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Metabolic pathways in Tetrahymena. Estimation of rates of the tricarboxylic acid cycle, glyoxylate cycle, lipid synthesis, and related pathways by use of multiple labeled substrates.

Abstract The incorporation of 14C into CO2 from ten labeled substrates: [1-14C]- and [2-14C]acetate; [1-14C]-, [3,4-14C]-, and [5-14C]glutamate; [1-14C]-, [4-14C]-, and [U-14C]aspartate, [1-14C]- and [U-14C]alanine was measured under steady state conditions in Tetrahymena pyriformis cultures at three different stages of growth. The rate of oxygen consumption and the incorporation of 14C from [1-14C]- and [2-14C]acetate into lipid and glycogen were measured under the same conditions. An isotopic and metabolic steady state model of carbon flow through the tricarboxylic acid cycle, the glyoxylate cycle, and the gluconeogenic and lipogenic pathways in Tetrahymena was developed. Fitting of the model to experimental data requires separation of mitochondrial and extramitochondrial pools of aspartate, oxalacetate, malate, and acetyl-CoA, as well as two pools of phosphoenolpyruvate. These structural requirements, necessary to fit the experimental data, were independently justified by information on enzyme compartmentation. By using the data in conjunction with the model quantitative estimates of the flux rates through the major pathways of intermediary metabolism were obtained. The pattern of intracellular flow of metabolites underlying the observed changes in glycogen content and lipid content with culture age are analyzed.