Comparison of the use of isotopic proline vs leucine to measure protein synthesis in cultured fibroblasts.
Compartmentation of the amino acid precursor pools for protein synthesis in cultured cells can substantially complicate measurements of synthesis rates. This is particularly true for nonessential amino acids such as proline, an amino acid often used in isotopic form to measure collagen synthesis. We have made a comparative study of this problem in cultured IMR-90 fibroblasts using isotopic proline and leucine to measure total protein and collagen synthesis. 3H-leucine in the extracellular (EC) medium equilibrates with tRNA-leucine at an EC concentration of 0.4 mM in both dividing and stationary cells. Thus, under these experimental conditions there is no complicating compartmentation of leucine for protein synthesis. Equilibration of EC and tRNA-bound 3H-proline, however, does not occur even when the EC concentration is in the mM range, based upon simultaneous measurements of synthesis rates using 3H-proline and 3H-leucine together. Furthermore, significant changes in EC proline concentration and specific activity occur over short time intervals (2 hr) if the initial EC proline concentration is below 0.2 mM. Thus, the use of isotopic proline to measure protein synthesis introduces substantial interpretive problems. Serum deprivation causes changes in both total collagen synthesis and the percent of protein synthesis devoted to collagen when measured with either 14C-leucine or 3H-proline. At the same time, isotopic proline remains the better choice for measuring percent collagen synthesis.
D002250:Carbon Radioisotopes; D002460:Cell Line; D003094:Collagen / Q000096:biosynthesis; D003470:Culture Media; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D014316:Tritium
D002250:Carbon Radioisotopes; D003470:Culture Media; D012346:RNA, Transfer, Amino Acyl; D014316:Tritium; D003094:Collagen; D011392:Proline; D007930:Leucine