Aqueous Solutions, Membranes,

Channels, and Pumps

(Old paradigm)
VERSUS
Protoplasm, Fully-Extended Proteins,
Structured Water, and Cardinal Adsorbents
(New paradigm)

A presentation of Dr. Gilbert Lings
Association-Induction Hypothesis

By Dr. John T. Zimmerman
Dr. Gilbert Lings
Association-Induction Hypothesis
explains:

1) Cell volume control (osmosis)
2) The differential outside/inside solute
concentrations of potassium and sodium ions
(potassium inside, sodium outside)
3) Semipermeable membranes (more
permeable to potassium ions than to sodium
ions)
4) The cellular resting potential difference (-70
mV inside)
This lecture is about a novel and extremely
important hypothesis of the living states
(they're two of them)
at both the cellular and below-cell level
called the Association-Induction
Hypothesis developed by Dr. Gilbert Ling.
The ASSOCIATION aspect of the
Association-Induction Hypothesis refers to
the association between water molecules
and the carbonyl (CO
-
) and imino (NH
+
) ends of
amino acid residues in polypeptide chains.
It also refers to the association of potassium ions
with alpha and gamma carboxyl (COOH
-
)
groups on the protein chains as well.
The INDUCTION aspect of the
Association-Induction Hypothesis refers to
ability of certain molecules to INDUCE a
change in the density of the electron
cloud surrounding certain charged
ions on the polypeptide chain and to have that
change propagated along a string of about
1,042 molecules long.
Living cells contain a large amount of water,
making up some 80% of the cell's weight,
though it could be as low as 50%
and as high as 90%.
The rest of the cell consists mostly of giant
proteins molecules (and in much smaller
amounts , the nucleic acids, DNA or RNA, and
carbohydrates like glycogen).
It is the nature and amounts of the cell
proteins that determine the
characteristics of living cells.
In turn the nature of the proteins
is dictated by the genetic information
carried in DNA and RNA.
The cell also contains an assortment of small
molecules and ions. Some of these small
molecules and ions like ATP are vital to life.
When a salt dissolves in water, it splits
into two oppositely charged particles or
ions, the positively-charged ion is called
a CATION and the negatively-charged
ion is called an ANION.
Most living cells spend their lives
in a salt-watery environment.

When common salt, or sodium chloride,
dissolves in water, the ionically-bonded
molecule splits into two charged
particles or ions, positively charged
sodium ions (Na
+
) and negatively
charged chloride ions (Cl
-
).
In the process of dissolution,
these ions take up a more or less
permanent coat of strongly-bound water
molecules and are then referred to
as hydrated sodium ions
and hydrated chloride ions.
The sodium-ion concentration in most living
cells is low, equal to about one tenth of that
in the fluid outside the cell. In contrast,
another univalent positively charged cation,
the potassium ion, though chemically very
similar to the sodium ion, distributes itself in
such a way that its concentration inside the
cells is some forty times higher than in the
surrounding medium (interstitial fluid).
The asymmetries in the distribution
of the
sodium ions (10X greater outside concentration)
and the
potassium ions (40X greater inside concentration)
are found in virtually all living cells.
How does the cell physiologist explain
this unusual pattern of distribution
of the potassium and sodium ions?

The mechanisms offered by the membrane-pump
theory and the association-induction hypothesis
are profoundly different.
In the membrane-pump theory, a living cell
represents essentially a bag-full of a water, an
aqueous solution of proteins,
a lot of potassium ions, a few sodium ions,
and other dissolved substances
in an aqueous solution.
With the membrane-pump theory,
the water inside the cell shows
no major difference from
normal liquid water bathing the cells.

