JBC, Vol. 252, Issue 5, 1589-1605, Mar, 1977
Quantitative analysis of the change of metabolite fluxes along the pentose phosphate and glycolytic pathways in Tetrahymena in response to carbohydrates
M. J. Borowitz, R. B. Stein and J. J. Blum
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...
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