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Date of Award

1982

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmacy

First Advisor

Francis Sayre

First Committee Member

Donald Shirachi

Second Committee Member

Alice S. Hunter

Third Committee Member

Charles W. Roscoe

Fourth Committee Member

Warren J. Schneider

Abstract

The effects of a physiologically balanced mixture of amino acids on the synthesis of proteins has been investigated. The roles of leucine and tryptophan, both implicated in the regulation of protein synthesis, were also studied. The balance of amino acids is important under specific rate-limiting conditions; the physiological balance "protects" the protein synthesizing system from the stressed condition of leucine limitation. Leucine is an important regulator of protein synthesis and the tryptophan effect on translation is dependent on the concentration of leucine. Thus tryptophan is a secondary regulator. The relative concentration of amino acids, described as balance, alterned the synthesis of protein in cell-free and intact cell culture experiments, when leucine was limiting. Both qualitative and quantitative differences were observed. The effect of the amino acid mixture decreased when the concentration of leucine was physiological. Two different components were sensitive to added leucine. This sensitivity was indicated by different kinetics; one component showed low Km and Vmax values while the other showed high Km and Vmax values. The initial rate of protein synthesis was first order with respect to leucine when it was limiting and mixed order when it was physiological. The effect of tryptophan on stimulation of protein synthesis was small in comparison to the effect of leucine, and was dependent on the ooncentration of leucine. The incorporation of leucine into protein was changed as the tryptophan concentration changed when leucine was limiting; synthesis of albumin was slightly stimulated. The ribosome distribution did not change as indicated by polysome analysis. The incorporation of leucine into protein did not change when· leucine was physiological and tryptophan was. varied. However, the ribosome distribution was altered. A low molecular weight inhibitor of protein synthesis was found in cell extracts which acted independently of amino acid or leucine concentrations. It could be partially removed by treatment with G-25 Sephadex, but has not been purified. Several nucleotide effects independent of amino acid concentration were also observed. ATP, at increasing concentrations, significantly depressed levels of synthesis and concentrations greater than 4 mM caused 100% inhibition of the protein synthesizing system. The phosphodiesterase inhibitor, theophyllin~ enhanced synthesis of albumin, although the cyclic nucleotide, cAMP, did not itself alter synthesis of protein. Finally, the concentrations of amino acids in plasma of C3HeB/FeJ mice were determined. The two amino acids examined in the protein synthesis experiments, tryptophan and leucine, were found to remain relatively constant, regardless of the fed or fasted condition of the animals, but showed changes with the age of the animals.

Pages

190

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