Association-induction Hypothesis Research Articles

Linking surface tension to water polarization with a new hypothesis: The Ling-Damodaran Isotherm.

Studying aqueous solutions of complex (bio)polymers is essential from both theoretical and practical perspectives. To understand the principles that govern the properties of these solutions is pivotal for the study of biological processes, considering that the most distinguished components of the cells are polymers (proteins, nucleic acids). These macromolecular aqueous systems, known as colloids, has raise the interest of scientists in recent years. It is known that several physicochemical properties deviate from ideal behaviour in this kind of solutions and that the physical state of water is different compared to its pure state. Particularly, the surface tension of such mixtures often shows a peculiar profile at semi-dilute and concentrated conditions. Here, we joined the colloidal concept of water polarization (proposed in the Association-Induction Hypothesis) with Damodaran's formalism for surface tension to theoretically derive a compelling mathematical model that explains the behaviour of polymer solutions. We measured the surface tension and osmolarity of different polyethylene oxide solutions and we used the ACDAN fluorescence probe to assess the water dipolar relaxation (polarization) in these mixtures. As a proof of concept, we also studied the influence of these polymer solutions on lipid interfaces. Our isotherm model explains the experimental observations with a unifying view that correlates with other measured properties, such as osmolarity and water dipolar relaxation. This provides a link between interfacial and bulk physicochemical properties of polymer solutions, also giving a new framework for studying the interaction of colloidal systems with lipid membranes interfaces.

Synthesis of theories on cellular powering, coherence, homeostasis and electro-mechanics: Murburn concept and evolutionary perspectives.

If evolution was/is a fact, a simplified/unifying approach to explain cellular physiology is warranted. Such a perspective should agree with the thermodynamic, kinetic, structural, and operational-probabilistic considerations; without invoking overt intelligence or determinism, and must enable a synthesis from chaos. In this regard, we first list salient theories in cellular physiology for (i) powering (generation of chemical/heat energy), (ii) coherence (interconnectivity and workability as a unit), (iii) homeostasis (metabolizing and expelling of unfamiliar/unwanted materials, maintaining concentration/volume), and (iv) cellular electrical-mechanical activities. While doing so, we discuss the scopes and limitations of (a) the classical active-site affinity and recognition-based modality of lock-key and induced-fit enzyme-catalytic mechanisms established by Fischer/Koshland, (b) membrane-pump hypothesis acclaimed by biologists-physicians and historically championed by the British Nobel-laureates like Hodgkin-Huxley-Katz-Mitchell, and (c) association-induction hypothesis advocated by physicists-physiologists from various parts of the world, for example, Gilbert Ling (China-USA), Gerald Pollack (USA), Ludwig Edelmann (Germany), Vladimir Matveev (Russia), and so on. We apply murburn concept (from "mured burning," capturing the thesis that one-electron redox equilibriums involving diffusible reactive species play vital roles in maintaining life order) to amalgamate several core cellular functions and further discuss the prospects for establishing the continuum of the principles of physics in biology.

The membrane potential arising from the adsorption of ions at the biological interface.

Membrane theory makes it possible to compute the membrane potential of living cells accurately. The principle is that the plasma membrane is selectively permeable to ions and that its permeability to mobile ions determines the characteristics of the membrane potential. However, an artificial experimental cell system with an impermeable membrane can exhibit a nonzero membrane potential, and its characteristics are consistent with the prediction of the Goldman-Hodgkin-Katz eq., which is a noteworthy concept of membrane theory, despite the membrane's impermeability to mobile ions. We noticed this troublesome facet of the membrane theory. We measured the potentials through permeable and impermeable membranes where we used the broad varieties of membranes. Then we concluded that the membrane potential must be primarily, although not wholly, governed by the ion adsorption-desorption process rather than by the passage of ions across the cell membrane. A theory based on the Association-Induction Hypothesis seems to be a more plausible mechanism for the generation of the membrane potential and to explain this unexpected physiological fact. The Association-Induction Hypothesis states that selective ion permeability of the membrane is not a condition for the generation of the membrane potential in living cells, which contradicts the prediction of the membrane theory. Therefore, the Association-Induction Hypothesis is the actual cause of membrane potential. We continued the theoretical analysis by taking into account the Association-Induction Hypothesis and saw that its universality as a cause of potential generation mechanism. We then concluded that the interfacial charge distribution is one of the fundamental causes of the membrane potential.