Nor are the small and large molecules and ions
inside the cell markedly different from similar
substances dissolved in normal liquid water.
With the membrane-pump theory,
cell proteins SUSPENDED in this normal liquid cell
water are themselves in their
so-called NATIVE STATE (a misnomer)
that is, a stable, and reproducible state, which a
protein assumes reproducibly in vitro when
purified by certain standard technical
procedures and dissolved in water.
However, this
so-called NATIVE STATE (a misnomer)
is NOT
the normal, natural state of proteins
found in living cells,
particularly cells in the
cooperative RESTING living state.
In the membrane-pump theory, an all-important
but very thin membrane, called the
CELL MEMBRANE or PLASMA MEMBRANE
encloses this bag of watery solution.
In the membrane pump theory, it is this very thin
membrane which determines the chemical
makeup and ionic distribution (potassium more
in the inside, sodium more on the outside)
of the cell.
The cell membrane accomplishes these tasks by
virtue of postulated critical diameters of rigid
membrane pores (or CHANNELS), which admit
small molecules and ions but bar larger ones
and by the ceaseless inward or outward
transportation of ions by a postulated energy-
consuming SODIUM-POTASSIUM PUMP
located in the cell membrane.
Then there are also pumps for the different
sugars, for the many different (free) amino
acids , many different positively charged
as well as negatively charged ions etc.
(For a partial list of the names of membrane
pumps postulated up to 1973, see Table 2 in
Ling et al, Annals of New York Academy of
Sciences, Vol. 204, pp.6-50, 1973).
Now we turn to the alternative theory,
the ASSOCIATION-INDUCTION HYPOTHESIS
developed by Dr. Gilbert Ling.
Everybody knows what some
raw hamburger feels like in your hands.
From its rich water content,
raw hamburger feels wet and moist.
Yet it is also quite different from a wet sponge.
Squeeze a wet sponge and water comes out.
Squeeze harder, more water comes out
until finally the sponge becomes almost dry.
If instead, you take some raw hamburger and try
to squeeze the water out from this water-rich
protein material, you will find that it is well nigh
impossible to squeeze any water out even after
the meat has been chopped into tiny pieces.
Even after spinning protein in a centrifuge at
1,000 times the force of gravity for 4 minutes,
water still remains in chopped-up muscle cells.
So this exceedingly simple experiment comprises
the first evidence showing, without ambiguity,
that the basic tenet of free water in the
membrane-pump theory is wrong.
The cell water cannot be normal liquid water.
Were the cell water truly normal liquid water, it
would have been extracted by squeezing or
even more so by centrifugation.
What should remain in squeezed hamburger or
centrifuged muscle fragments should be
nothing more than dried proteins
like a fully-squeezed out sponge.

But that does not happen while the cells are still
alive or close to being alive
as in fresh hamburger.
Our next question is to find out how water
(making up some 80% of the weight of the
fresh muscle (as well as other cells) can be
held so tenaciously inside the cell, resisting
centrifugation at 1,000-times gravity.
Since the cell is primarily water and proteins,
one naturally seeks an explanation in terms of
the interaction between the more mobile
water molecules and the more fixed proteins.
Theoretically speaking, all proteins have the
potential of reacting with
a large amount of water.
In reality, only some proteins interact with a large
amount of water "permanently.
One familiar water-retaining protein is gelatin,
the major ingredient of the powdered material
that comes in Jell-O packets.
Jell-O is almost all water and yet in Jell-O,
water can "stand up" as no normal
pure liquid water ever can.
This ability of the water in Jell-O to stand up
against gravity, which ice can also, indicates
that the water-to-water interaction in the Jell-O
water has been altered by the only other
component present, gelatin.
Why and how is this possible?
First, what is gelatin? Gelatin is a product of
"cooked" animal skin, hoof, horn, etc.

The main source material of gelatin
from these animal parts
is the protein known as collagen,
the major protein component
of our tendons and skin.
That gelatin is an unusual protein has been
known for a long time.
Thus the term COLLOID is its namesake.

