.1432-1033.1985.tb08621.x.
Ribonucleoprotein complexes (hnRNP) containing fragments of heterogeneous nuclear (hn)RNA and sedimenting at 35-40 S were isolated from the nuclei of HeLa S3 cells using the pH 8.0/diffusion technique. These hnRNP complexes are thought to be part of the hnRNA processing apparatus. The major protein components (core proteins) were identified by their constant ratios in native particles and in 35S hnRNP particles reconstituted in vitro. All of the core proteins, with one exception, show an increase in Mr on sodium dodecylsulfate (NaDodSO4)/polyacrylamide gels containing 8 M urea, indicative of secondary structure elements resistant to denaturation by NaDodSO4. The nine core proteins found by us are: A1 [Mr(NaDodSO4) 31 X 10(3)/Mr (urea) 38 X 10(3), apparent isoelectric point, pIapp 9.3], A2 (32.5 X 10(3)/39 X 10(3), 8.4), B1a (35.5 X 10(3)/41 X 10(3), 8.8), B1b (35.5 X 10(3)/44 X 10(3), 8.3), B1c (35.5 X 10(3)/43 X 10(3), 5.7) B2 (37 X 10(3)/42 X 10(3), 9.15), C1 (39 X 10(3)/46 X 10(3), 9.2), C2 (40.5 X 10(3)/45 X 10(3), 5.55) and C3 (38.5 X 10(3)/37 X 10(3), 4.8). Individual proteins were electroeluted from two-dimensional gels and their amino acid composition determined. Difference indices were calculated and show a group of closely related basic proteins (A1, A2, B1a, B1b, B2, C1), two related slightly acidic proteins (B1c, C2) and a distinct acidic member (C3). Two-dimensional analysis of tryptic fragments and one-dimensional separation of peptides after V8 protease treatment support these data. Peptide mapping of the proteins A1 and A2 from bovine and human cells yields identical fragments indicating a high degree of cross-species conservation. An additional protein (D4: 44 X 10(3)/55 X 10(3), greater than 9.5) was found, which preferentially associates with heavier, oligomeric hnRNP structures. Only traces of actin are present in the 35S hnRNP fraction. All core proteins are modified by charge. A large part of the charge isomers arises by phosphorylation, which has been shown by labeling with 32PO4 in vivo and with [gamma-32P]ATP in vitro. In vitro the phosphate transfer is mediated by an endogenous protein kinase associated with the 35S hnRNP complexes. The major core protein A1 exists in two conformeric forms (A1 and A1x) of which only A1x serves as phosphate acceptor in vivo.
采用pH 8.0/扩散技术，从HeLa S3细胞核中分离出含有不均一核（hn）RNA片段且沉降系数为35 - 40 S的核糖核蛋白复合物（hnRNP）。这些hnRNP复合物被认为是hnRNA加工装置的一部分。主要蛋白质成分（核心蛋白）通过其在天然颗粒和体外重构的35S hnRNP颗粒中的恒定比例来鉴定。除一种核心蛋白外，所有核心蛋白在含有8 M尿素的十二烷基硫酸钠（NaDodSO4）/聚丙烯酰胺凝胶上的Mr均增加，这表明存在对NaDodSO4变性有抗性的二级结构元件。我们发现的九种核心蛋白为：A1 [Mr(NaDodSO4) 31×10³/Mr (尿素) 38×10³，表观等电点，pIapp 9.3]，A2（32.5×10³/39×10³，8.4），B1a（35.5×10³/41×10³，8.8），B1b（35.5×10³/44×10³，8.3），B1c（35.5×10³/43×10³，5.7），B2（37×10³/42×10³，9.15），C1（39×10³/46×10³，9.2），C2（40.5×10³/45×10³，5.55）和C3（38.5×10³/37×10³，4.8）。从二维凝胶中电洗脱出各个蛋白质并测定其氨基酸组成。计算差异指数，结果显示有一组密切相关的碱性蛋白（A1、A2、B1a、B1b、B2、C1），两种相关的弱酸性蛋白（B1c、C2）和一个独特的酸性成员（C3）。胰蛋白酶片段的二维分析以及V8蛋白酶处理后肽段的一维分离支持了这些数据。来自牛和人细胞的蛋白质A1和A2的肽图谱产生相同的片段，表明高度的跨物种保守性。还发现了一种额外的蛋白质（D4：44×10³/55×10³，大于9.5），它优先与较重的寡聚hnRNP结构结合。在35S hnRNP组分中仅存在痕量的肌动蛋白。所有核心蛋白都有电荷修饰。大部分电荷异构体是由磷酸化产生的，这已通过体内用32PO4标记和体外用[γ-32P]ATP标记得到证实。在体外，磷酸转移由与35S hnRNP复合物相关的内源性蛋白激酶介导。主要核心蛋白A1以两种构象形式（A1和A1x）存在，其中只有A1x在体内作为磷酸受体。