Water as a Link between Membrane and Colloidal Theories for Cells.

This review is an attempt to incorporate water as a structural and thermodynamic component of biomembranes. With this purpose, the consideration of the membrane interphase as a bidimensional hydrated polar head group solution, coupled to the hydrocarbon region allows for the reconciliation of two theories on cells in dispute today: one considering the membrane as an essential part in terms of compartmentalization, and another in which lipid membranes are not necessary and cells can be treated as a colloidal system. The criterium followed is to describe the membrane state as an open, non-autonomous and responsive system using the approach of Thermodynamic of Irreversible Processes. The concept of an open/non-autonomous membrane system allows for the visualization of the interrelationship between metabolic events and membrane polymorphic changes. Therefore, the Association Induction Hypothesis (AIH) and lipid properties interplay should consider hydration in terms of free energy modulated by water activity and surface (lateral) pressure. Water in restricted regions at the lipid interphase has thermodynamic properties that explain the role of H-bonding networks in the propagation of events between membrane and cytoplasm that appears to be relevant in the context of crowded systems.

Critical analysis of explanations for cellular homeostasis and electrophysiology from murburn perspective.

Pursuits in modern cellular electrophysiology are fraught with disagreements at a fundamental level. While the membrane theory of homeostasis deems the cell membrane and proteins embedded therein as the chief players, the association-induction (or sorption/bulk-phase) hypothesis considers the aqueous phase of dissolved proteins (cytoplasm/protoplasm) as the key determinant of cellular composition and ionic fluxes. In the first school of thought, trans-membrane potential (TMP) and selective ion pumps/channels are deemed as key operative principles. In the latter theory, sorption-desorption dynamics and rearrangements of bulk phase determine the outcomes. In both these schools of thought, theorists believe that the macroscopic phase electroneutrality holds, TMP (whether in resting or in activated state) results solely due to ionic concentration differentials across the membrane, and the concerned proteins undergo major conformation changes to affect/effect the noted outcomes. The new entry into the field, murburn concept, builds starting from molecular considerations to macroscopic observations. It moots "effective charge separation" and intricate "molecule-ion-radical" electron transfer equilibriums as a rationale for ionic concentration differentials and TMP variation. After making an unbiased appraisal of the two classical schools of thought, the review makes a point-wise analysis of some hitherto unresolved observations/considerations and suggests the need to rethink the mechanistic perspectives.

Cellular metabolism and colloids: Realistically linking physiology and biological physical chemistry

Important concepts from colloidal physical chemistry such as coacervation, phase transitions, emergent properties and ionic association, are currently emerging in the lexicon of cellular biology, prompted mostly by recent experimental observations of liquid phase coexistence in the cell cytosol. Nevertheless, from an historical point of view, the application of these concepts in cell biology is not new. They were key concepts into the so-called protoplasmic doctrine, an alternative (and largely forgotten) approach to cell physiology. The most complete theory originating from this line of thinking was the Association-Induction Hypothesis (AIH), introduced by Gilbert N. Ling in 1962. The AIH, which envisions living cells as complex dynamical colloidal systems, provides ample theory and experimental evidence to call into question the now dominant view of living cells as fluid-filled vesicles. This review attempts to present and discuss the usefulness of the AIH to understand a series of experimental observations from our laboratory from living suspensions of the yeast Saccharomyces cerevisiae exhibiting glycolytic oscillations. Particularly, the AIH helped us integrate, in a mechanistic sense, the basis of a strong temporal coupling observed between ATP and a series of cellular properties such as intracellular water dipolar relaxation, intracellular K+ concentration, among many others, where the colloidal physical chemistry of the cell interior plays a fundamental role.

