Life at the Cell and Below-Cell Level. The Hidden History of a Fundamental Revolution in Biology
"Dr. Ling is one of the most inventive biochemist I have ever met."
This section is not a standard summary, reiterating what has been said earlier. It does that. But in addition, it also does something not possible until the whole story has been told—overviews. Then there are also bits and pieces of information, which are presented here for the first time, because their significance becomes evident only after the overviews have been drawn.
16.1 Early history
Chapter 1 begins with the discovery of the living cell. The inauguration of cell physiology soon followed. However, life in a broader context had captivated the minds of ancient philosophers long before the discovery of the living cell.
Thales (ca. 640-548 B.C.), reputedly the first natural philosopher of the West, chose water as the first principle of the cosmos.309 p 22; 526 Hippo of Samos (450 B.C.) perceived life as water.309 p 19 Heraclitus (500 B.C.) and Democritus (420 B.C.) considered the human body to be made up of not just water but earth and fire as well.309 p 34 p 56
Both Paracelsus (1493-1541) and Francis Bacon (1561-1626) saw life as fire. Paracelsus further alleged that something in the air is essential for both ordinary fire and Flama vitalis or living fire. In 1774, Joseph Priestley (1733-1804) captured that "something in the air" but did not know it at first. Instead, he thought it was a purer form of air, or dephlogisticated air.529 p 144 After a vigorous debate, Priestley gracefully and enthusiastically accepted the idea of his arch opponent, Antoine Lavoisier (1743-1794)527; 346
According to Lavoisier, what Paracelsus called living fire is respiration.527 Like ordinary fire, respiration entails not a loss of phlogiston from the combustible material—an idea which Georg Stahl (1659-1734) championed352 p 122 and was widely adopted at the time—but a chemical combination of the combustible material with what Priestley had discovered and, to which Lavoisier gave a different name, oxygen. Both ordinary fire and living fire consume oxygen and produce carbon dioxide.
Louis Pasteur (1822-1895) showed that life can also go on without oxygen-containing air.528 In an air-free or anaerobic environment, fermentation of plant cells produces alcohol, but glycolysis of animal tissues produces lactic acid.128 Extensive investigations by many talented biochemists paved the way for Lohmann's discovery of ATP in 1929272—as the end product of all energy metabolism, aerobic as well as anaerobic.
In 1941 Lipmann introduced his highly popular, though ill-fated, high-energy phosphate bond hypothesis.132 A mere 15 years later, Podolsky and Morales with the aid of more sophisticated methods of measurements than ones used earlier, found no extra energy stored in the phosphate bonds of ATP.133
The high-energy phosphate bond concept is thus the last of a succession of failed attempts to explain the energization of living activities. Engelmann's heat-engine theory for muscle contraction347 was disproved by Fick on thermodynamic grounds.383; 530 p 144 A.V. Hill's lactic acid theory for muscle contraction357 did not survive Lundsgaard's demonstration that iodoacetate-poisoned muscle can contract normally without producing lactic acid.372; 530 p 145 The disproof of the high-energy-phosphate bond concept left life sciences clueless on the pivotal issue of energization of physiological activities—altogether or only for a few years, depending on the inquirer's awareness of the AI Hypothesis.
Starting out as a part of Ling's fixed charge hypothesis (LFCH), the AI Hypothesis has offered a new theory of how ATP may energize life activities without violating the Law of Conservation of Energy—a law first discovered by physiologist-physicist Hermann von Helmholtz when he was barely 26 years old.352 p 213
But beyond the ideas of life as water and as fire, there were still other philosophical thoughts on life from ancient Greeks. Both Anaximenes (ca. 550 B.C.) and Aristotle (384-322 B.C.), for example, equated life with organization or form. Lamarck's Etatde chose mentioned in [14.2(1)] may be regarded as a sequel. So is Lepeschkin's live state.
However, motion, the most conspicuous expression of life, was not recognized as characterizing life at first. According to historian Thomas Hall, this neglect arose from the confusion from attributing all motions to life (psyche).309 p 267 Not until the 17th century did Descartes (1596-1650) dismiss psyche as the cause of animal and plant movements and begin the search for their true causes.309 p 268 In one direction this search merged with the trail begun with life as fire just discussed. In another direction, Dutrochet (1776-1847)—possibly the first recorded experimental cell physiologist in history—equated life with movement.
