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

TY - JOUR

T1 - Measurement of protein turnover rates by heavy water labeling of nonessential amino acids

AU - Busch, Robert

AU - Kim, Yoo-Kyeong

AU - Neese, Richard A

AU - Schade-Serin, Valerie

AU - Collins, Michelle

AU - Awada, Mohamad

AU - Gardner, James L

AU - Beysen, Carine

AU - Marino, Michael E

AU - Misell, Lisa M

AU - Hellerstein, Marc K

PY - 2006/5

Y1 - 2006/5

N2 - 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 (2H2O) 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 2H2O-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 2H2O 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 t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, 2H2O 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.

AB - 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 (2H2O) 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 2H2O-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 2H2O 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 t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, 2H2O 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.

KW - Amino Acids

KW - Animals

KW - Blood Proteins

KW - Brain

KW - Brain Chemistry

KW - Carbon Tetrachloride

KW - Collagen

KW - Deuterium Oxide

KW - Female

KW - Fibrosis

KW - Gas Chromatography-Mass Spectrometry

KW - Humans

KW - Isotope Labeling

KW - Kinetics

KW - Liver

KW - Male

KW - Mice

KW - Mice, Inbred C57BL

KW - Muscles

KW - Protein Biosynthesis

KW - Proteins

KW - Rats

KW - Rats, Sprague-Dawley

U2 - 10.1016/j.bbagen.2005.12.023

DO - 10.1016/j.bbagen.2005.12.023

M3 - Article

C2 - 16567052

SN - 0006-3002

VL - 1760

SP - 730

EP - 744

JO - Biochimica et biophysica acta

JF - Biochimica et biophysica acta

IS - 5

ER -