Sources of amino acids for protein synthesis during early organogenesis in the rat. 2. Exchange with amino acid and protein pools in embryo and yolk sac.
Tenth-day rat conceptuses were cultured in whole rat serum containing [3H]leucine and harvested after 24 or 48 h. Hydrolysates of the acid-precipitable fraction of embryo or yolk-sac homogenates were prepared and subjected to paper chromatography. Liquid scintillation counting of the separated amino acids showed that leucine was the only amino acid with above-background radioactivity. This established that radiolabel was not transferred from leucine to other amino acids in the cultured rat conceptus. Tenth-day rat conceptuses were cultured in whole rat serum containing [3H]leucine, as above. After 19 h, some conceptuses were harvested; other conceptuses were rinsed, transferred to culture medium without [3H]leucine, and after a further 24 h of culture the embryos and yolk sacs were harvested. A comparison of the protein-associated radioactivity of embryo and yolk sac before and after culturing for the further 24-h period showed that these structures quantitatively conserve radiolabelled leucine incorporated into their proteins. Further experiments involved culturing the rat conceptus for 24 h as above but in the presence of either [3H]leucine or [3H]leucine-labelled serum proteins. After harvesting the conceptuses, the specific radioactivity of [3H]leucine was determined in the acid-soluble and acid-precipitable fractions prepared from embryo and yolk-sac homogenates. The specific radioactivity of [3H]leucine in the acid-soluble fraction of embryos or yolk sacs from conceptuses grown in the presence of radiolabelled protein was about 120 per cent of that in the culture medium, while that in the acid-precipitable fractions was about 70 per cent of that in the culture medium. By comparison, the specific radioactivity of [3H]leucine in the acid-soluble fraction of embryos and yolk sacs from conceptuses grown in the presence of free [3H]leucine was only 3-4 per cent of that in the culture medium, while that in the acid-precipitable fraction was about 1 per cent of that in the culture medium. If our data on the fate of leucine incorporated into newly synthesized proteins of the early post-implantation rat embryo can be extrapolated to the other amino acids, they suggest that once amino acids have been incorporated into newly synthesized protein in embryo or yolk sac, they are not exchanged to any detectable extent with amino acid pools outside the conceptus. The results also provide independent confirmation of our earlier conclusion that the only significant source of amino acids utilized by the 10th-day embryo is protein taken up by the yolk sac and digested intracellularly.(ABSTRACT TRUNCATED AT 400 WORDS)
Beckman
D A
DA
Department of Pediatrics, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania.
Pugarelli
J E
JE
Koszalka
T R
TR
Brent
R L
RL
Lloyd
J B
JB
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002854:Chromatography, Paper; D004622:Embryo, Mammalian / Q000378:metabolism*; D005326:Fetal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007930:Leucine; D051381:Rats; D014316:Tritium; D015017:Yolk Sac / Q000378:metabolism*
D000596:Amino Acids; D001798:Blood Proteins; D005326:Fetal Proteins; D014316:Tritium; D007930:Leucine