It is the association-induction hypothesis, which
for the first time, offered an explanation for the
uniqueness of gelatin (as well as colloids) and
the "living substance" or protoplasm.
Proteins are long chain molecules.
However, unlike ordinary chains where each link
is just like another link, the proteins are chains
of some twenty different kinds of links,
called amino-acid residues which are amino acids
in a "joint" form. So in a way, the language of
life is spelled out not in a linear array of 26
alphabetic characters but in a linear array of
20 some amino-acid residues.
Each amino-acid component of the protein
(a long string of amino acid residues)
offers a pair of electrically charged or polar
groups between amino acids in the protein
chain, a negatively charged carbonyl oxygen
(CO
-
) carrying a "lone pair" of (negatively
charged) electrons and a positively-charged
imino (NH
+
) H atom,
which is lacking in one electron.
In most proteins, each CO
-
group is joined (or
hydrogen-bonded, or H-bonded) to the H
+

atom of the NH
+
group of the
third amino acid down the chain.
In this way, the protein chains assumes what is
known as the alpha-helix structure.
Both the polar NH
+
and CO
-
groups also have
affinity for water molecules.
The O end of the H
2
O water molecule can adsorb
onto the protein's NH
+
site; the H ends of the
H
2
O water molecules adsorb on to the O atom
of the protein's CO
-
site.
However, in most proteins in their so-called
native state, the NH
+
and CO
-
groups are joined
together intra-molecularly via H-bonds just
mentioned. Thus joined, they are unable to
interact with water. However, as first
pointed out by Ling in 1978, a large portion of
the gelatin chain cannot fold into the alpha-
helical folds because 54% of the amino acid
residues making up gelatin are either unable
(proline, hydroxyproline) or disinclined (glycine)
to assume the alpha-helical structure.
Accordingly, a large portion of the gelatin protein
molecules remains permanently in the
fully-extended conformation
just like the proteins in a living cell.

In this fully-extended conformation,
the polar CO
-
and NH
+
groups
are exactly properly spaced
and directly exposed to and they are free to
interact with, not just one layer,
but multiple layers of water molecules.


Water so polarized endows gelatin
with many of its unusual properties,
which it shares with living cells.

This is then the essence of what has been
known as the
Polarized Multilayer Theory of Cell Water
first introduced by Ling in 1965.
Parenthetically, by multiple layers,
of water this means no more than a few layers
(5, 6, or 7 layers of stacked-up water molecules)
on each protein chain (and there are hundreds of
such protein chains in a typical cell).

Stacking 5 to 7 layers of water molecules on top
of one another would be quite adequate
to account for all of the intercellular water
existing in the dynamic structure
of polarized multilayers
as proposed by the AI Hypothesis.

Since then, it has been fully established
that gelatin as well as similar long chain
organic molecules or polymers
that can maintain a linear chain of
fully-extended proteins,
which happen to have the properly spaced
CO
-
and NH
+
polar groups
will behave like gelatin and
like the protoplasm of living cells.

Water in all these model systems
and in the living cell shares
the property of maintaining,
at a lower concentration,
those molecules and hydrated ions
found at low levels in most living cells.

The most outstanding is the sodium ion
(lower on the inside than the
outside of the cell by a factor of 10).

In summary, according to the
association-induction hypothesis
all or virtually all the water in living cells
assumes the dynamic structure of
polarized multilayers.

Water assuming this dynamic structure
endows the living cells with many attributes
which had hitherto been assigned to other
(incorrect) causes.
Among these attributes is that of
maintaining a low concentration
of large (hydrated) ions like sodium,
sugars, and free amino acids.
An underlying assumption is
that some of the cell proteins
exist in the fully-extended conformation
even though, unlike gelatin, these
proteins do so only conditionally
(in the cooperative RESTING living state)
rather than permanently.
In other words, they do so only
when the cells are ALIVE.

What do we mean by being alive?
We will go on to that subject next.

It bears mentioning that the
membrane-pump theory
has not been able to produce
an answer to this simple
but basic question yet.
The major ingredients of living cells
are proteins, water, small molecules, some large
molecules like DNA
and ions (sodium, potassium, and chloride).