Glycolytic oscillations and intracellular K+ concentration are strongly coupled in the yeast Saccharomyces cerevisiae

We measured temporal oscillations of intracellular K+ concentration in yeast cells exhibiting glycolytic oscillations using fluorescence spectroscopy and microscopy methods. These oscillations showed the same period as those of glycolytic metabolites (NADH, ATP), indicating a strong coupling between them. We experimentally ruled out that oscillations originate in extra- or intracellular K+ fluxes and conclude that these oscillations arise from fluctuations in free and adsorbed states of K+ in the cell interior. Oscillations in K+ showed a strong dependence on ATP and the organization of the cell cytoskeleton. Our results challenge the widely held view that intracellular K+ predominantly exists in a free state. They can, however, be productively understood in terms of Gilbert Ling's Association-Induction hypothesis.

Coupled Response of Membrane Hydration with Oscillating Metabolism in Live Cells: An Alternative Way to Modulate Structural Aspects of Biological Membranes?

We propose that active metabolic processes may regulate structural changes in biological membranes via the physical state of cell water. This proposition is based on recent results obtained from our group in yeast cells displaying glycolytic oscillations, where we demonstrated that there is a tight coupling between the oscillatory behavior of glycolytic metabolites (ATP, NADH) and the extent of the dipolar relaxation of intracellular water, which oscillates synchronously. The mechanism we suggest involves the active participation of a polarized intracellular water network whose degree of polarization is dynamically modulated by temporal ATP fluctuations caused by metabolism with intervention of a functional cytoskeleton, as conceived in the long overlooked association-induction hypothesis (AIH) of Gilbert Ling. Our results show that the polarized state of intracellular water can be propagated from the cytosol to regions containing membranes. Since changes in the extent of the polarization of water impinge on its chemical activity, we hypothesize that metabolism dynamically controls the local structure of cellular membranes via lyotropic effects. This hypothesis offers an alternative way to interpret membrane related phenomena (e.g., changes in local curvature pertinent to endo/exocytosis or dynamical changes in membranous organelle structure, among others) by integrating relevant but mostly overlooked physicochemical characteristics of the cellular milieu.

Linking physiological mechanisms of coherent cellular behaviour with more general physical approaches towards the coherence of life

Schrödinger pointed out that one of the most fundamental properties of life is its coherent behaviour. This property has been approached from a physiological point of view by Ling in his 'association-induction hypothesis' and extended by Pollack (gel-sol theory), by Chaplin and by Kaivarainen (detailed studies of cellular water). The question of coherence has also been attacked from general physics in three independent approaches: from non-linear thermodynamics (Fröhlich), from quantum field theory (Del Giudice and his group) and from quantum mechanics (Davydov). In this paper all these approaches are unified. The emerging picture constitutes a new paradigm of life.

