Amino acid transport in cultured mammalian cells

Scott, Douglas Mackenzie (1975) Amino acid transport in cultured mammalian cells. PhD thesis, University of Glasgow.

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The studies reported in this thesis relate essentially to the transport of the acidic amino acid L-glutamate and the neutral amino acid L-alanine by monolayer cell cultures. The transport of these amino acids was examined in a number of cell lines of different species or tissue origin, but in most detail in the baby Syrian hamster kidney cell line BHK21-C13. Both amino acids were shown to be transported by energy, pH, temperature and Na+ dependent active transport systems capable of accumulating these amino acids to levels considerably greater than in the surrounding medium. L-glutamate was shown to be transported via a relatively high affinity, low capacity uptake system specific for the acidic amino acids L-glutamate, L-aspartate and certain of their analogues. L-alanine was transported via a separate, relatively low affinity, high capacity transport system of broad specificity. This system appeared capable of transporting all neutral L-amino acids with the exception of L-cysteine and L-cystine. This system however, showed a marked preference for L-alanine and the short polar unbranched neutral amino acids. In addition to this L-alanine preferring transport system a second neutral amino acid transport system of preferred substrates L-leucine and the branched chain aliphatic and the aromatic amino acids was indicated. Transport systems of similar specificity and characteristics to the acidic and L-alanine preferring transport systems of BHK21-C13 cells were also observed in a number of diploid or established cell lines of Syrian or Chinese hamster, mouse, sheep or human embryo. The characteristics of these systems did not appear to be altered following transformation by the DNA tumour virus SV-40 or Polyoma. The level of L-glutamate and L-alanine uptake was also examined in BHK21-C13 cells following different cell or growth conditions, in order to investigate possible factors Involved in the transport of their respective transport systems. These transport systems appeared to differ in their control mechanisms. L-alanine uptake was observed to be maximal in cells with low intracellular cyclic AMP levels, but reduced in cells with elevated levels of this cyclic nucleotide. L-glutamate however, appeared to be essentially Independent of intracellular cyclic AMP levels. Similar observations were made for a number of transformed or non-transformed cell lines. No evidence for control of the uptake of these amino acids by intracellular amino acid levels was provided by these studies, as the transport of neither of the amino acids was altered following treatments designed to alter cellular amino acid levels. Addition of various hormones or growth in their presence similarly had no effect on L-glutamate or L-alanine uptake, with the exception of insulin, which stimulated L-alanine uptake when added to cells maintained in low serum. In order to provide further evidence for the postulated acidic and L-alanine preferring transport systems, and possibly supply additional information about the specificity and nature of these uptake systems an attempt was made to isolate mutants defective in the transport of L-glutamate and L-alanine. Two major selection procedures were used, a) involving the selection for cells resistant to toxic amino acid analogues, and b) involving the selection for cells unable to transport radioactive amino acids. Such attempts however, proved unsuccessful. The results reported in this thesis have thus indicated the presence of active amino acid transport systems in cultured mammalian cells and provided information as to their possible mechanisms of control.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J A Pateman
Keywords: Physiology
Date of Award: 1975
Depositing User: Enlighten Team
Unique ID: glathesis:1975-72949
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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