lp6c Some High Lights of the Association-Induction Hypothesis

The association-induction hypothesis (AI Hypothesis) is a unifying, general theory of the living cell, the only one of its kind. The main hypothesis was presented in 1962 in a monograph, entitled "A Physical Theory of the Living State: the Association-Induction Hypothesis". The section on the physical state of water was added three years later ( Annals of the New York Academy of Sciences: Vol. 125, pp. 401-417, 1965).Although two versions, one short and one longer, are given below, I must emphasize that you must consult the full-length books, especially "A Revolution in the Physiology of the Living Cell" to obtain a full understnding.

Short version

Take away bones,horns, hoofs, storage fats and stomach-intestinal-bladder contents from a cow what remain are largely water and proteins. A large portion of this water/proteins is found in the muscle tissues, which , of course, are the source materials of hamburgers. Even though some 80% of the raw hamburger is water and most if not all the muscle cells in a raw hamburger have been broken up into pieces, try as you may, you will not be able to squeeze out that 80% water or even a fraction of it, like you can squeeze out water from a wet sponge.

This simple fact shows that living cells are not little sausages filled with a watery solution (as in the familiar textbook rendition of the living cell, see linkded page, lp6). If that were the case, all that water would be squeezed out when the sausages are broken up into little open-ended fragments and pressure applied. On the contrary, this simple fact also demonstrates that there is strong cohesion not only between the different components of the cell, that is, between proteins and water, but also among individual elements of the same components, that is, between proteins and proteins as well as between water and water. Indeed, this is a statement of one of the basic tenets of the association-induction hypothesis: association. In a more complete version of the cell, association with proteins and other intracellular macromolecules extends to smaller molecules and ions like potassium ions and adenosine triphosphate or ATP, the ultimate product of cell metabolism.

A cow can live; it can also die. What happens to the muscle of a dead cow? Obviously, it too eventually dies. But what does it mean for a living cell to die? To understand, we must first understand the concept of the physical state.

Compare liquid water and solid ice.Both are collective names of a bunch of water molecules. They differ from each other only in the specific physical state the collection of water molecules assume. In the solid ice state, the water-to-water interaction in space is more ordered and it does not change much in response to say gravity. In contrast, the water-to-water interaction in space in the liquid state is more flexible and it flows under gravitational force. In the association-induction hypothesis, being alive means that the components of the cell substance, proteins, water and small molecules and ions are associated in a specific spatial relationship and in the high (negative) energy-low entropy state, called the living state. The living state is what a physicist calls a cooperative state like solid ice and liquid water which also represent cooperative states, indicating that each state is well defined and discrete and that there are neighbor-to-neighbor interaction among the individual elements. Pure water can be in either the solid ice state or the liquid water state but not in a state in-between. So the system of cooperatively associated protein-water-small molecule assembly also tends to be in either the discrete living state or in an all-or-none manner shifts to the death state, but not exclusively so (see below).

The forces underlying the various modes of association are fundamentally electric in nature. Take the case of water-to-water interaction. Each water molecule represented by the formula, H2O, contains two hydrogen atoms and one oxygen atom, arranged in such a manner that one end of the water molecule is positively charged while the other end if negatively charged. In other words, each water molecule may be seen as a dipolar molecule. Basic laws of electrostatics dictate that there is a tendency for the positive end of one dipolar water molecule to attract and interact with the negative end of a neighboring water molecule. However, the interaction does not stop there because the proximity of a neighboring dipole also shifts the electron distribution of each water molecule and as a result, an induced dipole moment is created in both, by the mutual electrical polarization also called induction. This of course is an example of the second basic tenet of the association-induction hypothesis: induction.

Only induction is not present only between pairs of water molecules but among all the interacting protein-water-small molecules/ion assembly that makes up the living cell. Nor is induction just a means of enhancing the interaction among the different components of the cell. It also makes them into functionally coherent and discrete cooperative assemblies. And it makes it possible to shift an assembly from, say, one resting-living-cooperative state to an active-living-cooperative state--- by the on-or-off action of certain small but biologically potent molecules called cardinal adsorbents. The cardinal adsorbent par excellence is ATP.

ATP is the final end-product of the food materials we consume. At one time, biochemists thought ATP is unusual in that it contains a great deal of energy in special phosphate bonds. This idea turned out to be wrong. In time it became established that while ATP does not carry so-called high-energy phosphate bonds, ATP has powerful affinity for the protein it interacts with, thereby affirming the unique role of this compound as the queen of cardinal adsorbents in maintaining the resting living state and in shifting between alternate states. If one stops eating, ATP spent cannot be replenished. And without ATP, the cells die. And with that, the individual dies.

