Nature of the Inhibition of Protein Synthesis by Ethionine and Amino Acid Uptake in Early Sea Urchin Embryos

Abstract

The inhibition of protein synthesis by ethionine reported previously was found to be apparent, and ethionine inhibited only amino acid uptake like other usual amino acids. Even under such strong inhibition of the uptake, the syntheses of protein and DNA remained almost undiminished. The uptake of amino acid mixture by sea urchin embryos in the early cleavage stage was found to be carried out by active transport, since it was temperature-sensitive and was inhibited by 2,4-dinitrophenol. The uptake of an amino acid mixture or of single amino acids, e.g., valine, leucine and phenylalanine, was inhibited nonspecifically by an excess amount of other single amino acids added exogenously. Reflecting the inhibition of amino acid uptake, in vivo incorporation of amino acids into the protein fraction was apparently inhibited by excess amounts of other amino acids. As far as tested, the inhibition seems to be nonspecific and competitive for all amino acid species. The uptakes of leucine and phenylalanine were inhibited mutually by competition, with almost the same Km and Ki.In a previous paper (32), it was reported that lo-' M DL-ethionine almost completely inhibited in vivo protein synthesis by sea urchin embryos. A peculiar phenomenon, however, was then encountered: cell division was found to proceed almost normally under the strong inhibition of incorporation of exogenously added amino acids. Ethionine has been known to inhibit protein synthesis by degrading the polysomes ( 1 2 , 4 1-43) or causing the appearance of abnormal ribosomes (3), and also to interfere with the syntheses of DNA (24, 36) and RNA (3,37, 38, 43). Recent investigation has also demonstrated that ethionine inhibits the in vivo syntheses of these macromolecules in sea urchin embryos (14). If all of these observations are valid, one might conclude that the chemical processes for cell division in early development do not require protein synthesis, which is explicitly inconsistent with the present common knowledge.The present study was carried out to attempt to resolve these conflicts. The results indicate that the apparent inhibition of protein synthesis by ethionine is due to the inhibition of amino acid uptake by the presence of an excess amount of other amino acids, and represents a general case of competitive inhibition. MATERIALS AND METHODSMature eggs of the sea urchins, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus, were used. No detectable difference was found among these species. Fertilization was effected by