We have recently shown that rat liver 60 S ribosomal subunits active in protein synthesis can be reconstituted from inactive core particles lacking 30% of the total proteins, mainly LlOa, L12, L22, L24, A33 and the acidic phosphoproteins Pl-P2, obtained by treatment of 60 S subunits with dimethylmaleic anhydride [(1987) Eur. J. B&hem. 163, 15-201. In this study, an ethanol extract of the 60 S subunit which contains only Pl-P2 was also shown to be effective in reconstitution with the DMMA-core-particles: it strongly stimulated the EF-Zdependent GTP hydrolysis and, to a lesser extent, polyphenylalanine synthesis; like the DMMA wash it shifted the thermal denaturation curve of the DMMA-core particles towards that of control subunits. Prior dephosphorylation of the ethanol extract by alkaline phosphatase inhibited the reconstruction process.

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 Mr 95000, which were not artefactual for their amount varied when EF-2 was specifically ADP-ribosylatecl 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; 0988) Nature 334, 170-173]). Results of ADPribosylation and/or phosphorylation experiments with either unlabeled or labeled reagents ([14C]NAD and p2p]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 ADPribosylated derivatives.

Elongation factor 2 (eEF-2), which contains seven Trp residues, exhibited a tryptophan-characteristic intrinsic fluorescence with maximum excitation at 280 nm and an emission peak centered at 333 nm that suggested a hydrophobic environment of these tryptophans. Upon denaturation with 6 M guanidine hydrochloride, the maximum emission was shifted to 348 nm. Fluorescence quenching studies using acrylamide and iodide confirmed that the Trp residues were mainly buried in the native molecule and indicated an important heterogeneity, the fractional accessible fluorescence (fa) values being 0.50 and 0.25, respectively. Partial quenching of eEF-2 fluorescence by nucleotides proved the existence of an interaction of the factor in the absence of ribosomes, not only with GDP but also with GTP, nonhydrolyzable analogs, GMP, and adenylic, but not cytidylic, nucleotides. Saturating binding plots showed different maximal changes of fluorescence depending upon the nucleotides, from 6.4% with ADP to 24.5% with GDP, and suggested the existence of more than one binding site for each nucleotide. Among all the nucleotides tested, only GTP at saturating concentration modified the fa value obtained with acrylamide (-36%). The possibility that this modification is related to a conformational change of eEF-2 induced by GTP binding is discussed.

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