Heterogeneity of cardiac rat and human elongation factor 2

Elongation factor 2 (EF-2) catalyses the last step of the elongation cycle, translocation, in the course of protein biosynthesis. A system for analyzi ng post-translational modifications of EF-2, which is a single polypeptide of 857 amino acids, is reported and its application to cytosolic extracts o f cultured neonatal rat heart myocytes, neonatal and adult rat cardiac tiss ue, and extracts of human left ventricular myocardium is described. Compari ng different pH ranges in immobilized pH gradient-isoelectric focusing (IPG -IEF), a range of pH 3 -10 and 4 - 9 resulted in a highly defined and repro ducible resolution of six different EF-2 variants of all extracts in the fi rst dimension. These six variants were detected by the "imaging plate" (pho sphor radiation image sensor) after specific labeling with Pseudomonas exot oxin A catalyzed [P-32]ADP-ribosylation. This finding could be confirmed in Western blot analysis with a specific polyclonal rabbit antibody. Using tw o-dimensional polyacrylamide gel electrophoresis (2-D-PAGE), five to six EF -2 variants could be demonstrated in all extracts. By application of a seco nd IPG indicator strip to the 2-D gel, they could be aligned with correspon ding spots in a silver-stained 2-D separation of human myocardial tissue, r evealing that the EF-2 variants belong to the group of low-abundance protei ns.