Equilibrium and steady-state models of the coupling between the amino acid gradient and the sodium electrochemical gradient in mouse ascites- tumour cells.
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ABSTRACT:
SUBMITTER: Philo RD
PROVIDER: S-EPMC1185986 | biostudies-other | 1978 Sep
REPOSITORIES: biostudies-other
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Project description:1. The fluorescent dye 3,3'-dipropyloxadicarbocyanine was used to show that the tumour cells absorbed 2-aminoisobutyrate, glycine, L-leucine and L-isoleucine and certain other amino acids electrogenically. The Km values with respect to amino acid concentration ([A]o), obtained from the fluorescence assays, varied through the above series from 0.8 to 26 mM, with Vmax. fairly constant. 2. Similar Km values described the uptake of the 14C-labelled amino acids in five instances where this was measured. 3. Each amino acid lowered the membrane potential (E) by 10-20 mV when its cellular concentration ([A]i) had reached a steady value and [A]o was 10mM. In these experiments energy metabolism was maintained by glycolysis, 2,4-dinitrophenol was present and cellular respiration was inhibited. The corresponding net flow of amino acid through the Na+ symport was deduced by making use of the fact that the depolarization an amino acid initially caused was roughly proportional to the net influx of amino acid itself. 4. The steady-state depolarization was attributed to the presence of a leak pathway for the amino acid with a rate coefficient PA. As assayed in the absence of Na+, PA was about 5-fold larger for isoleucine than for glycine. 5. Direct estimates of Vmax./PA were similar to those inferred from the extent of depolarization in the steady state and [A]i. 6. A mathematical model was used to predict [A]i/[A]o in term of the measured values of [Na]o, [Na]i, E, Km and Vmax./PA. The predicted and observed values agreed fairly well when [A]o was 1 mM or 10 mM. 7. [A]i/[A]o varied from about 2.5 for 10 mM-isoleucine to 30 for 1 mM-2-aminoisobutyrate when delta microNa, expressed as a ratio, was ostensibly in the range 19-43. 8. The concentration of 2-aminoisobutyrate from a 0.1 mM solution in the presence or absence of ouabain was consistent with the model, whereas the concentration of isoleucine from a 0.1 mM solution exceeded the predicted values 2-5-fold. 9. The tumour cells concentrated 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid by a non-electrogenic mechanism, with which isoleucine may also interact.
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Project description:1. To deplete them of ATP the tumour cells were starved at 37 degrees in a Ringer solution containing 33m-equiv. of Na(+)/l., 131m-equiv. of Li(+)/l., 2mM-sodium cyanide and 0.1mm-ouabain. The cellular content of K(+) was largely replaced by Li(+), but cellular [Na(+)] remained near 33m-equiv./l. 2. The addition of 12mm-glycine to the system caused cellular [Na(+)] to increase, during the next 4min., by about 4m-equiv./l., so that it slightly exceeded extracellular [Na(+)]. This occurred in parallel with the absorption of glycine. 3. The cellular K(+) content fell by an amount representing about 10% of the amount of Na(+) absorbed. 4. The results provide a clear demonstration that the flow of glycine into the cells is linked to a parallel movement of Na(+); K(+) appears to play a facultative role in the carrier system, whereas Li(+) is almost inert. 5. The effects produced by glycine were not reproduced by l-arabinose.
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