Letters to the Editor

LETTERS TO THE EDITOR Dear Sir: Although most scientists, at least in pubUc and official responses, do indeed try to be fair about rival formulations, the real pragmaticjudgment is reflected in the paths of research they actually pursue [I]. On this basis Lmg and associates righdy feel ignored, for in proportion to the total workers in die field diey are probably far fewer dian were Priesdey and feUow phlogistonists at the end ofdie eighteenth century. Thus, although Ling did publish a paper in thefournal of Physiobgy in 1978 [2], the Science Citation Index through February 1982 lists only 13 citations of this paper, and of these, six have Ling as the first author and two Negendank. Of the remaining five, two citations are from journals not in our library, one favors the association-induction hypothesis [3], one presents evidence against the hypothesis [4], and die last uses die reference to support the statement that die "large gradient in potassium . . . appears to be related to die presence of oxygen-dependent energy supply" [5]. By comparison, a paper on the reaction mechanism ofdie sodium pump published that year [6] has received 68 citations in the same period. The resolution of conflicts among rival hypotheses may not be based on congruity with ultimate trutii (still waiting to be revealed) or even in terms of the weight of available evidence (often against new formulations) [7]. More important , I think, is the perceived fruitfulness ofone formulation as a better guide to further studies and understanding. Each day every scientist must decide what next to do, and an essential role of any formulation is in selecting die next experiment from die universe of possibilities. Few would doubt diat binding is involved in cation disposition, and popular formulations of cellular calcium homeostasis include bodi pumps and binding, with the latter concept supported, and the formulation in turn strengthened, by the identification of calciumbinding proteins, leading to still further studies. Where die association-induction hypodiesis currendy fails may be considered (depending on one's vision of die future) as not necessarily due to the actual absence ofthe proposed high-capacity potassium-binding sites but as the inability to guide an effective search for diem if they do exist. REFERENCES 1. Robinson, J. D. The sodium pump and its rivals: an example of conflict resolution in science. Perspect. Biol. Med. 25:486-495, 1982. Permission to reprint a letter printed in diis section may be obtained only from die audior. Perspectives in Biology andMedicine, 26, 3 · Spring 1983 | 509 2.Ling, G. N. Maintenance of low Na+/high K+ levels in resting muscle cells./. Physiol. 280:105-123, 1978. 3.Edelmann, L. Potassium binding sites in muscle. Histochemistry 67:233-242, 1980. 4.Brown, A. D., and Sturtevant,J. M. State ofwater in extremely halophilic bacteria./. Membr. Biol. 54:21-30, 1980. 5.McKinley, B. ?.; Houtchens, B. ?.; and Janata, J. Continuous monitoring of interstitial fluid potassium during hemorrhagic shock in dogs. Crit. Care Med. 9:845-851, 1981. 6.Karlish, S. j. D.; Yates, D. W.; and Glynn, I. M. Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase studied widi formycin nucleotides . Biochim. Biophys. Acta 525:252-264, 1978. 7.Feyerabend, P. Against Method. London: Verso, 1978. Joseph D. Robinson Department ofPharmacology State University of New York Syracuse, New York 13210 Dear Sir: I read widi great interest Robinson's [1] essay "The Sodium Pump and Its Rivals: An Example ofConflict Resolution in Science." Having done so, I was led to read notonly the letters by Ling and Negendank [2] and Catchpole [3] but also die original article [4] diat sparked off die exchanges. Robinson has made out a good case for comparing the conflict between the sodium pump and the association-induction hypothesis of die present century with the oxygen/ phlogiston controversy of die late eighteenth. He has argued diat die position of Ling is analogous to diat ofJoseph Priesdey who, by die turn ofdiat century, was almost alone in upholding die phlogiston theory. Ling, on die other hand, has been able to attract collaborators and is dius not alone. This, I believe, has sòme interesting implications. Ling and Negendank [2] as...

Multiple fractions of sodium exchange in human lymphocytes.

Human lymphocytes contain a large, saturable fraction of K+ that exchanges slowly with K+ in the external medium, and a small non-saturable fraction that exchanges rapidly. We determined whether or not Na+ exchanges in a similar manner with external Na+. Cells were pre-equilibrated to ensure absence of net ion movements. Efflux was studied by loading with 22Na and transferring without washing to a non-labeled medium. Influx was studied by transferring to labeled medium and separating large samples of cells at 6,000g. There are fast, intermediate, and slow fractions of Na+ exchange, with half-times of 2, 14, and 120 minutes. At normal external K+, most cells Na+ exchanges rapidly, while at lower external K+ the Na+ that replaces cell K+ exchanges slowly. Parellel sources of fast and slow fractions, such as extracellular ones and subpopulations of cells, were ruled out by simultaneous 42K and 22Na fluxes and by a quantitative analysis of the combined K+ and Na+ content and flux data over a range of external K+ and Na+ levels. Five possible models of ion fluxes occurring in series were considered. Surface matrix, surface binding sites, and cytoplasmic channels with rapid nuclear exchange were eliminated as sources of the fast fractions. Therefore, the fast fractions of K+ and Na+ must reflect the permeability of the surface membrane. This left only two possible sources of the slow fractions. One, a subcellular compartment (e.g., nucleus), was eliminated by the combined content and flux data. We conclude that the slow fractions of ion flux are rate-limited by adsorption onto and desorption from cellular macromolecules. The data support the association-induction hypothesis and are understood by reference to two fundamental concepts: that of rapid solute exclusion from cell water existing in a polarized state; and that of solute accumulation limited by adsorption onto fixed anionic sites within the cell.

