...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >ATP nucleotidylation of creatine kinase.
【24h】

ATP nucleotidylation of creatine kinase.

机译：肌酸激酶的ATP核苷酸化。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.
机译：肌酸激酶（CK）会自动掺入来自[gamma32P] ATP的放射性标记，因此据报导会被自动磷酸化。此外，与正常的脑酶相反，阿尔茨海默氏病脑匀浆中的CK显示活性大大降低，[32P] 8N3ATP取消了光标记，[γ32P] ATP也未检测到放射性标记的自动掺入。出乎意料的是，我们对人脑和纯净CK的研究表明，[α32P] ATP，[γ32P] ATP，[α32P] ADP，[2,8H3] ATP，[γ32P] 2'，3'-O-（2,4 ，6-三硝基苯基）-ATP和[gamma32P]二苯甲酮-gammaATP都将放射性标记自动结合到CK中，效率很高。这证明了γ-磷酸和2'和3'羟基不参与共价键，并且所有三种磷酸，ATP分子的核糖和碱基在自掺入（核苷酸化）后均被保留。用NaIO3处理以破坏2'-3'连锁会导致放射性标记的完全丧失，这表明核苷酸化会导致C1'位置的核糖环打开。在37摄氏度下，随着[alpha32P] ATP的增加而进行的核苷酸化作用将使k0.5约为125 microM，并在340 microM核苷酸处饱和。拷贝数的8-10％的修饰在饱和时发生，并且CK活性被抑制到大致相同的程度。低微摩尔水平的天然底物（如ADP，ATP和磷酸肌酸）会大大降低[alpha32P] ATP核苷酸的形成。相反，AMP，GTP，GMP，NADH和肌酸并不能有效地减少核苷酸。当用胰蛋白酶处理[α32P] ATP核苷酸化或[α32P] 8N3ATP-光标记的CK时，会生成单一的相同的放射性标记的肽（V279-R291），该肽在反相HPLC和Tris-tricine电泳时开始迁移。 ATP的存在可防止86％的核苷酸被糖基化。我们得出的结论是，CK通过在C1'位置的修饰而在活性位点被核苷酸化，并且不会发生该酶的自磷酸化。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号