adp核糖化组蛋白H1。埃利希-腹水-肿瘤细胞核分离及部分鉴定。

H. C. Braeuer, P. Adamietz, U. Nellessen, H. Hilz
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引用次数: 16

摘要

在埃利希腹水肿瘤细胞的分离细胞核中形成的组蛋白H1和(adp -核糖)n之间的偶联物,使用高氯酸萃取、离子交换和硼酸-纤维素色谱法纯化了未修饰的H1。分离的偶联物占总蛋白结合adp核糖残基的0.6%,约占组蛋白H1群体的1%。酸/尿素凝胶的电泳分析显示,多个组分的迁移速度比未修饰的组蛋白H1慢。它们可以通过蛋白质染色或[3H] adp核糖残基的荧光成像可见。邻近的条带似乎因一个adp核糖残基而彼此不同。在大多数制剂中发现平均链长为2-3。用碱或磷酸二酯酶处理去除adp -核糖基,将条带移至未修饰的组蛋白H1的位置。在高分辨率的凝胶上,能带分裂成双峰,代表不同程度的磷酸化。在非adp核糖基化的对照组中也观察到相同的微观异质性。这表明组蛋白H1的磷酸化对adp -核糖基转移的受体性质没有显著影响。对(adp -核糖)n蛋白连锁的稳定性研究表明,约20%的adp -核糖由一个NH2OH/ naoh敏感键连接,70%由一个耐NH2OH/ naoh敏感键连接,剩余的10%显然由一个额外的耐naoh键连接。用n-溴琥珀酰亚胺切割(adp -核糖)n-组蛋白H1偶联物,并对这两个片段进行凝胶电泳分析,结果显示到目前为止,大多数adp -核糖残基(可能在多个位点)连接到c端片段。此外,大部分缀合物只在c端片段或n端片段上携带adp -核糖基。
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ADP-ribosylated histone H1. Isolation from Ehrlich-ascites-tumor-cell nuclei and partial characterization.
Conjugates between histone H1 and (ADP-ribose)n, formed in isolated nuclei of Ehrlich ascites tumor cells, were purified free of unmodified H1 using perchloric acid extraction, ion-exchange and boronate-cellulose chromatography. The isolated conjugates comprised 0.6% of the total protein-bound ADP-ribose residues, and about 1% of the histone H1 population. Electrophoretic analysis in acid/urea gels revealed the presence of multiple components migrating slower than unmodified histone H1. They could be made visible by staining for protein or by fluorography of the [3H]ADP-ribose residues. The neighboring bands appeared to differ from cach other by a single ADP-ribose residue. In most preparations a mean chain length of 2–3 was found. Removal of the ADP-ribosyl groups by treatment with alkali or phosphodiesterase shifted the bands to the position of unmodified histone H1. On higher-resolving gels the bands split into doublets representing different degrees of phosphorylation. The same microheterogeneity was also observed with the non-ADP-ribosylated control. This indicated that phosphorylation of histone H1 did not significantly influence the acceptor properties for ADP-ribosyl transfer. Studies on the lability of the (ADP-ribose)n protein linkage showed that about 20% of the ADP-ribose was linked by an NH2OH/NaOH-sensitive bond, 70% by an NH2OH-resistant/NaOH-sensitive bond, and the residual 10% apparently by an additional, NaOH-resistant bond. Cleavage of the (ADP-ribose)n- histone H1 conjugates by N-bromosuccinimide and gel eleetrophoretic analysis of the two fragments revealed that by far the most ADP-ribose residues were linked (presumably at multiple sites) to the C-terminal fragment. Furthermore, a large fraction of the conjugates carried ADP-ribosyl groups exclusively either at the C-terminal fragment or at the N-terminal fragment.
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