Abstract
High continuous hydrostatic pressure is known to inhibit the total cellular protein synthesis. In this study, our goal was to identify pressure‐regulated proteins by using two dimensional gel electrophoresis and mass spectrometry. This analysis showed that under 30 MPa continuous hydrostatic pressure the biosynthesis of eukaryotic elongation factor‐2 (eEF‐2) was inhibited both in HeLa carcinoma and T/C28a4 chondrocytic cell lines. Western blot analysis of HeLa cells revealed that the cellular protein level of eEF‐2 decreased by 40%–50% within 12 h of the pressure treatment. However, the steady‐state mRNA level of eEF‐2 was not affected by the pressure. Cycloheximide addition after 4 h‐pressure treatment suggested that the half‐life of eEF‐2 protein was shorter in pressurized cells. eEF‐2 is responsible for the translocation of ribosome along the specific mRNA during translation, and its phosphorylation prevents the ribosomal translocation. Therefore, increased phosphorylation of eEF‐2 was considered as one mechanism that could explain the reduced level of protein synthesis in pressurized HeLa cell cultures. However, Western blot analysis with an antibody recognizing the Thr56‐phosphorylated form of eEF‐2 showed that phosphorylation of eEF‐2 was not elevated in pressurized samples. In conclusion, the inhibition of protein synthesis under high pressure occurs independent of the phosphorylation of eEF‐2. However, this inhibition may result from the decrease of cellular eEF‐2 protein. © 2004 Wiley‐Liss, Inc.