Potassium binding sites in muscle: electron microscopic visualization of K, Rb, and Cs in freeze-dried preparations and autoradiography at liquid nitrogen temperature using 86Rb and 134Cs.

Normal frog sartorius muscles and muscles in which a major portion of the intracellular K+ was reversibly replaced by Rb+ or Cs+ were frozen, freeze-dried and embedded without chemical fixation or staining. Dry-cut sections of these preparations reveal striation patterns with higher contrast than those of wet-cut sections of the same preparation. The results suggest that in the living state the alkali metal ions are mainly localized in the A bands and Z lines of myofibrils. This idea is confirmed by a new autoradiographic technique by means of which the distribution of Rb+ and Cs+ in frozen-hydrated single muscle fibers has been investigated. The findings support the association-induction hypothesis according to which most cell K+ and other alkali-metal ions are not free in cell water but are adsorbed to beta- and gamma-carboxyl groups of cell proteins.

Fast and slow fractions of K+ flux in human lymphocytes

Potassium influx and efflux were studied in human peripheral blood lymphocytes equilibrated over a wide range of external K+ levels. The absence of a net ion movement throughout the flux study was established, trapped space was measured with polyethylene glycol, and cells were separated from incubation media without exposure to any washing solution. There are both rapid and slow cellular fractions of 42K influx and efflux, and half-times of exchange of around 2 minutes, and 400 minutes, respectively. The rapid component is identical in magnitude to the smaller non-saturable component of cell K+, while the slow component is identified with the larger, sigmoidal, saturable component of cell K+ that was previously shown to follow a cooperative adsorption isotherm. These results support the association-induction hypothesis, which predicts (a) a rapid fraction of K+ flux due to equilibration of ion within cell water existing in a state of polarized multilayers, and (b) a slower component of K+ flux limited by adsorption onto, or desorption from, fixed anionic sites existing throughout the cell. K+ influx, as a function of external K+, showed a triphasic relation with a peak around 1 mM K+ex, then a trough around 4mM K+ex, and then a gradual rise. This relation was readily explained, in terms of the association-induction hypothesis, by the cooperative interaction between, and ion occupancy of, fixed anionic sites that adsorb K+ or Na+.

Potassium-sodium distribution in human lymphocytes: description by the association-induction hypothesis.

Human lymphocytes were equilibrated for 48 hours over a wide range of external potassium levels, and their contents of potassium, sodium, and water determined. As external potassium rose from zero, cell potassium rose steeply in a sigmoidal fashion, reached half-saturation at 0.4 mM esternal potassium, and then saturated at 129 mmoles/kg cells. The saturable cell potassium exchanged mole-for-mole with sodium. Analysis of the saturable components by a statistical-mechanical adsorption model demonstrated a cooperative intraction between sites determining equilibrium potassium-sodium distribution. Superimposed upon the saturable fraction of cell potassium was a smaller one that was non-saturable with increasing external potassium to at least 64 mM, and that, when expressed as mmoles/liter cell water, existed in a ratio to external potassium of 0.6. The results strongly support the association-induction hypothesis, which predicts a small non-saturable component of ions determined by exclusion from oriented cell water and a cooperative interaction between sites throughout the cell that associate with potassium or sodium.

Rate of potassium-sodium exchange by human lymphocytes: prediction of the cooperative adsorption model.