In the conventional membrane-pump theory,
all these ingredients exist as part of
a DILUTE AQUEOUS SOLUTION.
In contrast, according to the
association induction hypothesis,
proteins, water, and much of the
small molecules and ions are closely ASSOCIATED
or bonded together
and maintain themselves
in a high-(negative) energy and
a highly-ordered or low-entropy state
called the cooperative RESTING living state.
A cell maintained at its
cooperative RESTING living state is ALIVE.
Most individuals know that
matter exists in three different states:
a gas, a liquid, or a solid.
Water, therefore, exists as
gaseous water (water vapor)
liquid water, or
solid water (ice).
However, the liquid water state has
two different sub-states: unstructured (as in
normal liquid water) and structured (as found
inside the cell). The multilayered structured
water is due to adsorption of the water
molecules to the carbonyl (CO-) and imino
(NH+) polypeptide bonds.

Thus structured water (inside of a cell)
can be considered as
a state of water somewhere
in between normal liquid water and ice.
Water inside cells is somewhat structured.
But water in the solid state is totally structured.

Now water and ice comprise the same
water molecules represented as H
2
O.

As mentioned before, these molecules exist in
different physical states, which we call
respectively the liquid state and the solid state.

Note that each of these states specifies the
relationship between individual H
2
O molecules
in characteristic space and time coordinates.
In ice, water molecules are rigidly fixed in space
and move relatively little in time.
Water molecules in liquid water are much more
mobile and move about more freely with time.
Similarly, the cooperative RESTING living state
specifies interactions
among the individual components
of the living substance of closely-associated
proteins, water, small molecules, and ions in
relatively fixed-space-and-time coordinates.
In particular, special emphasis is on their mutual
electronic interactions which provide the basis
for their existence in what physicists call
"cooperative states" in which there are
near-neighbor interactions among
the individual components of the assembly.

To maintain the
cooperative RESTING living state,
interaction with certain key
small molecules like adenosine triphosphate
(ATP) is vital.

When the cell is deprived of its supply of ATP, the
cell dies and the protoplasm enters into
another state, called the DEAD state.
This is permanent and irreversible!

In the cooperative RESTING living state,
cell proteins cause the bulk of cell water
to exist in the dynamic structure of
polarized multilayers.

Water assuming that dynamic structure
shows reduced solvency for and partly excludes
large hydrated molecules and ions like sodium
large molecules like sucrose, and certain small
molecules like free amino acids.
The cell proteins also offer their
singly and negatively charged
beta- and gamma-carboxyl groups (COOH
-
)
to adsorb preferentially
on a one ion-one site basis
hydrated potassium ions
(over the sodium ion, for example).
Since there is a high concentration of beta- and
gamma-carboxyl groups carried on intracellular
proteins, the potassium ion concentration in
living cells is as a rule much higher (40 times
higher) than in the surrounding medium.



However, there is a
hugely larger number of
negatively-charged beta- and gamma-carboxyl
groups (COOH-) on the fully-extended cell
protein molecules than there are adsorbed
potassium
+
ions to neutralize them.



It is for this reason (more negatively charged
anionic sites on intercellular proteins than
positively charged potassium cations) that the
inside of the cell is
-70 millivolts negative.

It is NOT due to the operation of a hypothesized
sodium-potassium pump that the inside of the
cell is negative.

Sodium ions being unable
to compete successfully
against the smaller hydrated potassium ion
for these charged carboxyl (COOH
-
) groups,
or adsorption sites, while the cell is in the
cooperative RESTING living state,
remain largely in the extracellular water
and therefore the sodium ion exists at a much
lower level (10 times lower) inside the cell
than in the extracellular fluid.

