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 Ling’s

Association-Induction Hypothesis

By Dr. John T. Zimmerman

Dr. Gilbert Ling’sAssociation-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 thethird 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 H2O water molecule can adsorb onto the protein's NH+ site; the H ends of the H2O 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 thefully-extended conformation

just like the proteins in a living cell.

In this fully-extended conformation,the polar CO- and NH+ groupsare 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 gelatinwith many of its unusual properties,

which it shares with living cells.

This is then the essence of what has beenknown as the

Polarized Multilayer Theory of Cell Waterfirst 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

adequateto account for all of the intercellular water

existing in the dynamic structureof polarized multilayers

as proposed by the AI Hypothesis.

Since then, it has been fully establishedthat gelatin as well as similar long chain

organic molecules or polymersthat can maintain a linear chain of

fully-extended proteins,which happen to have the properly spaced

CO- and NH+ polar groupswill behave like gelatin and

like the protoplasm of living cells.

Water in all these model systemsand in the living cell sharesthe 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 theassociation-induction hypothesis

all or virtually all the water in living cellsassumes the dynamic structure of

polarized multilayers.

Water assuming this dynamic structureendows the living cells with many attributes which had hitherto been

assigned to other (incorrect) causes.

Among these attributes is that ofmaintaining a low concentration

of large (hydrated) ions like sodium,sugars, and free amino acids.An underlying assumption isthat some of the cell proteins

exist in the fully-extended conformationeven though, unlike gelatin, theseproteins do so only conditionally

(in the cooperative RESTING living state)rather than permanently.

In other words, they do so onlywhen the cells are ALIVE.

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

It bears mentioning that themembrane-pump theory

has not been able to producean answer to this simplebut basic question yet.

The major ingredients of living cellsare proteins, water, small molecules, some

large molecules like DNAand ions (sodium, potassium, and chloride).

In the conventional membrane-pump theory,

all these ingredients exist as part ofa DILUTE AQUEOUS SOLUTION.

In contrast, according to theassociation induction hypothesis,proteins, water, and much of the

small molecules and ions are closely ASSOCIATED or bonded together

and maintain themselvesin a high-(negative) energy and

a highly-ordered or low-entropy statecalled 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 samewater molecules represented as H2O.

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 H2O

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 interactionsamong the individual componentsof 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 arenear-neighbor interactions among

the individual components of the assembly.

To maintain thecooperative RESTING living state,

interaction with certain keysmall 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 ofpolarized multilayers.

Water assuming that dynamic structureshows reduced solvency for and partly

excludeslarge hydrated molecules and ions like sodium large molecules like sucrose, and certain small molecules like free amino

acids.

The cell proteins also offer theirsingly and negatively charged

beta- and gamma-carboxyl groups (COOH-)to adsorb preferentially

on a one ion-one site basishydrated 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 aHowever, there is ahugely larger number ofhugely larger number of

negatively-charged 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 unableto compete successfully

against the smaller hydrated potassium ionfor these charged carboxyl (COOH-) groups,or adsorption sites, while the cell is in the

cooperative RESTING living state,remain largely in the extracellular waterand therefore the sodium ion exists at a much lower level (10 times lower) inside

the cellthan 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

propagatedshort-range interactions.

In the association-induction hypothesis,it is this basic electronic mechanism,

which not only permits suchkey 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 andthe 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-calledhigh-energy-phosphate bonds.

However, there is no doubt that ATPis 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 itscentral role in polarizing

the protein-water-ion systemthus maintaining the assembly

in the cooperative RESTING living state.

Note also, the concept of the"living state"

despite its occasional plebeian usageby other investigators,

is uniquely a concept of theAssociation-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

andhighly ordered, low entropy state.

The transition into the dead statespecifies what is dead.

In the dead state, water and ionsare to a large extent liberated andexist 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

calledthe 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 concentratedoutside the cell than inside) and

to travel down their electrical gradientto enter the the unstructured cell water,

thus reversingthe potassium and sodium concentrations.

End of lecture as of 8/24/2005