13 Oscillations in Yeast Glycolysis
215
will also reduce the slow decline in intracellular ATP [74]. Oscillations in intracellular
K
+ concentration can also be measured as shown in Fig. 13.1e using the potassiumsensitive fluorescent probe PBFI [43]. Interestingly, these measurements suggest
that the concentration of free intracellular potassium ion is on the order of 20 mM or
below [47], which contrasts with the up to 300 mM total intracellular concentration
of this ion in yeast [72]. This suggests that most intracellular potassium ion may be
bound to proteins and other negatively charged groups in the cell [47].
Recently, our group has explored a new class of fluorescent dyes, which has
allowed us to measure a new and hitherto overlooked physical property linked to glycolytic oscillations. These dyes are the so-called 6-acyl-2-(dimethylamino)naphtalene
(DAN) probes, which are polarity-sensitive molecules that can be used to measure
the dynamics of intracellular water. These probes, originally introduced by G. Weber
[39, 70], were designed to measure dipolar relaxation times of the environment
(water in particular) [6, 39, 70]. When these probes are added to yeast cells with
oscillating glycolysis it was shown that their fluorescence oscillates synchronously
with NADH fluorescence (Fig. 13.1f) [64, 65]. This suggests that the dynamics of
water and metabolism (glycolysis) are tightly coupled as will be discussed further in
Sect. 13.3.
A useful way to analyse multiple simultaneous time series from oscillating reactions is to perform phase plots of the variables. Such phase plots are shown in Fig. 13.2
for some of the data shown in Fig. 13.1. From these phase plots we may infer, e.g.,
that intracellular ATP, K
+ concentration and ACDAN GP (see Sect. 13.3) all oscillate
in phase.
Other variables that have been measured in oscillating glycolysis in intact yeast
cells include carbon dioxide production [55], heat flux [63], temperature and cell
volume [66] (see Sect. 13.4).
The fact that so many different and seemingly unrelated intensive and extensive
thermodynamic variables were found to oscillate in synchrony suggest that numerous
cellular processes, and not just enzymes in the glycolytic pathway, contribute to the
mechanism of the oscillations.
13.3 The Dynamics of Intracellular Water Modulate
Glycolytic Oscillations
13.3.1 Macromolecular Crowding and the Dynamics
of Intracellular Water
Water is the most abundant component of the cell and plays important roles in many
cellular processes as a solvent for macromolecules, small molecules and ions, but
also as a substrate in hydrolysis reactions. Traditionally, intracellular water has been
considered to be in a liquid state [27, 69]. In fact, essentially all previous biochemical
models of glycolytic oscillations assume mass action kinetics, which is only strictly
215
will also reduce the slow decline in intracellular ATP [74]. Oscillations in intracellular
K
+ concentration can also be measured as shown in Fig. 13.1e using the potassiumsensitive fluorescent probe PBFI [43]. Interestingly, these measurements suggest
that the concentration of free intracellular potassium ion is on the order of 20 mM or
below [47], which contrasts with the up to 300 mM total intracellular concentration
of this ion in yeast [72]. This suggests that most intracellular potassium ion may be
bound to proteins and other negatively charged groups in the cell [47].
Recently, our group has explored a new class of fluorescent dyes, which has
allowed us to measure a new and hitherto overlooked physical property linked to glycolytic oscillations. These dyes are the so-called 6-acyl-2-(dimethylamino)naphtalene
(DAN) probes, which are polarity-sensitive molecules that can be used to measure
the dynamics of intracellular water. These probes, originally introduced by G. Weber
[39, 70], were designed to measure dipolar relaxation times of the environment
(water in particular) [6, 39, 70]. When these probes are added to yeast cells with
oscillating glycolysis it was shown that their fluorescence oscillates synchronously
with NADH fluorescence (Fig. 13.1f) [64, 65]. This suggests that the dynamics of
water and metabolism (glycolysis) are tightly coupled as will be discussed further in
Sect. 13.3.
A useful way to analyse multiple simultaneous time series from oscillating reactions is to perform phase plots of the variables. Such phase plots are shown in Fig. 13.2
for some of the data shown in Fig. 13.1. From these phase plots we may infer, e.g.,
that intracellular ATP, K
+ concentration and ACDAN GP (see Sect. 13.3) all oscillate
in phase.
Other variables that have been measured in oscillating glycolysis in intact yeast
cells include carbon dioxide production [55], heat flux [63], temperature and cell
volume [66] (see Sect. 13.4).
The fact that so many different and seemingly unrelated intensive and extensive
thermodynamic variables were found to oscillate in synchrony suggest that numerous
cellular processes, and not just enzymes in the glycolytic pathway, contribute to the
mechanism of the oscillations.
13.3 The Dynamics of Intracellular Water Modulate
Glycolytic Oscillations
13.3.1 Macromolecular Crowding and the Dynamics
of Intracellular Water
Water is the most abundant component of the cell and plays important roles in many
cellular processes as a solvent for macromolecules, small molecules and ions, but
also as a substrate in hydrolysis reactions. Traditionally, intracellular water has been
considered to be in a liquid state [27, 69]. In fact, essentially all previous biochemical
models of glycolytic oscillations assume mass action kinetics, which is only strictly