The Elongation Factor 2 Content of Mammalian Cells

Abstract

Abstract A method is described for quantitatively assaying elongation factor 2 (EF-2), the enzyme that catalyses the translocation of polypeptidyl tRNA on eucaryotic ribosomes. Tissue homogenates are centrifuged in the presence of 0.5 m salt, which removes EF-2 from ribosomes, and charcoal, which adsorbs endogenous NAD. The soluble extract is incubated with 0.18 m histamine, activated diphtheria toxin, and [32P]NAD of known specific activity, and the resulting acid-insoluble [32P]ADP-ribosyl EF-2 is counted. The EF-2 content of different tissues is closely correlated with the number of ribosomes with an average ratio of about 1.2 molecules per ribosome. On centrifuging sucrose homogenates of tissues, only the excess of EF-2 over the number of ribosomes remains in the supernatant fraction.