The equilibrium parameters of potassium-sodium distribution in human lymphocytes, determined experimentally in the preceding study (Negendank and Shaller, '79), were incorporated into a stochastic treatment of the cooperative adsorption model in order to predict the kinetics of "active" potassium-sodium exchange. The rate of uptake of potassium, in potassium-depleted, sodium-loaded cells, is complex and deviates markedly from simple exponential functions. The sigmoid form of the exchange data closely followed the predicted curve. This result enhances one's confidence in the usefulness and applicability of the cooperative adsorption model, and adds further support to the association-induction hypothesis as a coherent theory of cell physiology.

Maintenance of low sodium and high potassium levels in resting muscle cells.

1. Previous work has shown that a frog sartorius muscle consists of parallel cells running all the way from one end of the muscle to the other and that amputation of one end of the muscle is not followed by regeneration of a new cell membrane. If now only the cut end of the amputated muscle is exposed to a Ringer solution in which the solutes 42K and 22Na act as radioactive labels and the rest of the cell is suspended in air, we have what is described as an effectively membraneless open-ended cell or EMOC preparation. In this case the only remaining anatomically intact plasma membrane and pumps are made nonfunctional by the removal of 'sources' for inward pumps and 'sinks' for outward pumps. 2. The healthy region of a frog sartorius muscle EMOC preparation continues to accumulate labelled K+ to a level higher than that in the Ringer solution and to exclude labelled Na+ to a level below that in the Ringer solution, much as a normal uncut muscle does in its normal environment. The differences were reduced by inclusion of ouabain in the medium. 3. The diffusion coefficient of Na+ in the normal muscle cytoplasm at 25 degrees C was measured using two methods. The average diffusion coefficient measured was 2.07 X 10(-6) cm2/sec, roughly 1/6 that of the diffusion coefficient of Na+ in a 0.1 N-NaCl solution. 4. The data obtained are discussed in terms of the association-induction hypothesis. In this theory asymmetrical solute distribution, basically an expression of a non-energy consuming metastable equilibrium state, is the result of specific combinations of two opposing mechanisms: adsorption which raises the level of the intracellular solute; and exclusion from cell water which tends to lower it.

238 - Two opposing theories of the cellular electrical potential: a quarter of a century of experimental testing

The fundamental differences between the Hodgkin-Katz ionic theory of cellular electric potential and the surface adsorption theory of cellular electric potential (an integral part of the association-induction hypothesis) were summarized and compared with results of experiments designed to test these theories that had accumulated over a period of more than a quarter of a century.

Potassium Accumulation Frog Muscle: The Association-Induction Hypothesis Versus the Membrane Theory

Potassium Accumulation Frog Muscle: The Association-Induction Hypothesis Versus the Membrane Theory

Response : Potassium Accumulation Frog Muscle: The Association-Induction Hypothesis Versus the Membrane Theory

<i>Response</i> : Potassium Accumulation Frog Muscle: The Association-Induction Hypothesis Versus the Membrane Theory

The physical state of water and ions in living cells and a new theory of the energization of biological work performance by ATP.

In this article, the key concepts of the association-induction hypothesis and their experimental verifications were reviewed: According to this hypothesis, the bulk of cell water exists in the state of multilayers polarized and oriented by the backbones of certain proteins existing in an extended state. The major intracellular cation, K+, is also adsorbed but singly and on different protein sites (i.e.,β &γ-carboxyl groups). The living protoplasm of protein, ion and water represents a three-dimensional cooperative assembly under the control of certain cardinal adsorbents, of which ATP is a prime example. Association of ATP with the cardinal site of a key protein may produce a cooperative change in the electron distribution of the protein with consequent change of the state of ion and water adsorption. Such an alteration in the physical state of water leads to a change in the solubility of various solutes in this water as well as permeability of various solutes (e.g., Na) through this water. In contrast to the conventional concept, the role of ATP is not to provide a package of energy held in a special chemical bond. Rather, as a cardinal adsorbent, ATP acts by means of its specific electronic interaction with the protein molecules, maintaining the protein—ion—water system at a higher energy state. Dephosphorylation of ATP leads to a cooperative shift of the electron state of the whole assembly to a lower energy state. Restoration to the original resting state follows the resynthesis of ATP (where energy is injected into the system) and its readsorption on the cardinal site.