title: Regulation of amino acid metabolism in the cellular slime mould Dictyostelium discoideum
Creators: name: Iqbal, Aftab
Publisher: name: University of Glasgow
Year: 1984

description: The thesis begins with a description of cellular slime moulds and briefly reviews growth and differentiation of D. discoideum and the changes occurring during differentiation. Proteins are a major nutrient during growth and differentiation of D. discoideum Ax2. The proteinases and peptidases involved in the breakdown of protein and peptides, and some amino acid metabolising enzymes are described. The effects of glucose and amino acids on differentiation are discussed and the introduction concludes with a brief description of transport systems used by the slime mould namely pinocytosis and phagocytosis. The aims of the experimental work were four fold. A) To develop and define different growth systems where the rates of amino acid utilization by the cells were different. B) To determine if rates of amino acid utilization could be altered by growth conditions. C) To measure the rates of amino acid utilization and the effect of metabolites on the utilization of amino acids. D) To determine if rates of amino acid metabolism were related to the levels of amino acid metabolising enzymes in the cells. Axenic liquid medium (ALM), either with or without glucose, at 86mM, was used to grow the cells. The following results were obtained. 1) In the absence of glucose. a) During growth of the cells, the pH of the medium increased. The yield of the cells obtained increased as the initial pH of the medium was decreased within the pH range 6.0 - 7.0. b) Growth of the cells was limited by pH change of the medium. With the pH of the culture maintained at 6.2 the cells grew to a xviii density of 6.6 x 106 cells/ml. In the absence of pH control, the cell density reached 2.8 x 106 cells/ml. c) Ammonia was produced during growth. An ammonia concentration of 19 mM was achieved during growth to a density of 6.6 x 106 cells/ml. Cultures growing to a lower cell density produce proportionally less ammonia. 2. In the presence of glucose. a) The yield of the cells increased to 1.2 x 107 cells/ml and the pH change associated with growth was reduced. The final density was much less susceptible to the pH at which the medium was prepared (routinely 6.2). b) Control of medium pH at 6.2 marginally increased the final cell density to 1.5 x 10 cells/ml. c) During growth ammonia production was reduced to about half the value obtained in the absence of glucose. 3. Fructose, added to ALM produced similar results to glucose with respect to growth yield, pH change and ammonia production. 4. Galactose, added to ALM reduced the growth rate and diminished the yield to values less than the control value. Galactose had little effect on ammonia production. 5. Pyruvate, added to ALM had a small effect. It increased the yield by 25% and decreased the ammonia production by 20%. 6. The addition of ammonium chloride (up to 20 mM), sodium and potassium phosphate {up to 28.76 mM) and 2(N-morpholino) ethane sulphonic acid (up to 20 mM) all reduced the growth rate and the yield of the cells. 7. In washed cell suspensions. a) Glucose, added to the cell suspensions, reduced ammonia production to an extent comparable to its effect during growth. b) By using the techniques of ammonia estimation, amino acid analysis, radiolabelled substrate utilization and oxygen uptake, amino acid breakdown was measured. In general, cells grown in the absence of glucose had a greater ability to catabolise amino acids. c) Among the individual amino acids, whose rates of degradation were measured, arginine was the most rapidly catabolised followed by lysine and tyrosine. The pattern was independent of the medium in which the cells were grown. d) The rate of amino acid catabolism, measured by ammonia production or 14C- carbon dioxide production, was reduced by the addition of a range of compounds to washed cell suspensions. e) In washed cell suspensions prepared from cells grown in the absence of glucose, glucose at 10 and 20 mM stimulated the breakdown of extracellular amino acids. A similar effect was produced by 2 deoxy D-glucose and trehalose but not by other additives. These results are discussed in terms of stimulation of uptake but reduction in metabolism. f) Galactose, pyruvate, phosphate, and glucose in ALMg cells and glucose and 2 deoxy D-glucose above 20 mM in ALM cells, all reduced amino acid metabolism. The data are considered in relation to metabolic and osmotic effects on the cells. 8. The activities of several amino acid catabolising enzymes were measured in cells grown in the presence and absence of glucose. Cells grown in the absence of glucose possessed higher levels of activity and this correlates with their increased ability to catabolise amino acids.