With the AI hypothesis continual energy
consumption by a sodium-potassium pump is
not needed to maintain the high potassium,
low sodium ion distribution in living cells as is
required by the membrane-pump theory.
Here again one finds another profound difference
between the AI Hypothesis and the membrane
pump theory, which requires a continuous
supply of energy just to keep the ions and
molecules where they are and at the
concentrations they are found---a requirement
that permitted a set of crucial experiments
which has unequivocally disproved the
membrane-pump theory.
Thus far we have dealt with the
"associative" aspect of
the association-induction hypothesis.
Equally important is the "inductive " aspect, or
electrical polarization.
Thus in the AI Hypothesis, the living cell is
essentially an electronic machine,
where the electronic perturbations
are not carried out through long-range ohmic
conduction of free electrons along electric wires
but by a falling-domino-like propagated
short-range interactions.


In the association-induction hypothesis,
it is this basic electronic mechanism,
which not only permits such
key components,
referred to as cardinal adsorbents,
to sustain the protoplasm
of closely associated proteins-ion-water system
in its normal cooperative RESTING living state.

It also provides the mechanism for cardinal
adsorbents to control the reversible shifts
between the cooperative RESTING living state
and the cooperative ACTIVE living state.

The cardinal adsorbent par excellence is the
ultimate metabolic product
of the combination of the food we eat and
the oxygen in the air we breathe,
adenosine triphosphate (ATP).
This ubiquitous and crucial small molecule,
ATP, was once wrongly believed
to carry extra energy in the so-called
high-energy-phosphate bonds.

However, there is no doubt that ATP
is strongly adsorbed
on certain key sites (cardinal sites)
on cell proteins and the adsorption energy upon
these proteins is what gives ATP its energy,
not the high-energy phosphate bonds.
Indeed, the adsorption energy of ATP on the
muscle protein, myosin,
even exceeds what was once
(wrongly) assigned as a
high energy phosphate bond
and this high adsorption energy
fits like hand in glove in its
central role in polarizing
the protein-water-ion system
thus maintaining the assembly
in the cooperative RESTING living state.
Note also, the concept of the
"living state"
despite its occasional plebeian usage
by other investigators,
is uniquely a concept of the
Association-Induction Hypothesis.
Being in the living state specifies what is living.
In the cooperative RESTING living state,
all the major components exist in their closely
associated high (negative) energy and
highly ordered, low entropy state.
The transition into the dead state
specifies what is dead.
In the dead state, water and ions
are to a large extent liberated and
exist as free water and free ions,
with a large entropy gain (more disorganization).
In death, the proteins enter an internally
neutralized alpha-helix or beta-sheet state.
As already mentioned, there is no corresponding
concept of what is living and what is not living
in the membrane-pump theory.
There is a third state, which is uniquely different
from either the dead state or the
cooperative RESTING living state and it is called
the cooperative ACTIVE living state.

This is INDUCED by the adsorption of certain
electron DONATING cardinal adsorbents or
electron WITHDRAWING cardinal adsorbents
onto so-called CARDINAL SITES
on protein molecules (receptor sites).
In 1957, Dr. Ling described the result
of a theoretical model in which selectivity for
K
+
and for Na
+
could be REVERSED,
as in the nerve or muscle action potential.
This phenomenon was found to be a result from
a difference in the electron density (or c-value).

At low electron density and
a relatively low c-value
the cell water is structured,
potassium is adsorbed onto alpha and gamma
carboxyl sites, and K+ is preferred over Na+
in the structured cell water.
In contrast at a relatively high c-value
This ion selectivity is reversed and
Na+ is preferred over K+.
The reason for this is there is a loss of
structured water intervening between the
negatively-charged carboxyl groups
and the Na+ ions.
With the loss of structured water,
caused by a relatively high c-value,
sodium ions are therefore allowed to
travel down their concentration gradient
(of being 10 times more concentrated
outside the cell than inside) and
to travel down their electrical gradient
to enter the the unstructured cell water,
thus reversing
the potassium and sodium concentrations.


End of lecture as of 8/24/2005