CHARACTERIZATION OF CHEMICAL AND ENZYMATIC ACID-LABILE PHOSPHORYLATION OF HISTONE H-4 USING P-31 NUCLEAR MAGNETIC-RESONANCE
CHARACTERIZATION OF CHEMICAL AND ENZYMATIC ACID-LABILE PHOSPHORYLATION OF HISTONE H-4 USING P-31 NUCLEAR MAGNETIC-RESONANCE
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DOI：
10.1021/bi00515a055
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发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
SMITH, RA
中科院分区：
文献类型：
--
作者：
FUJITAKI, JM;FUNG, G;SMITH, RA
31 P NMR用于研究组蛋白H4的酸不稳定磷酸化。31 P NMR检测体外酶促磷酸化H4中的磷酸化组氨酸残基。激酶的来源是来自再生大鼠肝脏或步行者-256癌肉瘤的细胞核。当再生的大鼠肝脏是源，31 P NMR光谱上的变性磷酸化蛋白质表现出共振在5.3 ppm相对于85%的Pi外部参考。该峰与标准π的化学位移很好地对应。在类似条件下扫描磷酸组氨酸。十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）证实了酸不稳定性。当激酶的来源是步行者-256癌肉瘤时，31 P NMR谱在4.9 ppm处含有共振，其与标准τ-256很好地对应。磷酸组氨酸在相同条件下运行。H4的化学磷酸化是通过使用氨基磷酸二钾，特异性磷酸化的咪唑部分的组氨酸在中性pH值。SDS-PAGE证实酸不稳定性，和蛋白质水解产物的高压液相色谱产生磷酸组氨酸。在结构化状态下的化学磷酸化的H4的31 P NMR显示在4.8和7.3ppm处的2个峰，线宽分别为9和55 Hz。这些共振表明H4的两个组氨酸残基（His-18和His-75）被磷酸化，后者相对不动，而前者在溶液中相对游离。H4的化学磷酸化肽片段的31 P NMR研究，即，H4（1-23）和H4（38-102）证实了这种H4结构模型。
31P NMR is used to investigate acid-labile phosphorylation of histone H4. 31P NMR detects phosphorylated histidine residues in in vitro enzymatically phosphorylated H4. The source of kinase is nuclei from either regenerating rat liver or Walker-256 carcinosarcoma. When regenerating rat liver is the source, 31P NMR spectroscopy on the denatured phosphorylated protein exhibits a resonance at 5.3 ppm relative to an 85% Pi external reference. This peak corresponds well with the chemical shift of standard .pi.-phosphohistidine scanned under similar conditions. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) confirms acid lability. When the source of kinase is Walker-256 carcinosarcoma, the 31P NMR spectrum contains a resonance at 4.9 ppm which corresponds well with standard .tau.-phosphohistidine run under the same conditions. Chemical phosphorylation of H4 was accomplished by using dipotassium phosphoramidate which specifically phosphorylated the imidazole moiety of histidine at neutral pH. SDS-PAGE confirms acid lability, and high-pressure liquid chromatography of protein hydrolysates yields phosphohistidine. 31P NMR of chemically phosphorylated H4 in a structured state reveals 2 peaks at 4.8 and 7.3 ppm with line widths of 9 and 55 Hz, respectively. These resonances indicate that both histidine residues of H4 (His-18 and His-75) are phosphorylated, the latter relatively immobile and the former relatively free in solution. 31P NMR studies on chemically phosphorylated peptide fragments of H4, i.e., H4(1-23) and H4(38-102), confirm this model of H4 structure.