Biosynthesis of Gramicidins and Tyrocidines in Cell-Free Preparations from Bacillus brevis^*

Further Aspects of Gramicidin and Tyrocidine Biosynthesis in the Cell-free System of Bacillus brevis*

“…3). As in radioactivity measurements (Uemura et al, 1963), the antibiotic activity remained associated with the ribosomal fraction. It may be seen that the omission of the gramicidin group of amino acids from the incubation mixture had no effect on tyrocidine synthesis.…”

“…-The biochemicals and radioactive amino acids were those previously described (Uemura et al, 1963). In addition, the following substances were employed: hog kidney D-amino acid oxidase and snake venom L-amino acid oxidase, both from Worthington Biochemical Corporation; puromycin from Nutritional Biochemicals Corporation; and chloramphenicol from Parke Davis and Company.…”

“…Use was also made of the variety of B. brevis (ATCC 9999) which forms gramicidin S . Cellular Components.-A sonicate of washed B. brevis ce!ls was prepared and freed of debris, as previously described (Uemura et al, 1963). This extract, referred to as the complete sonicate system, was used in some incubation experiments.…”

“…This extract, referred to as the complete sonicate system, was used in some incubation experiments. In other instances, ribosomes plus the 140,000 X g supernatant phase were employed instead (Uemura et al, 1963).…”

“…Amino Acid Incorporation into Peptides and Protein.-The assay method utilized either the complete sonicate, or the ribosomal-140,000 x g supernatant system, with isotopic leucine or valine in the standard reaction mixture (Uemura et al, 1963). The latter contained MgCln, ATP, glutathione, phosphoenolpyruvate, pyruvic kinase, phosphate buffer, and amino acid mixture.…”