Aqueous Solutions, Membranes,Channels, and Pumps(Old paradigm)VERSUSProtoplasm, Fully-Extended Proteins,Structured Water, and Cardinal Adsorbents(New paradigm)

A presentation of Dr. Gilbert LingsAssociation-Induction Hypothesis

By Dr. John T. Zimmerman

Dr. Gilbert LingsAssociation-Induction Hypothesisexplains: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 theAssociation-Induction Hypothesis refers to the association between water moleculesand 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 theAssociation-Induction Hypothesis refers to ability of certain molecules to INDUCE achange in the density of the electroncloud surrounding certain chargedions 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 thecharacteristics of living cells.In turn the nature of the proteins is dictated by the genetic informationcarried 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 livesin 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 lesspermanent coat of strongly-bound water molecules and are then referred toas hydrated sodium ionsand 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 distributionof thesodium ions (10X greater outside concentration)and thepotassium ions (40X greater inside concentration)are found in virtually all living cells.

How does the cell physiologist explainthis unusual pattern of distributionof 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 substancesin an aqueous solution.

With the membrane-pump theory,the water inside the cell showsno major difference fromnormal 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 theirso-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, thisso-called NATIVE STATE (a misnomer)is NOTthe normal, natural state of proteinsfound in living cells,particularly cells in the cooperative RESTING living state.

In the membrane-pump theory, an all-important but very thin membrane, called theCELL 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 PUMPlocated in the cell membrane.

Then there are also pumps for the different sugars, for the many different (free) amino acids , many different positively chargedas 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 HYPOTHESISdeveloped by Dr. Gilbert Ling.

Everybody knows what someraw 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 outuntil 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 proteinslike a fully-squeezed out sponge.

But that does not happen while the cells are still alive or close to being aliveas 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 mobilewater molecules and the more fixed proteins.

Theoretically speaking, all proteins have the potential of reacting witha 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 normalpure 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 gelatinfrom these animal partsis the protein known as collagen,the major protein componentof 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 of20 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 conformationjust 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 thePolarized 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 waterexisting in the dynamic structureof polarized multilayersas proposed by the AI Hypothesis.

Since then, it has been fully establishedthat gelatin as well as similar long chainorganic molecules or polymersthat can maintain a linear chain offully-extended proteins,which happen to have the properly spacedCO- and NH+ polar groupswill behave like gelatin andlike 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 ionsfound 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 hypothesisall or virtually all the water in living cellsassumes the dynamic structure ofpolarized 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 concentrationof large (hydrated) ions like sodium,sugars, and free amino acids.An underlying assumption isthat some of the cell proteinsexist 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 theoryhas 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 thesmall molecules and ions are closely ASSOCIATED or bonded togetherand maintain themselvesin a high-(negative) energy anda highly-ordered or low-entropy statecalled the cooperative RESTING living state.A cell maintained at itscooperative RESTING living state is ALIVE.

Most individuals know thatmatter exists in three different states:a gas, a liquid, or a solid.Water, therefore, exists as gaseous water (water vapor)liquid water, orsolid water (ice).

However, the liquid water state hastwo 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 asa state of water somewherein 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 statespecifies 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 amongthe 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 waterto 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 chargedbeta- and gamma-carboxyl groups (COOH-)to adsorb preferentiallyon 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 ahugely larger number ofnegatively-charged beta- and gamma-carboxyl groups (COOH-) on the fully-extended cell protein molecules than there are adsorbedpotassium+ 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 successfullyagainst 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 ofthe 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 perturbationsare 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 protoplasmof closely associated proteins-ion-water systemin 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 productof 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 believedto carry extra energy in the so-calledhigh-energy-phosphate bonds.

However, there is no doubt that ATPis strongly adsorbedon 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 ahigh energy phosphate bondand this high adsorption energyfits like hand in glove in itscentral role in polarizingthe protein-water-ion systemthus maintaining the assemblyin 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 SITESon protein molecules (receptor sites).

In 1957, Dr. Ling described the resultof a theoretical model in which selectivity forK+ 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 anda relatively low c-valuethe 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-valueThis 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 groupsand 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) andto 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