Development and characterization of cell-free amino acid incorporation systems from <u>Cellvibrio gilvus</u>

Abstract

A ribosomal system has been derived from Cellvibrio gilus which is active with respect to amino acid incorporation into hot trichloroacetic acid-insoluble material. Optimal incorporation systems showed a requirement for ribosomes, pH 5 enzyme protein, ATP plus ATP-generating system, magnesium ion, ammonium ion, and GTP. ¹⁴C-phenylalanine incorporation was stimulated by poly U in the presence of t-RNA which has been derived from this bacterium. The t-RNA had some activation effect on poly U-directed ¹⁴C-phenylalanine incorporation, whereas no effect on natural m-RNA primed ¹⁴C-leucine incorporation was observed. A complementary unlabeled amino acid mixture inhibited ¹⁴C-leucine incorporation. ¹⁴C-leucine incorporation was greatly inhibited by RBAase and puromycin, moderately inhibited by chloramphenicol, but not effected significantly by DNAase and actinomycin D. The optimum pH and temperature for ¹⁴C-leucine incorporation were 7.8 and 300° C, respectively. Polyamiaes (putrescine, spermine, and spermidine) had a slight stimulatory effect on poly U-directed.¹⁴C-phenylalanine incorporation, but no effect on ¹⁴C-Leucine incorporation.