Studies of the protein synthetic activity of lysates from HeLa cells incubated in hypertonic medium

Abstract

When HeLa S-3 cells are incubated with medium made hypertonic by adding NaCl, protein synthesis is inhibited. When the cells are returned to normal conditions protein synthesis is restored. To study the molecular mechanism of this regulation of protein synthesis, lysates were prepared from HeLa S-3 cells grown in minimum essential medium (normal, N); from cells which were incubated with additional (100 mM) NaCl (hypertonic, H); and from cells which were treated similarly in hypertonic medium and then restored to isotonic conditions (hypertonic-isotonic, H-I). Lysates of H cells exhibited reduced endogenous protein synthesis. Studies with mixed lysates from H and N cells implicated that the H lysate did not contain a soluble, non-labile macromolecule (> 10 000 daltons) with an inhibitory activity upon the protein synthesis. Analysis by Edman reaction of H lysates showed a reduced incorporation of [ 35S]methionine into N-terminal suggesting that the initiation step of protein synthesis was affected. However, sucrose gradient analysis indicated that lysates of H cells were still able to form 80-S initiation complexes with [ 35S]methionyl-tRNA Met i. The block in initiation was not complete. The lesion could not be reversed by adding post-ribosomal supernatant or a ribosomal salt wash from N cells to ribosomes from the H cells. The data show that the ribosomal fraction is primarily involved in the inhibition.