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L. F. Olsen and A. Lunding
is almost constant and only shows significant variation in response to changes in
temperature or in cell density [1] or to partially replacing H 2 O by D 2 O [64]. The
reason for this robustness of frequency is not known and cannot be reproduced by
most mathematical models of the pathway. While it is generally accepted that the
enzyme phosphofructokinase plays a central role in the mechanism responsible for
the oscillations, recent evidence has shown that also a number of enzymes and proteins, normally considered unrelated to glycolysis, contribute to the mechanism [61,
65].
Thus, glycolytic oscillations seem to involve a plethora of cellular processes. Here,
we will present new evidence that the dynamics of intracellular water also plays an
important role in the mechanism of the oscillations. These results seem to be general
and may be carried over to other oscillatory processes and ultimately suggest that
intracellular water may have a key role in cell metabolism and signal transduction in
cells. The function of the glycolytic oscillations is not known, but nevertheless they
have been preserved throughout evolution. Several roles of the oscillations have been
proposed, e.g. that they constitute an inevitable side effect in the tradeoff between
hard robustness and efficiency [13] or they increase the thermodynamic efficiency of
glycolysis [60]. Here we propose an alternative function for the oscillations, namely
that they serve to maintain the cell in a state of constant low entropy.
13.2 Variables Measured in Oscillating Glycolysis
Oscillations in glycolysis are typically measured as oscillations in the autofluorescence of NADH. Most other metabolites in glycolysis are optically “silent” and therefore cannot be measured in real time, but only after quenching of cell metabolism,
extraction of cell content and subsequent analysis by off-line assays [22, 40, 41, 59].
NADH oscillations in intact cells may be observed using fluorescence spectroscopy
to measure oscillations in cell suspensions [9, 23] or fluorescence microscopy [4,
12] to measure oscillations in single cells [12]. A typical experiment which shows
oscillations of NADH fluorescence in a suspension of yeast cells can be seen in Fig.
13.1a. Oscillations in NADH fluorescence are induced by addition of first glucose
and then 60 s later potassium cyanide to a suspension of starved yeast cells.
However, more recently time-resolved measurements of other variables such as
intracellular pH [19, 46], mitochondrial membrane potential [2], intracellular potassium ion [47] and water dynamics [64, 65] have been made. For example, it has long
been known that mitochondria contribute to the regulation of glycolytic oscillations
[3], and more recently oscillations in mitochondrial and other intracellular membrane potentials were directly measured using membrane potential-sensitive fluorescent dyes and shown to oscillate synchronously with NADH [2]. Figure 13.1 shows
simultaneous measurements of NADH, intracellular ATP, mitochondrial membrane
L. F. Olsen and A. Lunding
is almost constant and only shows significant variation in response to changes in
temperature or in cell density [1] or to partially replacing H 2 O by D 2 O [64]. The
reason for this robustness of frequency is not known and cannot be reproduced by
most mathematical models of the pathway. While it is generally accepted that the
enzyme phosphofructokinase plays a central role in the mechanism responsible for
the oscillations, recent evidence has shown that also a number of enzymes and proteins, normally considered unrelated to glycolysis, contribute to the mechanism [61,
65].
Thus, glycolytic oscillations seem to involve a plethora of cellular processes. Here,
we will present new evidence that the dynamics of intracellular water also plays an
important role in the mechanism of the oscillations. These results seem to be general
and may be carried over to other oscillatory processes and ultimately suggest that
intracellular water may have a key role in cell metabolism and signal transduction in
cells. The function of the glycolytic oscillations is not known, but nevertheless they
have been preserved throughout evolution. Several roles of the oscillations have been
proposed, e.g. that they constitute an inevitable side effect in the tradeoff between
hard robustness and efficiency [13] or they increase the thermodynamic efficiency of
glycolysis [60]. Here we propose an alternative function for the oscillations, namely
that they serve to maintain the cell in a state of constant low entropy.
13.2 Variables Measured in Oscillating Glycolysis
Oscillations in glycolysis are typically measured as oscillations in the autofluorescence of NADH. Most other metabolites in glycolysis are optically “silent” and therefore cannot be measured in real time, but only after quenching of cell metabolism,
extraction of cell content and subsequent analysis by off-line assays [22, 40, 41, 59].
NADH oscillations in intact cells may be observed using fluorescence spectroscopy
to measure oscillations in cell suspensions [9, 23] or fluorescence microscopy [4,
12] to measure oscillations in single cells [12]. A typical experiment which shows
oscillations of NADH fluorescence in a suspension of yeast cells can be seen in Fig.
13.1a. Oscillations in NADH fluorescence are induced by addition of first glucose
and then 60 s later potassium cyanide to a suspension of starved yeast cells.
However, more recently time-resolved measurements of other variables such as
intracellular pH [19, 46], mitochondrial membrane potential [2], intracellular potassium ion [47] and water dynamics [64, 65] have been made. For example, it has long
been known that mitochondria contribute to the regulation of glycolytic oscillations
[3], and more recently oscillations in mitochondrial and other intracellular membrane potentials were directly measured using membrane potential-sensitive fluorescent dyes and shown to oscillate synchronously with NADH [2]. Figure 13.1 shows
simultaneous measurements of NADH, intracellular ATP, mitochondrial membrane