16.2 The membrane (pump) theory
The low resolving power of early microscopes might have misled Theodor Schwann, the accepted founder of the Cell Theory, into the belief that cells are typically membrane-enclosed bodies of dilute solutions. Schwann's error was quickly corrected from the microanatomists's perspective. Less fortunate were the cell physiologists, who were misled into the same erroneous generalization. But this mistake has stayed on.
The membrane theory, often attributed to Wilhelm Pfeffer, was the first general theory of cell physiology. (For doubts about the validity of this assignment, see Chapter 3). On the assumptions—as Schwann had assumed before—that all living cells are membrane-enclosed dilute solutions and that the enclosing cell membrane is permeable to water but permanently impermeable to substances like sucrose and NaCl, proponents of this theory could explain, or so it seemed at the time, four classic cell physiological manifestations of the resting living cell: (i) selective permeability, (ii) selective solute accumulation and exclusion, (iii) cell volume control and (iv) electric potentials.
The membrane theory's apparent strength in simplicity is also its fatal weakness. When Nasonov, Aizenberg and Kamnev demonstrated that the cell membrane is in fact permeable to sucrose and galactose, the paradigm of cells as membrane-enclosed dilute solution faced grave difficulties.
In the crisis thus created, the sodium pump was installed. However, one (sodium) pump is not enough. To keep the cell afloat, an ever-lengthening list of pumps has been introduced. Yet the sodium pump at the cell membrane alone would require at least 15 to 30 times the total energy available to the cell—under rigorously controlled conditions and with the assumption that the cell needs no energy at all beyond pumping sodium—the membrane theory was in serious trouble again, this time with no easily-discernable escape route in sight.
The energy shortage, however, is far from being the only unfavorable evidence against the membrane-pump-theory. It may be the most persuasive one, because the issue is simple and the inconsistency demonstrated between theory and facts staggering. However, a part of the original membrane theory has survived, namely, the existence of some sort of a covering protoplasmic skin or cell membrane, as Pfeffer advocated. But even this surviving part requires drastic modifications from the one widely accepted. Nor is the new cell membrane semipermeable (according to the original van't Hoff definition) and the seat of osmotic activities. A summary of these modifications required will be presented in [16.6(3.2)] to come.
16.3 Early protoplasm-oriented cell physiologists and their contributions
At the turn of the 20th century, a number of adventuresome investigators came forth with alternative concepts on the living cell. Fischer, Moore, Roaf, Lepeschkin, Nasonov, Ernst all the way down to Troshin agreed on one point. What the advocates of the membrane (pump) theory had considered as reflecting the attributes of the cell membrane originates (often) from the bulk-phase protoplasm.
More specifically, the efforts of these early protoplasm-oriented cell physiologists were focussed on two of the four subjects of cell physiology mentioned above: solute distribution and volume control. Left largely un-tackled by these investigators are cell permeability and electric potentials. Nasonov went so far as to deny the existence of a cell membrane (hence selective membrane permeability) or a resting potential 86 p 164, 178 (Chapter 8).
These early protoplasm-oriented cell physiologists often chose gelatin as an inanimate model for protoplasm. They believed that protoplasm and gelatin are both colloids. Lepeschkin and Troshin argued that living cells represent the special form of colloids known as coacervates. Overton presented evidence that at least a part of cell water exists as imbibition water ("Quellungswasser").
That adsorption could provide a mechanism for the accumulation in cells of solutes like K+ at a level higher than in the surrounding medium was first suggested in 1908 independently by Benjamin Moore & Herbert Roaf together77 and by Martin Fischer alone.78 In addition, Fischer invoked the "partition law" to account for solutes found at concentration lower than that found in the external medium. This was an important new insight in cell physiology at that time; but Fischer did not exploit it beyond the short comment he made in a lengthy document on edema.78 Later, Troshin welded these twin concepts—adsorption and partial exclusion—into the two-term Troshin equation (Equation Al in Appendix 1). And he demonstrated that the distribution of various nonelectrolytes in a complex coacervate (of gelatin and gum arable) and in living cells obeys this equation.
