3. CELLULAR ASPECTS OF ACTIVE TRANSPORT
191
the question must be asked whether the number of molecules is so small
as to make their isolation and identification impossible. On the basis of
the calculation made by Solomon et al. (207) concerning the kinetics
of ouabain inhibition of Κ transport, one site for every million square
angstroms of cellular surface is postulated, while Glynn (208) estimates
only one-tenth of this number of sites to be present. Thus 5 kg. of cells
must be collected in order to obtain 1 /xmole of a molecule carrying the
active site.
For yeast cells Conway et al. (209) estimate from the rubidiumpotassium competition that 130 μeq of a cation carrier should be found
in 1 kg. of centrifuged cells.
If one postulates that inorganic ions are passing the membrane
through aqueous channels, it is possible to estimate from water flow
measurements the density of these channels. From the figures obtained,
the amount of molecules responsible for the specific permeability characteristics of the membrane can be calculated.
It appears that about 0.02% of the total area of the membrane can
be attributed to "pore" surface. This should be divided into patches of
4 A. radius, i.e., for 1 cm.
2 0.4 Χ 10
13 patches. If each patch contains 1
molecule carrying the transport site(s), 0.4 Χ 10
13 molecules can be
extracted from 1 cm
2 of membrane surface. Since 1 cm.
2 corresponds
to ca. 1.10
6 cells, this means that ca. 150 /xmoles could be extracted from
1 kg. of cells.
This estimation gives, of course, the total number of molecules carrying active sites lining a water-filled "pore" in the membrane.
The next question to be asked is whether or not the transport system
has the properties of an enzyme. A priori it is not necessary to assume
that the active site is an integral part of an enzyme molecule. The fact
that a molecule is able to bind a substance in one phase and to release
it in another does not describe an enzyme. However, when we are
dealing with active transport, we know that energy must be fed into the
transporting system. There is good experimental evidence to show that
ATP is the energy source. The most conclusive experiments on this
matter have been made by Hodgkin and his colleagues (210-213) on the
squid giant axon. The endogenous arginine phosphate and ATP levels
are depleted by poisoning the axon with 2 mM cyanide. As a result Na
efflux as measured with Na
22
, diminishes. The subsequent injection of
arginine phosphate, phosphoenol pyruvate, ATP, or ADP restores the
Na efflux. Hodgkin concludes therefore that high-energy phosphate compounds are necessary for active transport of cations in the squid giant
axon. If this conclusion is correct, part of the transport system must act
as an adenosine triphosphatase.
191
the question must be asked whether the number of molecules is so small
as to make their isolation and identification impossible. On the basis of
the calculation made by Solomon et al. (207) concerning the kinetics
of ouabain inhibition of Κ transport, one site for every million square
angstroms of cellular surface is postulated, while Glynn (208) estimates
only one-tenth of this number of sites to be present. Thus 5 kg. of cells
must be collected in order to obtain 1 /xmole of a molecule carrying the
active site.
For yeast cells Conway et al. (209) estimate from the rubidiumpotassium competition that 130 μeq of a cation carrier should be found
in 1 kg. of centrifuged cells.
If one postulates that inorganic ions are passing the membrane
through aqueous channels, it is possible to estimate from water flow
measurements the density of these channels. From the figures obtained,
the amount of molecules responsible for the specific permeability characteristics of the membrane can be calculated.
It appears that about 0.02% of the total area of the membrane can
be attributed to "pore" surface. This should be divided into patches of
4 A. radius, i.e., for 1 cm.
2 0.4 Χ 10
13 patches. If each patch contains 1
molecule carrying the transport site(s), 0.4 Χ 10
13 molecules can be
extracted from 1 cm
2 of membrane surface. Since 1 cm.
2 corresponds
to ca. 1.10
6 cells, this means that ca. 150 /xmoles could be extracted from
1 kg. of cells.
This estimation gives, of course, the total number of molecules carrying active sites lining a water-filled "pore" in the membrane.
The next question to be asked is whether or not the transport system
has the properties of an enzyme. A priori it is not necessary to assume
that the active site is an integral part of an enzyme molecule. The fact
that a molecule is able to bind a substance in one phase and to release
it in another does not describe an enzyme. However, when we are
dealing with active transport, we know that energy must be fed into the
transporting system. There is good experimental evidence to show that
ATP is the energy source. The most conclusive experiments on this
matter have been made by Hodgkin and his colleagues (210-213) on the
squid giant axon. The endogenous arginine phosphate and ATP levels
are depleted by poisoning the axon with 2 mM cyanide. As a result Na
efflux as measured with Na
22
, diminishes. The subsequent injection of
arginine phosphate, phosphoenol pyruvate, ATP, or ADP restores the
Na efflux. Hodgkin concludes therefore that high-energy phosphate compounds are necessary for active transport of cations in the squid giant
axon. If this conclusion is correct, part of the transport system must act
as an adenosine triphosphatase.