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The mechanism of biosynthesis of the gramicidin and tyrocidine peptides
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|Title:||The mechanism of biosynthesis of the gramicidin and tyrocidine peptides|
|Authors:||Bodley, James William|
|Abstract:||Recently a cell-free system (from Bacillus brevis ATCC 8185) was prepared and preliminarily characterized by I. Uemura, K. Okuda, and T. Winnick (Biochem., 2, 719, 1963) which catalyzed the formation of gramicidin and tyrocidine as well as protein. In collaboration with these authors, and more recently with P. R. Adiga, we have made a detailed investigation of the mechanism of synthesis of these pep tides and compared it to protein formation using the above system. Enzymes which activate the various constituent amino acids (including D-forms and L-ornithine) of gramicidins and tyrocidines, as measured by PPi-ATP exchange, were found in ~. brevis extracts. Experiments with purified s-RNA revealed that the L-amino acids were well utilized for peptide formation, via the aminoacyl-s-RNA pathway, as in protein biosynthesis. However, D-amino acids were poorly incorporated into s-RNA and seemed to require a different type of carrier, associated with a soluble cell constituent distinct from pH 4.8 precipitable protein. After charging this substance with labeled or unlabeled D-amino acids, marked effects on the rate of incorporation of C14 into polypeptide could be observed upon addition of ribosomes. By using mixed preparations from two different strains of B. brevis, it could be shown that the control of specificity of peptide synthesis resided chiefly in the soluble phase of the system, rather than in the particulate fraction. This controlling factor could be distinguished from the activating enzymes and phenol-extractable RNA in these experiments. Chloramphenicol (10 pg/ml) and puromycin (100 pg/ml) were found to inhibit polypeptide and protein synthesis more than 98%, with either the cell-free system or with growing cultures. The crude particulate preparation which was required for peptide and protein formation has been separated into fractions which sediment at 40,000 x g, and 140,000 x g. The larger particles, which probably represent fragments of cell membrane, could accept newly synthesized peptide (but not protein) from the second (ribosomal) fraction. The latter, with maximum synthesizing activity at 5 x 10-3 MMg++ concentration, was found to consist predominantly of 50 S particles, but with a minor 32 S component. Sucrose gradient experiments revealed that the 50 S ribosome functioned in both protein and peptide formation, whereas the 32 S particle was active only in the latter process. At 5 x 10-5 MMg++, the C14-1abeled 50 S ribosomal particle appeared to break down into 27 Sand 18 S sub-units. Labeled protein was associated only with the larger fragment, while radioactive peptide was found in both subunits. The separate fragments lacked intrinsic protein-synthesizing ability, but some measure of peptide synthesis could be achieved when the 18 S particle was assayed. Both sub-units could recombine at higher Mg++ concentrations to restore peptide and protein synthesizing activity. We tentatively postulate that the 50 S ribosome is composed of one 27 S and two 18 S sub-units, and that the 32 S ribosome is a dimer of 18 S particles.|
Thesis (Ph. D.)--University of Hawaii, 1964.
Bibliography: leaves -109.
ix, 109,  l mounted graphs, tables
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|Appears in Collections:||Ph.D. - Biomedical Sciences (Biochemistry)|
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