The protoplasm-oriented cell physiologists suffered a severe setback from Hill's reputedly conclusive evidence—bolstered by the later demonstration of free K+ by Hodgkin and Keynes—that cell water is normal and cell K+ is free. The inability of early colloid chemists to refute Hill's claim, to provide a more cogent definition of colloids (and coacervates) beyond molecular size, and to explain the underlying difference between gelatin and other proteins, might have also weakened the cause for the colloid-oriented cell physiologists in the eyes of some fellow-scientists at least—as seen in the following verbatim quotation:
"Aside from technical difficulties, protein chemistry has also suffered from the abortive effects of an ill-founded idea, which could be called the colloid concept (ital. original)."538 p 4
16.4 Ling's fixed charge hypothesis (LFCH)
Before its collapse, the membrane theory was bolstered by the eminent theories of dilute solutions including those of Arrhenius, Debye, Hückel, van't Hoff and others. In contrast, the early protoplasm-oriented cell physiologists had no intellectual pillars like these on which to lean. Both Carl Ludwig and Martin Fischer openly lamented the poverty of relevant physico-chemical knowledge. However, by the time I began my career as a cell physiologist, things had improved considerably. Statistical mechanics, protein chemistry, colloid chemistry, polymer chemistry had either already entered a stage of maturity or were in the process of doing so. It was these new advances in knowledge, which made possible what had been denied my forbears—the option of dreaming up possible physico-chemical mechanisms for cell physiology, and eventually of putting forth a general physicochemical theory of life itself, the association-induction hypothesis.
In addition, useful experimental techniques also came into existence at just the right time. Examples are the radioactive tracer technology and the Gerard-Graham-Ling capillary microelectrode. Thus, the disproof of the sodium pump hypothesis on the basis of energy considerations (Chapter 12) relied on the employment of both of these tools.
After the introduction of LFCH (Ling's fixed charge hypothesis) in 1952, it took me ten more years to develop and publish the association-induction (AI) hypothesis. For the convenience of summarizing, I sort the theory into two parts: association and induction. I shall begin with association.
The most abundant components of the living cell in terms of mass are water first and proteins next. The most abundant components in terms of number are water first and K+ second. Association of K+ with proteins is the central theme of LFCH presented in Chapter 10; association of water with proteins is that of the PM theory presented in Chapter 11.
The first theoretical question the LFCH raised and attempted to answer concerns the act of association itself. Why should K+, for example, engage in (close-contact) association with fixed anionic groups, when we know that at the same ionic strength K+ does not associate with similar anionic groups in a free aqueous solution? To the best of my knowledge, the LFCH has published (possible) answers to this question for the first time in history.
Two mechanisms have been offered for the enhanced close-contact association with site fixation, respectively electrostatic and kinetic, (i) Overlapping electric field reduces the entropy of dissociation and increases association, (ii) Fixation of the adsorption site nullifies half or more of the potentially-effective bombardments by solvent molecules, which would have torn apart an associated pair of free ions or non-ionic particles in a solution. The electrostatic mechanism enhances the association of only particles bearing opposite electric charges. The kinetic mechanism suffers no such restriction; it applies to all associated molecules, bearing net electric charges or not. By the time the LFCH was published, Kern's striking experimental demonstration of the enhanced counterion association with charge fixation had been around for four years.469
The LFCH also offered: (1) another historical first in the form of a quantitative mechanism for the selective adsorption (hence accumulation in cells) of K+ over Na+ (2) the salt-linkage hypothesis to account for prior repeated failures to demonstrate selective K+ adsorption on isolated proteins and (3) an answer to the question why K+ is lost on cell death—built on the still-to-be-fully-evolved theory of ATP function as the principal cardinal adsorbent.
The essence of the LFCH was presented within an hour's time at a Symposium held in Baltimore in 1952. It has taken me and others more than 40 years to complete its experimental confirmations to be summarized next.
With the then-newly-introduced EMOC setup,
I demonstrated that the seat of selective K+ accumulation (and Na+
exclusion) in frog muscle cells is the bulk-phase cytoplasm rather than the
cell membrane (and postulated pumps located in the cell membrane). The adsorbed
state of cell K+ as well as the nature and locations of the adsorption sites
have been established by six sets of mutually supportive evidence: (i) the low intracellular electrical conductance, (ii) the
strongly reduced mobility of cell K+ (iii) the altered X-ray
absorption fine edge structure of cell K+, (iv) the widely different
activity coefficients of K+ measured in different cell types with
an intracellular K+-specific microelectrode, (v) the obedience of
intracellular K+ to the prediction of a Langmuir
isotherm, demonstrating close-contact, one-on-one adsorption, (vi) the
identification of (most of) the K+-adsorbing sites as β- and
γ-carboxyl groups carried respectively on aspartic and glutamic
residues on myosin in frog muscle cells.
