Quantitative analysis of intermediary metabolism in Tetrahymena. Cells grown in proteose-peptone and resuspended in a defined nutrient-rich medium.

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Tetrahymena pyriformis were grown to early-stationary phase and resuspended in a defined mixture containing glucose, fructose, ribose, glycerol, acetate, pyruvate, bicarbonate, glutamate, and hexanoate, with only one substrate labeled with 14C in any flask. Incorporation of label into CO2, glycogen, RNA, alanine, glutamate, glycine, lipid glycerol, and lipid fatty acids was measured 20, 40, and 60 min after the start of the incubation. To develop a model suitable for quantitative analysis of the data, it was necessary to join two preceding models, one for carbohydrate-metabolizing cells and one for acetate-metabolizing cells, eliminating the over-simplified sections of each. Equations were written and programmed for a digital computer to allow computation of the amount of label expected to be incorporated into any of the products measured for any given set of steady state flux values in the metabolic network. The model formed by simply joining the two preceding models did not yield satisfactory agreement with the complete data obtained in the present study, although each partial set of data could be fit well by the appropriate partial model. Analysis of the ways in which the model failed to yield good fits to the data indicated that another pool of P-enolpyruvate, of pyruvate, and of acetyl-CoA had to be added at the junction of the two models. The presence of such poolte into fatty acids as compared to the incorporation of label from glucose into fatty acids. A new model was therefore constructed which differed from the preceding model only in its structural organization at the level of P-enolpyruvate, pyruvate, and acetyl-CoA. The model is consistent with all known information on the compartmental structure of metabolism in Tetrahymena, on enzyme localization, and on the enzyme complement of this cell. Over 70 measurements of label incorporation into products were made at each time. These, plus a large number of "limit" measurements which constrain any possible solutions, were in sufficient excess of the 39 independent flux values to permit a stringent assessment of the model. A set of flux values was found which yielded a good fit to the data. These flux values therefore provide a quantitative description of metabolite flux in the intact cell during the slow adaptation to the nine-substrate mixture. The rates of utilization of glucose, fructose, glycerol, and ribose were in the ratio of about 10:1:0.33:0.16, i.e. fairly similar to the ratio observed with carbohydrate-metabolizing cells. Initial flux through phosphofructokinase is about 160 nmol/10(6) cells.h, increasing over 3-fold during tje jpir incubation. Initial flux through fructose-1,6-diphosphatase is about 110 nmol/10(6) cells.h and also increases almost 3-fold during the incubation. Thus net flux is glycolytic and increases 4-fold during the hour with a large amount of futile cycling at this step...