Abstract
A metabolic scheme of glycolysis and the pentose phosphate pathway has been constructed, assuming that the reactions occur in a single compartment. From this scheme, equations are written for a system in metabolic and isotopic steady state. These allow computation of the specific activity of every carbon atom of all the intermediates of the glycolytic and pentose phosphate pathways and consequently of the flux of carbon along each step of these pathways. A sufficiently large number of well distributed measurements of incorporation of radioactive label from different positions of several substrates into intermediates or products must be made to determine all the fluxes. This is done by choosing a set of metabolic fluxes, calculating incorporation with the aid of a computer, and then manipulating the flux rates until the computed incorporations match the data. The model is used in this paper to analyze the metabolism of the protozoan Tetrahymena pyriformis. The metabolic scheme of the model is consistent with all available information on the enzyme complement of this ciliate. Cells grown to transition phase in proteose/peptone medium were inoculated into a mixture of glucose (6 mM), fructose (6 mM), ribose (3 mM), and glycerol (3 mM) and incubated for 1 h. In each of these experiments, one of the following labeled substrates was present: [1-, 2-, 6-, or U-14C]glucose; [1- or U-14C]fructose; [1- or U-14C]ribose; [1(3)-or 2-14C]glycerol. The incorporation of label from these substrates into CO2, lipid, glycogen, and RNA was measured. In contrast to earlier studies on the metabolism of 2- and 3-carbon substrates by Tetrahymena, the rate of incorporation of label from some substrates into some products (e.g. from [1-14C]glucose into CO2) changed during the incubation. To treat these time-dependent data within the framework of the steady state model, the 1-h incubation was divided into three 20-min intervals; within each of these, the rates of incorporation were approximately constant, as required for a steady state system. Measurements of the pool sizes of glucose-6-P and fructose-6-P showed that only slow changes in pool sizes occurred after the first 5 min of incubation and indicated that the system was effectively in a metabolic and isotopic steady state throughout most of the incubation. The finding that a low concentration of cycloheximide prevented the acceleration of 14CO2 production from labeled glucose suggests a role for protein synthesis in the slow adaptation to carbohydrate addition and supports the quasi-steady state treatment of this system. The expected incorporation into each product was computed for trial sets of 1, independent flux rates. A set of flux values was found which yielded a good fit to the 29 measurements made for each interval. These flux values therefore constitute a quantitative description of temporal changes in carbon flow along the glycolytic and pentose phosphate pathways during the 1st h of adaptation to the carbohydrate mixture...
Highlights
A metabolic scheme of glycolysis and the pentose phosphate pathway has been constructed, assuming that the reactions occur in a single compartment
Choice of Substrates and Experimental Design-Since a large number of measurements is needed in order to determine the 18 independent flux rates with a minimum of ambiguity, it was decided to use a total of 10 labeled substrates in the standard mixture of glucose (6 mM), fructose (6 mM), ribose
Less than 2% of the ribose, glycerol, and fructose, and less than 10% of the glucose was consumed during the course of a l-h incubation, so that the concentration of each of the four substrates remained essentially constant during the course of these experiments
Summary
A metabolic scheme of glycolysis and the pentose phosphate pathway has been constructed, assuming that the reactions occur in a single compartment. Some general properties of this model and its application to other systems are described in detail in Appendix I, in miniprint format.g The model, relatively free of simplifying assumptions, accounts for the metabolism of the substrates glucose, fructose, ribose, and glycerol, in an expanded scheme of glycolysis, glycogen synthesis, the pentose phosphate pathway, and an abbreviated tricarboxylic acid cycle. In contrast to the earlier work on acetyl-CoA metabolism in Tetruhymenu, in the present study of carbohydrate metabolism, some of the observed rates of “C incorporation into products from labeled substrates were time-dependent This called into question the use of a model predicated on a metabolic steady state. Of the 22 independent parameters in this scheme, 4 were set equal to zero (V’,;, V,,, V,., and V,); justification for this is discussed in detail below
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