20
Chapter 2
Now, the intracellular pH is usually greater than the typical pH of the medium in penicillin cultivations.
Equation (4) then indicates that there is a higher total concentration of the acid inside the cells than in the
extracellular medium. Using this equation Henriksen
et al.
(1998) calculated the concentration ratio at
different extracellular pH values and an intracellular pH of 7.2.
For an extracellular pH of 6.5 the
accumulation is low (about 2.3-fold), whereas at an extracellular pH of 5.0 the accumulation is high
(about
100
-fold).
For a given total extracellular acid concentration, the concentrations of both forms of the acid on each
side of the cytoplasmic membrane can be calculated using Eq. (1), from which the mass flux of acid
across the membrane can be calculated using Eq. (2.3). Because the net outflux of dissociated acid equals
the net influx of undissociated acid, the result of acid transport is a net influx of protons, which have to
be re-exported by the cytoplasma membrane bound ATPase in order to maintain a constant intracellular
pH. If it is assumed that the export of each proton requires the expenditure of one ATP by the ATPase
reaction, Henriksen
et al.
(1998) calculated that the ATP consumption resulting from this futile cycle
amounts to 0.15 mmol of ATP (g DW
)'1
h
' 1
at an extracellular pH of 6.5 and an intracellular pH of 7.2.
This is a low value compared with other non-growth-associated processes that also consume ATP (see
Section 5.2). However, at an extracellular pH of 5.0 the ATP loss is about 7 mmol of ATP (g DW
)'1
h
'1
(again with an intracellular pH of 7.2), which is a significant drain of cellular free energy. It is thus seen
how the maintenance of acid concentration gradients across the plasma membrane contributes to the
decoupling of ATP generation and ATP consumption used strictly for biosynthetic demands.___________
2.L2.2 Facilitated Diffusion
In the cytoplasmic membrane there are a number of carrier proteins that allow specific compounds
to be transported passively, but considerably faster than by free diffusion across the membrane.
This process is referred to as facilitated diffusion, and this transport mechanism is typical for fungi,
but much rarer for bacteria - thus glycerol is the only substrate which is known to enter
E. coli
by
facilitated diffusion (Ingraham
et al.,
1983). Facilitated diffusion resembles free diffusion since
transport occurs only in the downhill direction of a concentration gradient. The compound can enter
the membrane only if there is an available free carrier, and the rate of the transport process therefore
follows typical saturation-type kinetics; i.e., at low concentrations the rate is first order with respect
to the substrate concentration, whereas it is zero order at high concentrations (see Example 2.2).
The most important substances transported by facilitated diffusion are glucose and other sugars in
fungi.
Example 2,2, Facilitated diffusion
A substrate is transported across a (lipid) membrane by a carrier molecule, which is present in the
membrane either in free form (concentration
e
) or bound to the substrate (concentration
c„e).
The free
substrate concentration just inside the membrane at z =
0
is
=
K c
a, and at the other face of the
membrane, at
z
=
d
the concentration is
c„b = Kycb
(see figure below).
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