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L. F. Olsen and A. Lunding
valid in dilute solutions. However, due to the molecular crowding of the cytoplasm
caused by the high intracellular concentrations of biological macromolecules [16,
17, 32, 38, 54, 73] the state of intracellular water may be different from that in dilute
solution. The overall concentration of macromolecules in the cytoplasm amounts to a
volume fraction of 40%, and this and the large polar surface areas of intracellular lipid
membranes may facilitate the binding of water molecules and restrict their motion,
challenging the view that intracellular water is mainly in a fluid state [14, 21, 75].
This creates a gap in our current understanding of metabolism and its regulation,
mainly because enzymes may have different properties in the crowded environment
inside a cell [15, 53]. In some cases this macromolecular crowding is referred to as an
“excluded volume effect” [15] where crowding has no effect on the physical-chemical
properties of water. However, recent studies using molecular dynamics simulations
have indicated that the mobility of water molecules is very heterogeneous and that a
large fraction of intracellular water molecules is essentially immobilised in long-lived
water bridges between proteins [37].
13.3.2 Coupling of Dynamics of Intracellular Water
to Glycolysis
The discovery of biomolecular condensates has spurred new interest in intracellular
water [7]. These phase-separated concentrates of proteins and nucleic acids may
offer additional cellular compartmentalization on top of that provided by organelles
and may provide a special aqueous environment for temporal and spatial control of
cellular biochemistry [62]. Interestingly—and relevant here—the liquid-like droplets
may show viscous fluid dynamics and their apparent viscosity is ATP-dependent [10].
Recently it was found that the dynamics of intracellular water has a strong influence
on glycolytic oscillations [64]. For example adding increasing concentrations of D 2 O
(up to 50%) to a suspension of yeast cells results in a decrease in the frequency of
the oscillations. As stated above the oscillation frequency is otherwise generally
insensitive to changes in experimental conditions [11, 57]. The effect of D 2 O is not
due to its physicochemical properties (e.g. density and viscosity) because ACDAN
and PRODAN show exactly the same emission peaks in pure H 2 O and D 2 O [6, 64].
Instead, the effect of D 2 O on the frequency of glycolytic oscillations was interpreted
as a secondary isotope effect [64], i.e. the presence of deuterium affects the rates of
physicochemical processes even if deuterated bonds are not themselves involved [68].
Further evidence that the solvent (H 2 O or D 2 O) is involved in the mechanism
of oscillation comes from the use of the so-called DAN (6-acyl-2-(dimethylamino)
naphtalene) probes, specifically ACDAN, PRODAN and LAURDAN [64]. These
probes were developed as polarity sensitive fluorescent probes to study nanosecond
relaxation processes in biological systems [70]. LAURDAN is, because of its long
hydrophobic side chain, not soluble in water, whereas the two other probes (ACDAN
and PRODAN), because of their shorter side chains, are very soluble in water and
L. F. Olsen and A. Lunding
valid in dilute solutions. However, due to the molecular crowding of the cytoplasm
caused by the high intracellular concentrations of biological macromolecules [16,
17, 32, 38, 54, 73] the state of intracellular water may be different from that in dilute
solution. The overall concentration of macromolecules in the cytoplasm amounts to a
volume fraction of 40%, and this and the large polar surface areas of intracellular lipid
membranes may facilitate the binding of water molecules and restrict their motion,
challenging the view that intracellular water is mainly in a fluid state [14, 21, 75].
This creates a gap in our current understanding of metabolism and its regulation,
mainly because enzymes may have different properties in the crowded environment
inside a cell [15, 53]. In some cases this macromolecular crowding is referred to as an
“excluded volume effect” [15] where crowding has no effect on the physical-chemical
properties of water. However, recent studies using molecular dynamics simulations
have indicated that the mobility of water molecules is very heterogeneous and that a
large fraction of intracellular water molecules is essentially immobilised in long-lived
water bridges between proteins [37].
13.3.2 Coupling of Dynamics of Intracellular Water
to Glycolysis
The discovery of biomolecular condensates has spurred new interest in intracellular
water [7]. These phase-separated concentrates of proteins and nucleic acids may
offer additional cellular compartmentalization on top of that provided by organelles
and may provide a special aqueous environment for temporal and spatial control of
cellular biochemistry [62]. Interestingly—and relevant here—the liquid-like droplets
may show viscous fluid dynamics and their apparent viscosity is ATP-dependent [10].
Recently it was found that the dynamics of intracellular water has a strong influence
on glycolytic oscillations [64]. For example adding increasing concentrations of D 2 O
(up to 50%) to a suspension of yeast cells results in a decrease in the frequency of
the oscillations. As stated above the oscillation frequency is otherwise generally
insensitive to changes in experimental conditions [11, 57]. The effect of D 2 O is not
due to its physicochemical properties (e.g. density and viscosity) because ACDAN
and PRODAN show exactly the same emission peaks in pure H 2 O and D 2 O [6, 64].
Instead, the effect of D 2 O on the frequency of glycolytic oscillations was interpreted
as a secondary isotope effect [64], i.e. the presence of deuterium affects the rates of
physicochemical processes even if deuterated bonds are not themselves involved [68].
Further evidence that the solvent (H 2 O or D 2 O) is involved in the mechanism
of oscillation comes from the use of the so-called DAN (6-acyl-2-(dimethylamino)
naphtalene) probes, specifically ACDAN, PRODAN and LAURDAN [64]. These
probes were developed as polarity sensitive fluorescent probes to study nanosecond
relaxation processes in biological systems [70]. LAURDAN is, because of its long
hydrophobic side chain, not soluble in water, whereas the two other probes (ACDAN
and PRODAN), because of their shorter side chains, are very soluble in water and