in case D was submitted to gel filtration on Sephadex G-25, this effect was lost (data not shown).
To optimize culture age, the growth rate of A. fumigatus was monitored by measuring glucose consumption (which correlates with biomass generation as confirmed by measuring also intracellular ATP). According to Fig. , the best yield for in vitro translation purposes was obtained at mid-log phase, 16 -20 h postinoculation.
Having selected conditions of growth and culture age, optimization of the assay was undertaken. Filtration of the lysate through Sephadex G-25 led to a sevenfold increase in the activity (from 5800 to 40,000 cpm/mg of protein) as expected since the endogenous inhibitors are removed by this chromatography. Maximum activity was obtained when K Ο© and Mg 2Ο© concentrations were 150 and 10 mM, respectively, at pH 7.4. For the nucleotide triphosphate system, optimal results were obtained with 450 M ATP, 100 M GTP, and 24 mM phosphocreatine. The addition of exogenous tRNA (up to 50 g/ml) contributed to an increase in the activity of the system. Under such optimal conditions the time course of the assay was kept linear for 30 min.
Several protein synthesis inhibitors were tested in the assay and proved to inhibit the reaction with different efficiencies as determined by the concentration of the compound that causes 50% of inhibition with respect to the control without compound (IC 50 ): anisomycin, hygromycin, verrucarin A, puromycin, and cycloheximide yielded IC 50 s of 0.05, 0.1, 0.2, 1.0, and 2.0 g/ml, respectively. This validates the assay not only for its use in the search of new anti-A. fumigatus molecules, but also as a useful tool for dissecting the molecular mechanisms involved in the translational machinery of this pathogenic fungus.