Development and characterization of cell-free amino acid incorporation systems from Cellvibrio gilvus
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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 sup>14C-phenylalanine incorporation, whereas no effect on natural m-RNA primed sup>14C-leucine incorporation was observed. A complementary unlabeled amino acid mixture inhibited sup>14C-leucine incorporation. sup>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 sup>14C-leucine incorporation were 7.8 and 300Â° 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.
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