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Title: Amino acid interactions in intracellular pool formation and protein synthesis in cultured mammalian cells
Author: Page, Catherine Mary
ISNI:       0000 0001 3463 1730
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1983
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(1) To survive and evolve all cells must make protein. Therefore, the fundamental mechanism controlling the intracellular availability of amino acids, from witch proteins are made, has far-reaching consequences, as all life is dependent on it. It is to the problem of intracellular availability of amino acid that this thesis is addressed. (2) Traditionally, theories of amino acid uptake, i.e. transport of amino acids through specific sites in the impermeable cell membrane, are based on hypotheses which make a number of assumptions which may not be correct, not least is the notion that the cell membrane is impermeable to these hydrophobic substances. (3) Amino acid interactions were examined in cultured mammalian cells in a steady-state growth phase by means of competition experiments. Uptake of amino acids into a defined intracellular acid-extractable pool and their incorporation into acid-precipitable material, were the parameters measured. (4) Generally, inhibition of uptake of an amino acid was of more non-specific character than specific character when the concentration of the same or other amino acids was increased up to 100-fold, with a few not/able exceptions e.g. the interactions of Gly and Ser. (5) In contrast, incorporation of amino acids into protein in the same cells, was highly specific but could be significantly inhibited under certain conditions of amino acid imbalance, where the total number of other amino acids reduced the flux of labelled molecules at the selection mechanism for loading onto tRNA. Thus at 10-5m a labelled amino acid was not limiting for protein synthesis in other-wise normal medium, but, in medium containing an additional 10-2m competing amino acid, its flux was reduced and this could limit protein synthesis. (6) In work dealing with amino acid interactions the ability of amino acids to exchange with pre-existing molecules in the pool was investigated Using raised, levels of amino acids to examine the effect on uptake and incorporation of others an 'overshoot phenomenon' was observed and has been further analysed. This, an unusually rapid uptake of an amino acid apparently against its gradient under definite conditions i.e. when a heavy dose or preload of one amino acid was followed by a return to physiological growth medium containing that amino acid in a labelled form, was first observed by Heinz, 1954 the overshoot was transient and attributed to a rapid incursion of labelled molecules exchanging with amino avoids in the acid- extractable pool and the simultaneous discharge of that pool as the system came to a new steady-state. (7) Amino acid analysis of preloaded cells in which to observe the above phenomenon showed that the total concentration of amino acids in the acid-extractable pool shows small fluctuations under these conditions, e.g. when Leu is preloaded at 10 - 3m the pool size increases by 3% above unpreloaded controls, and with a, 10 -2m Leu preload the pool size decreases by about 25 % but the amino acid composition of the pool is altered in response to changes in the external medium. (8) Overshoot did not always occur when a different labelled amino acid was used to challenge the preloaded amino acid, suggesting that interactions between amino acids may be more specific than competition experiments indicated. Preloading experiments where not designed to look at amino acid entry into the acid-extractable pool but rather to focus on the interactions between amino acids within the pool. Here specificities may be due to the stability of amino acid complexes within the pool and the ease with which they are displaced by other amino acids. (9) The ways in which amino acid interactions have been investigated throughout this study have been discussed in terms of conventional theories of amino acid movement across cell membranes as opposed to flux pathways. Conventional theories have many shortcomings and although flux pathways are more' in agreement with results presented here, points still remain which have yet to be clarified. On this basis an explanation of overshoot can be offered which for the first time is feasible and thermodynamically sound. (10) Amino acid pathways into, through and out of cells are very complex and only the surface has been scratched in this thesis. Throughout, pains have been taken to maintain cells within a complete medium to avoid the pitfalls of using cells in balanced salt solutions with only single amino acids present, experimental conditions which are grossly unphysiological.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry