Measurement of protein turnover rates by heavy water labeling of nonessential amino acids

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 ( 2H 2O) labeling can safely maintain stable body water 2H en...

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Published in:	Biochimica et biophysica acta Vol. 1760; no. 5; pp. 730 - 744
Main Authors:	Busch, Robert, Kim, Yoo-Kyeong, Neese, Richard A., Schade-Serin, Valerie, Collins, Michelle, Awada, Mohamad, Gardner, James L., Beysen, Carine, Marino, Michael E., Misell, Lisa M., Hellerstein, Marc K.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01-05-2006
Subjects:	Amino Acids - metabolism Animals Blood Proteins - analysis Blood Proteins - metabolism Brain - metabolism Brain Chemistry Carbon Tetrachloride - toxicity Collagen - analysis Collagen - metabolism Deuterated water Deuterium Oxide - administration & dosage Deuterium Oxide - pharmacokinetics Female Fibrosis Gas Chromatography-Mass Spectrometry Humans Isotope Labeling - methods Kinetics Liver - drug effects Liver - metabolism Male Mass isotopomer distribution analysis Mice Mice, Inbred C57BL Muscles - chemistry Muscles - metabolism Protein Biosynthesis Protein synthesis/turnover Proteins - analysis Proteins - metabolism Rats Rats, Sprague-Dawley Stable isotope labeling Gas chromatography/mass spectrometry 2H 2O Mass isotopomer distribution analysis PFB BSA AAs LDL 2H VLDL NEAAs m/z Stable isotope labeling ApoB Protein synthesis/turnover Deuterated water PBS tRNA PTFE EDTA GCRC SEC SDS-PAGE PFBBr PMSF NCI PCI MIDA GC/MS Ig ELISA
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Summary:	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 ( 2H 2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C–H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so 2H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of 2H 2O-labeled rodent tissue proteins that metabolic 2H flux into C–H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By 2H 2O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl 4 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, 2H 2O 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.
ISSN:	0304-4165 0006-3002 1872-8006
DOI:	10.1016/j.bbagen.2005.12.023