Amino acid incorporation into interior protein sites by purified rat liver systems

Abstract

M ETHODS have recently been described for the preparation of metabolically active RNP-particles2 from rat liver microsomes [2, 61. These particles readily incorporate labeled amino acids into protein in the presence of a soluble enzyme fraction from rat liver and a nucleoside triphosphate generating system. The particles have been used also in incorporation experiments with “S-RNA”-bound %-amino acids as the labeled precursor [a]. In the presence of soluble liver enzymes and GTP [l] the bound amino acids are rapidly transferred from the S-RNA to particles and incorporated into protein. In experiments with free RNP-particles from peas \Vebster has recently observed that labeled S-RNA-bound amino acids become incorporated only into the N-terminal ends of the growing protein chains [8]. The same was true when free, labeled amino acids were used in incubation systems with pea particles in the absence of a full amino acid complement [9]. The experiments to be described here show that the RNP-particles from liver differ from the pea seedling particles in these respects. Irrespective of whether the labeled amino acids or S-RNA-bound amino acids were added to the incubation system singly, or in the presence of other amino acids, they became incorporated mainly into interior protein sites. Experimental.-The following kinds of incorporation systems were used: (I) Livers from 70-80 g rats were perfused in situ with 0.15 M KC1 and homogenized with 2.5 volumes of an ice-cold medium containing 0.25 M sucrose, 0.025 M KCl, 0.01 M MgCl, and 0.035 M Tris. The homogenate was centrifuged at 12,000 xg for 8 minutes. To each incubation tube 0.8 ml of this mitochondria-free homogenate was added together with 1.3 pmoles of ATP, 13 pmoles of PEP and 0.125 pmoles of 14C-L-valine (8.0 pc/,umole). The tubes (final volumes 1.5 ml) were incubated for 40 minutes (35°C). (2) 1vashed RNP particles were prepared from rat liver microsomes as in previous experiments [2]. The particles were incubated for 30 minutes with 0.5 ml of the