Binding assays

All samples in binding assays were tested in triplicate. For the saturation binding experiment, 100 nM rabbit eEF1A2 was mixed with several concentrations of [¹⁴C]-plitidepsin (ranging from 0.1 to 4 μM) in 45 mM Hepes-KOH pH 7.5, 5 mM magnesium acetate, 75 mM potassium chloride, 1 mM DL-dithiothreitol and 5% (v/v) DMSO in the presence of 1 μM Gpp(NH)p in a final volume of 500 μL. After 1 h incubation at room temperature, 450 μL were withdrawn, filtered through GF/C filters (Millipore, Bedford, CA) and washed three times with the same buffer used for the incubation. Filters were then removed, dried and their radioactivity was finally counted as a measurement of total (specific and non-specific) bound drug. An aliquot from each of the [¹⁴C]-plitidepsin solutions used to prepare each sample was counted in triplicate to determine the actual amount of total radioligand present in each case and such value was considered for data processing. Scintillation counting was transformed into concentration values considering the specific activity of the radioligand and the sample volume. The concentration of bound radioligand was related to the total amount of radioligand in the sample using the expression derived by Swillens¹² to account for ligand depletion when the concentration of protein was similar to that of radioligand:

where B_T is the concentration of total (specific and non-specific) bound plitidepsin, L_T is the total concentration of plitidepsin in the sample, K_D is the dissociation constant, B_max is the maximum amount of drug bound to the protein and α is a parameter corresponding to the ratio between non-specifically bound ligand and free ligand (which in fact accounts for the dependency of non-specific binding with ligand concentration).

For dissociation kinetics, 1 μM [¹⁴C]-plitidepsin was mixed with 100 nM rabbit eEF1A2 in the same buffer as above for 1 h and either DMSO to 1% or unlabeled plitidepsin to reach 10 μM in 1% DMSO were added to the mixture whose final volume was 500 μL. At the selected times after this last addition, 400 μL of each sample were withdrawn and processed as above.

For the fractionation process, tested samples (fractions from either the subcellular fractionation or the chromatography eluates) were mixed with 500 nM [¹⁴C]-plitidepsin in 45 mM Hepes-KOH pH 7.5, 5 mM magnesium acetate, 75 mM potassium chloride, 1 mM DL-dithiothreitol and 5% (v/v) DMSO in the presence or absence of 10 μM plitidepsin, in a final volume of 500 μL. After 1 h incubation at room temperature 400 μL of each tube were withdrawn and processed as described above.