Measurement of protein turnover rates by heavy water labeling of nonessential amino acids
Measurement of protein turnover rates by heavy water labeling of nonessential amino acids
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DOI：
10.1016/j.bbagen.2005.12.023
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发表时间：
2006-05-01
影响因子：
3
通讯作者：
Hellerstein, Marc K.
中科院分区：
文献类型：
--
作者：
Busch, Robert;Kim, Yoo-Kyeong;Hellerstein, Marc K.
使用同位素标记的氨基酸（AAS）对蛋白质合成的体内测量受到AA池的异质性的阻碍，并且对于缓慢的周转蛋白，长期标记的难度和费用。连续的口服重水（（H2O）-H-2）标记可以安全地维持稳定的体水H-2富集数周或数月。 H-2代谢纳入非必需AAS（NEAA）的C-H键，从而将其纳入蛋白质中。没有发生翻译后标签交换，因此原理H-2纳入了蛋白质NEAAS，报告了蛋白质合成。在这里，我们通过（HO）-H-2-O-2标记的啮齿动物组织蛋白的质量同位素分布分析（MIDA）表明，代谢H-2通量用于ALA，GLY或GLN用于蛋白质合成的C-H键是几乎完成。通过（H2O）-H-2标记啮齿动物的标记，量化了骨和肌肉混合蛋白的周转率，并检测到CCL4刺激肝胶原蛋白合成。还测量了几种人血清蛋白的动力学，重现已发表的t（1/2）估计值。不同蛋白质之间的高原富集在不同的蛋白质之间。在色谱或电泳上分离的蛋白质中等量足以进行动力学分析。总之，（H2O）-H-2标记允许通过上升到平原方法对体内蛋白质更新的敏感，定量，操作简单的测量，尤其是对于具有缓慢的本构转换的蛋白质。 （c）2006 Elsevier B.V.保留所有权利。
In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water ((H2O)-H-2) labeling can safely maintain stable body water H-2 enrichments for weeks or months. H-2 is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, So H-2 incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of (HO)-H-2-O-2-labeled rodent tissue proteins that metabolic H-2 flux into C-H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By (H2O)-H-2 labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl4 was detected. Kinetics of several human serum proteins were also measured, reproducing published t(1/2) estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, (H2O)-H-2 labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover. (c) 2006 Elsevier B.V. All rights reserved.