Theodore Winnick

Gramicidin S messenger RNA: I. Isolation and characterization

Gramicidin S, the cyclic decapeptide characteristic of Bacillus brevis no. 9999 from the American Type Culture Collection was produced when pH 4·8 supernatant solution from ruptured cells of this strain was incubated with ribosomes and pH 4·8 precipitate from the American Type Culture Collection no. 8185 (Dubos) variety of the organism, which does not normally synthesize this polypeptide. The factor determining the nature of the polypeptide produced was extracted from B. brevis 9999 supernatant solution with phenol, and shown to be sensitive to RNase. Upon elution from a DEAE-Sephadex column, this RNA separated into two major fractions. Both were required for gramicidin S synthesis, in the presence of RNase-treated supernatant solution from B. brevis 9999. However, only the first fraction was necessary for synthesis in the presence of untreated supernatant solution from B. brevis 8185. Evidence is presented that this RNA acted as the template for the synthesis of gramicidin S.

Some metabolic and enzymic experiments with α-fluoro-β-alanine

Abstract dl -α-Fluoro-β-alanine was found to be moderately toxic to rats, mice and chicks. The toxicity was reduced by the simultaneous administration of aminooxyacetic acid. Following injection of the fluoroamino acid into rats, a considerable proportion of the unaltered compound was recovered in the tissues and urine. The fluoro-analog was not utilized for transamination by a liver-enzyme preparation. It was a fairly good substrate for carnosine synthetase in vitro, but was poorly incorporated into skeletal muscle dipeptides in rats and chicks. The α-fluoro-β-alanine was not active for pantothenic acid synthetase of Escherichia coli. It supported growth of a β-alanine-dependent strain of Saccharomyces cerevisiae only when used at high concentrations. By employing fluoro-[3H]β-alanine under these conditions, it was possible to demonstrate labeling in the coenzyme A fraction of yeast.

EFFECT OF ANTIBIOTICS AND RIBONUCLEASE ON POLYPEPTIDE AND PROTEIN BIOSYNTHESIS IN DIFFERENT STRAINS OF BACILLUS BREVIS.

The effect of chloramphenicol, puromycin, and ribonuclease (EC 2.7.7.16) on polypeptide and protein biosynthesis was tested in growing cultures and in cell-free systems of three different strains of Bacillus brevis. With all three strains of the organism, the peptide-synthesizing ability was completely or severely inhibited under conditions which caused total or marked blockage of protein formation. The results support our previous conclusion, based on studies with ribosomal and soluble cellular components, that gramicidins, tyrocidines, and gramicidin S molecules are all synthesized by a pathway which resembles that of protein biogenesis.

Biosynthesis of growth hormone in ribosomal preparations of bovine anterior pituitary tissue.

Abstract A ribosomal-pH 5 enzyme system prepared from bovine anterior pituitary glands was found to be active in the incorporation of isotopic amino acids into growth hormone and mixed proteins. A small scale modification of an existing procedure was employed for isolation of the labeled hormone, following incubation of the cell-free biosynthetic system in the presence of required cofaetors. The radioactive product was indistinguishable from authentic growth hormone standards by three physical criteria: Sephadex gel filtration, polyacrylamide disc gel electrophoresis, and sucrose density gradient centrifugation. In addition, it displayed immunological activity by binding specific antibody.

Biosynthesis of malformin in washed cells of Aspergillus niger.

Abstract Washed mycelium of Aspergillus niger , incubated in buffer solution, was found to incorporate suitable 14 C-labeled amino acids efficiently into the cyclic peptide, malformin, as well as into protein. Optimum conditions were developed for the assay of this process. Evidence for the specificity of the biosynthesis was that only component amino acids of the malformin molecule were utilized. The chemical identity of the isolated malformin fraction was confirmed by paper chromatography and by altered paper electrophoresis following oxidation with performic acid.