Ahn Y S, Choi Y C, Goldknopf I L, Busch H
Biochemistry. 1985 Dec 3;24(25):7296-302. doi: 10.1021/bi00346a041.
A 125-kilodalton (kDa) phosphoprotein was isolated from nucleoli of Novikoff hepatoma cells in the presence of various inhibitors of proteases, alkaline phosphatase, and RNase. This protein was the most highly phosphorylated protein found thus far in the nucleolus. The half-life of [32P]phosphate in the 125-kDa phosphoprotein was approximately 60 min. Amino acid analysis of the protein showed it had a high serine content (15.5 mol %), a high glutamine plus glutamic acid content (15.5 mol %), and a high lysine content (10.3 mol %). Phosphoserine was the only phosphorylated amino acid identified. After alkaline hydrolysis of the 32P-labeled protein, ribonucleotides were found which accounted for approximately 8.5% of the [32P]phosphate. After cytidine 3',5'-[32P]diphosphate ([32P]pCp) labeling by RNA ligase, several oligoribonucleotide sequences were purified including GGGCOH and GGGGCOH. The binding of oligonucleotides to peptides was stable under denaturing fractionation conditions including 6 M urea treatment and incubation at 100 degrees C for 10 min in sodium dodecyl sulfate and beta-mercaptoethanol. Furthermore, when nucleotide-peptide complex was treated with ribonuclease T2 followed by snake venom phosphodiesterase, the junctional nucleotide pCp was released. These results suggest that one or more ribonucleotides are covalently bound to the 125-kDa phosphoprotein.
在存在各种蛋白酶、碱性磷酸酶和核糖核酸酶抑制剂的情况下，从诺维科夫肝癌细胞的核仁中分离出一种125千道尔顿（kDa）的磷蛋白。该蛋白是迄今为止在核仁中发现的磷酸化程度最高的蛋白。125-kDa磷蛋白中[32P]磷酸盐的半衰期约为60分钟。对该蛋白的氨基酸分析表明，它具有较高的丝氨酸含量（15.5摩尔%）、较高的谷氨酰胺加谷氨酸含量（15.5摩尔%）和较高的赖氨酸含量（10.3摩尔%）。磷酸丝氨酸是唯一鉴定出的磷酸化氨基酸。对32P标记的蛋白进行碱性水解后，发现了核糖核苷酸，其占[32P]磷酸盐的约8.5%。在用RNA连接酶进行胞苷3',5'-[32P]二磷酸（[32P]pCp）标记后，纯化了几个寡核糖核苷酸序列，包括GGGCOH和GGGGCOH。在变性分级分离条件下，包括6M尿素处理以及在十二烷基硫酸钠和β-巯基乙醇中于100℃孵育10分钟，寡核苷酸与肽的结合是稳定的。此外，当核苷酸-肽复合物用核糖核酸酶T2处理，随后用蛇毒磷酸二酯酶处理时，连接核苷酸pCp被释放。这些结果表明一个或多个核糖核苷酸与125-kDa磷蛋白共价结合。