A Longer Version

The AI Hypothesis is far too extensive a theory for me to attempt a presentation here of all the major aspects of the whole theory. Instead, I present only some selected high lights. Even in this modest attempt , some knowledge on the physico-chemical nature of a living cell, and the membrane-pump theory is necessary, and I shall accordingly begin with these two subjects.

Living cells are the basic unit of all life, ranging from minute single-celled organisms, like bacteria (e.g., E. coli) and protozoa (e.g., amoeba) to giant many-celled organism like ourselves. Physico-chemically speaking, all living cells share similar basic attributes. They all 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 and RNA and carbohydrates like glycogen). It is the nature and amounts of the cell proteins that determine the characteristics of living cells---although proteins are 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.

Most living cells spend their lives in a watery environment. When common salt, or sodium chloride, dissolves in water, it splits into two charged particles or ions, the positively charged sodium ion (Na+ ) and negatively charged chloride ion (Cl-).And 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, for example, as hydrated sodium ion and hydrated chloride ion.

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 ion, the potassium ion, though chemically very similar to the sodium ion, distributes itself in such a way that the concentration in the cells is some forty times higher than in the surrounding medium. Indeed, as our knowledge of the cell expands, we find that this asymmetry in the distribution of the sodium ion and potassium ion is found in virtually all living cells. How does cell physiologist explain this unusual pattern of distribution of the sodium ion? The mechanisms offered by the membrane-pump theory and the association-induction hypothesis are profoundly different. (See Linked page lp6a, for disproofs of the membrane-pump theory. For anchor to this linked page, see Home-page just above "A Unifying Validated New Alternative" )

In the membrane-pump theory, a living cell represents essentially a bagful of a water solution of proteins and other dissolved substances(see linked page, lp6 for the historic origin of this basically erroneous idea). 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 substance dissolved in normal liquid water. Cell proteins dissolved in this normal liquid cell water are themselves in their native state---that is, a stable, and reproducible, which a protein assumes reproducibly in vitro when purified by certain dtandard technical procedures and dissolved in water. An all-important membrane, called the cell membrane or plasma membrane encloses this bag of watery solution. Although this cell membrane is so thin that one cannot see it even with the best of light microscope; nonetheless, in the membrane pump theory, it is this very thin membrane which determines the chemical makeup of the cell either by virtue of postulated critical diameters of rigid membrane pores (or channels, see linked page lp16) which admit small molecules and ions but bar larger ones; or by the ceaseless inward or outward transportation by postulated energy-consuming specific pumps located in the cell membrane..

The best known example among these hypothetical membrane pumps is the sodium pump, which constantly pumps sodium ion out of the cell in spite of constant inward leakage, so that the sodium-ion concentration is at all times kept to a steady low level often a tenth to a fifth of that in the surrounding medium. It is also believed by some proponents of the membrane-pump theory at least, that this sodium pump does not just pump sodium ion out, it also pumps potassium ion in, thereby keeping the potassium ion concentration forty times higher than outside. For this reason, the sodium pump has also been known as the sodium-potassium pump.

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). I now turn to the alternative theory, the Association-Induction Hypothesis.

Everybody knows what a raw hamburger is like. From its rich water content, it resembles a wet sponge. Yet it is also quite different from a wet sponge. Squeeze a wet sponge, water comes out. Squeeze harder, more water comes out until finally the sponge becomes almost dry. If instead, you take a raw hamburger and try to squeeze the water out from this water-rich material, you will find that it is wellnigh impossible to squeeze any water out even after the meat has been chopped into tiny pieces. Indeed we carried on this line of inquiry in a more rigorously controlled manner.

Thus instead of squeezing the cut-up muscle by hand, we utilized centrifugation. As you know, it is by means of centrifugation, that water is extracted from wet laundry in a washing machine. Only in the muscle experiment, we made sure that every muscle cell had been cut into short segments with both ends open---which do not regenerate a new membrane, see linked page, lp6a{3}--- and subjected them to a centrifugal force of 1000 times gravity. Thus after centrifuging for 4 minutes, all the water found in between the muscle cells are completely extracted. Yet water from the inside the broken cells remains inside the cells (See Ling and Walton in Science, Volume 191, pp.293-295, 1976).

