The initiation of haemoglobin synthesis in rabbit reticulocytes.
1. The incorporation of labelled valine by rabbit reticulocytes into the N-terminal position of nascent haemoglobin was investigated by deaminating the nascent peptides with nitrous acid and isolating labelled alpha-hydroxyisovaleric acid and valine after acid hydrolysis. 2. The amount of radioactivity in alpha-hydroxyisovaleric acid relative to that in valine indicated the presence of 12.3% N-terminal valine having a free amino group. This high value suggests that most if not all nascent peptides contain valine in the N-terminal position. 3. Cell-free preparations containing reticulocyte ribosomes and pH5 enzymes incorporated alpha-hydroxy-[(14)C]isovaleryl-tRNA (where tRNA refers to transfer RNA), which was obtained by deamination of [(14)C]valyl-tRNA from yeast or liver with nitrous acid, into both soluble and nascent protein. 4. When the soluble protein was chromatographed on CM-cellulose, radioactivity was found to be associated with both the alpha-and beta-globin chains. 5. The kinetics of hydrolysis of [(14)C]valine, was also investigated. Most of the material was hydrolysed rapidly at pH10, but a minor component that was relatively stable appeared to be present to the extent of about 10% of the total valyl-tRNA. Valine was, however, the only hydrolysis product detected by paper chromatography. 6. It is concluded that chain initiation in haemoglobin synthesis involves valine as the N-terminal amino acid and that the amino group of nascent protein is probably not substituted.
D000818:Animals; D002247:Carbon Isotopes; D002845:Chromatography; D002854:Chromatography, Paper; D006454:Hemoglobins / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D009573:Nitrites / Q000494:pharmacology; D012343:RNA, Transfer / Q000096:biosynthesis; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D014633:Valine / Q000378:metabolism; D015003:Yeasts
D002247:Carbon Isotopes; D006454:Hemoglobins; D009573:Nitrites; D012343:RNA, Transfer; D014633:Valine