Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
OPTIMUM CONDITIONS FOR TYROCIDINE SYNTHESIS BY PURIFIED TYROCIDINE SYNTHETASE: SUBUNIT STRUCTURE AND STIMULATION BY TRITON
Abstract
Particle free extracts from B. brevis 8185 catalyze the production of tyrocidine. The enzyme complex consists of heavy, intermediate, and light subunits with molecular weights of 440,000, 230,000 and 100,000 daltons, respectively. The (NH(,4))(,2)SO(,4) precipitation is critical to obtain active preparations. These studies established that a factor present in the light subunit fraction obtained from a 0-40% (NH(,4))(,2)SO(,4) fraction is required in addition to the purified subunits to obtain tyrocidine synthesis. With highly purified heavy and intermediate subunits and the less purified light subunit, containing the required factor or factors, a specific activity of 0.085 nmol tyrocidine/min-mg was achieved. A new growth procedure of B. brevis 8185 which gives a more synchronous growth and higher cell densities with very reproducible enzyme content was developed. Ultragel 34 gives better resolution of the three subunits by gel filtration chromatography than the substances previously used. Exchange activity remaining after the action of trypsin (10 minutes) on the different amino-acid dependent ATP-PPi exchange activities of the heavy subunit varied from 2% for ornithine to 42% for leucine. The synthesis of tyrocidine is more sensitive to the action of trypsin than are the most sensitive of the amino acid activation sites since this activity was reduced 90% while the ornithine and leucine exchange activities were not markedly decreased. Ornithine stimulates ATP-PPi exchange activity in E. coli lysyl tRNA synthetase. Thus the occurrence of ornithine exchange activity in low molecular weight fractions may not indicate that the heavy subunit had been broken down to subunits of 70,000 to 90,000 daltons as previously reported. Triton X-100 (0.2% w/v) stimulates tyrocidine synthesis 2.5 fold. Polyethylene glycols, PEG-4000, PEG-6000, Brij 35, Lubrol WX, and Tritons X-45, 165, 405 and N-101 stimulated tyrocidine synthesis. Zwittergent 3-14, W.S.35, egg yolk phosphatidylcholine, Tween 80, lipids, egg yolk phosphatidylethanolamine, phosphatidylserine, and asolection showed little or no stimulation. Preliminary experiments suggested that Triton X-100 may increase the stability of the interaction between the subunits.
Subject Area
Biochemistry
Recommended Citation
PARKIN, DAVID WARREN, "OPTIMUM CONDITIONS FOR TYROCIDINE SYNTHESIS BY PURIFIED TYROCIDINE SYNTHETASE: SUBUNIT STRUCTURE AND STIMULATION BY TRITON" (1982). ETD collection for University of Nebraska-Lincoln. AAI8306498.
https://digitalcommons.unl.edu/dissertations/AAI8306498