So this exceedingly simple experiment adds yet another set of evidence showing without ambiguity that the basic tenet of free water in 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 along with the indisputably normal liquid water (held in between the muscle cells), which is quantitatively squeezed out. What remains would 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.

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 1000-time gravity. Since the cell is primarily water and proteins, one naturally seeks explanation in terms of interaction between the more mobile water molecules and the more fixed proteins.

Now 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 powered material that comes in Jello packets. As you know, when the content of this packet is heated in water and chilled, you have the colorful, transparent, quivering and delicious Jello. Jello is almost all water and yet in Jello, water can "stand up" as no normal pure liquid water ever can. This ability of the water in Jello to stand---which ice can also---indicates that the water-to-water interaction in the Jello water has been altered by the only other component present, gelatin. Why and How?

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. But 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 the similarity of which to gelatin gel has also a long historical background).

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 linear arrays of 26 alphabets but in linear arrays of 20 some amino-acid residues. Each amino-acid component of the protein (an amino acid residue) offers a pair of electrically charged or polar groups to the protein chain, a negatively charged carbonyl oxygen (CO) carrying a "lone pair" of (negatively charged) electrons and a positively-charged imino group (NH) H atom. 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 water molecule can adsorb onto the protein's NH site; the H ends of the 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 ( see Ling et al. Physiol. Chem. Phys. 10: 87, 1978, see also Ling, " A Revolution in the Physiology of the Living Cell, Krieger, 1992, pp. 81-92), 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 helical structure. Accordingly, a large portion of the gelatin molecule remains permanently in the fully-extended conformation.

In this fully-extended conformation, the polar CO and NH groups are directly exposed to, and 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 and mentioned above.

Parenthetically, by multiple layers, I mean no more than few layers on each protein chain. That would be quite adequate to account for all the cell 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 properly spaced polar groups will behave like gelatin and the protoplasm of living cells. Water in all these model systems and 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.

In summary, according to the association-induction hypothesis all or virtually all 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 protein do so only conditionally 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 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 and small molecules and ions. In the conventional membrane-pump theory, all these ingredients exist as part of a solution. According to the association induction hypothesis, proteins, water and much of the small molecules and ions are closely associated and maintained in a high-(negative)energy and low-entropy state called the living state. A cell maintained at its living state is alive.

Now water and ice comprise the same water molecules represented as H2O. These molecules exist in different physical states which we call respectively liquid and 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 little in time. Water molecules in liquid water are more mobile and move about more freely with time.

Similarly, the living state specifies interactions among the individual components of the living substance of closely associated proteins, water and small molecules and ions in 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 is near-neighbor interaction among the individual component of the assembly. To maintain the cooperative living state, interaction with certain key small molecules like ATP is vital. When the cell is deprived of its supply of ATP, the cell dies and the protoplasm enters into another state, the dead state.

In the 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 large hydrated molecules and ions like sodium. The cell proteins also offer their singly and negatively charged beta- and gamma-carboxyl groups to adsorb preferentially--- on a one ion-one site basis---- 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 than in the surrounding medium. Sodium ions being unable to compete successfully against the potassium ion for these charged groups, or adsorption sites, while the cell is in the living state, remain largely in the cell water and exists at lower level than in the surrounding medium. Continual energy consumption is not needed to maintain the high potassium, low sodium ion distribution in living cells. 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 experiment which has unequivocally disproved the 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 interaction. In the association-induction hypothesis, it is this basic electronic mechanism which not only permits such key component, referred to as cardinal adsorbents, to sustain the protoplasm---of closely associated proteins-ion-water system---in its normal resting living state. It also provides the mechanism for cardinal adsorbents to control the reversible shifts between active and resting state. The cardinal adsorbent par excellence is the ultimate metabolic product, ATP.

This ubiquitous and crucial small molecule was once wrongly believed to carry an 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. Indeed, the adsorption energy of ATP on the muscle protein, myosin, even exceeds what was once (wrongly) assigned as phosphate bond energy and this high adsorption energy fits like hand in glove in its central role in polarizing the protein-water-ion system maintaining the assembly in the living state.

Note also, the concept of the "living state", despite its occasional plebeian usage by other investigators, is uniquely a concept of the AI Hypothesis. Being in the living state specifies what is living. Transition into the dead state specifies what is dead. In the living state, all the major components exist in their closely associated high (negative) energy and low entropy state. 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. In death, the proteins enter an internally neutralized state.

As already mentioned, there is no corresponding concept of what is living and what is not living in the membrane-pump theory.