Heterogeneity of native rat liver elongation factor 2

The high heterogeneity of native rat liver EF‐2 prepared from either 105000 x g supernatant or microsome high‐salt extract was detected by two‐dimensional equilibrium isoelectric focusing‐SDS‐polyacrylamide gel electrophoresis in the presence of 9.5 M urea. Five spots were always detected, all of M r 95000, which were not artefactual for their amount varied when EF‐2 was specifically ADP‐ribosylated by diphtheria toxin in the presence of NAD+, and/or phosphorylated on a threonine residue by a Ca2+/calmodulin‐dependent protein kinase (most likely Ca2+/calmodulin‐dependent protein kinase III described by others [(1987) J. Biol. Chem. 262, 17299–17303; (1988) Nature 334, 170–173]). Results of ADP‐ribosylation and/or phosphorylation experiments with either unlabeled or labeled reagents ([14C]NAD and [32P]ATP) strongly suggest that our preparation contained native ADP‐ribosylated and native phosphorylated forms which could be estimated at about 20% and 40% of the whole EF‐2. Phosphorylated and ADP‐ribosylated forms of EF‐2 could be ADP‐ribosylated and phosphorylated, respectively, but a native form both ADP‐ribosylated and phosphorylated was not detected. Our results also suggest the existence of a minor native form of EF‐2 and of its phosphorylated and ADP‐ribosylated derivatives.