16.5 The polarized multilayer (PM) theory of cell water
In the PM theory, virtually all cell water assumes a dynamic structure different from that in normal liquid water. This departure originates from interaction of cell water with a matrix of fully-extended protein chains. The carbonyl (CO) and imino (NH) groups of these fully-extended protein(s) offer properly-spaced, alternatingly negatively-charged N sites and positively-charged P sites. Together, these N and P sites adsorb a layer of (oppositely-oriented) water molecules or dipoles. The water molecules thus oriented and polarized, in turn, polarize and orient a second layer of water molecules and this continues until all the cell water is oriented and polarized.
A prediction of the PM theory is that proteins, which for structural reasons (e.g., gelatin) or in response to certain denaturants (e.g., urea, guanidine HC1, NaOH), exist at least in part in a fully-extended conformation, behave like the postulated fully-extended protein in living cells. In contrast, proteins in what is conventionally called native conformation,107 p 37, n5 will not behave this way, or do so weakly. This theoretical perception led to the classification and deployment of two kinds of models. Extrovert models like gelatin imitate the (postulated) fully-extended proteins in living cells; introvert models, including most of the so-called native proteins, do not or do so weakly.
Another prediction of the PM theory is that water in living cells and in the presence of extrovert models suffers motional restrictions. (In contrast, the presence of introvert models has less or no impact.) This prediction has been put to world-wide testing. Thus (i) NMR studies of the correlation times of water protons; (ii) ultra-high frequency dielectric studies of (Debye) reorientation time of (whole) water molecules and (iii) quasi-elastic neutron scattering studies of the rotational diffusion coefficient of water molecules unanimously confirmed the predictions of greater motional restriction of water molecules (and their protons) in both living cells and in aqueous solutions of extrovert models than in plain liquid water.
In addition, more detailed confirmation of the predicted similar behaviors of living cells and extrovert models and dissimilar behaviors of introvert models extend to a host of other attributes of cell water as well. They include osmotic activity, freezing point depression, vapor sorption at near saturation vapor pressure but above all, (partial) solute exclusion.
Based on a quantitative theory of solute distribution introduced in 1993, I demonstrated how a small difference of only 126 cal/mole—over and above the enormous normal water-to-water interaction energy (heat of vaporization) of 9.7171 kcal/mole—is able to explain quantitatively the phenomena of sucrose-, and Na+-exclusion from living cells, which have interested cell physiologists from the very beginning.
The PM theory of solute distribution, and in particular, the size-rule, also explain why and how the historic mistake was committed in regarding the equal distribution of urea and ethylene glycol as definitive proof that cell water and K+ are both free in living cells.
In harmony with the concept that what the bulk-phase protoplasm is and does, protoplasm elsewhere in the cell can also be and do, the PM theory also offers a new theory of the physico-chemical makeup of the cell membrane (a name accepted not without reservation because this structure may not have a sharp delineating boundary on its inner side as in all true membranes). In the new model, the continuous phase of the cell membrane—like the continuous phase of the bulk-phase cytoplasm—is not lipids or phospholipids but multilayers of polarized and oriented water. This new model of the cell membrane, in agreement with facts, is virtually permeable to everything but with vastly different rates of permeation. Nor does this membrane have much to do with the osmotic activities of the cell, which are primarily expressions of the bulk-phase protoplasm (as witnessed by the normal osmotic behaviors of cells without an intact membrane.)
A series of mutually-supportive findings confirms this theory of (polarized-oriented) water membrane. They include the demonstration that the diffusion of water in frog ovarian egg and giant barnacle muscle fiber is bulk-phase limited. That is, the rate of diffusion of water through the cell membrane is the same as that through the bulk-phase cytoplasm, both orders of magnitude faster than water diffusion through a phospholipid bi-layer. Valinomycin (10-7 M), which increases 1000-fold the K+ permeability of bilayers of (authentic) phospholipids bilayers, has no detectable influence on the K+ permeability of frog voluntary muscle, frog ovarian egg, giant squid axon, human lymphocytes and mouse-liver mitochondrial inner membrane.
Finally, the permeation of water and six non-electrolytes through a water-loaded cellulose-acetate membrane quantitatively matches the permeation rates through an (inverted) living frog skin of the same substances at the same temperatures (0°, 4° or 25°C). The cellulose acetate membrane, which, like the frog skin, is virtually impermeable to sucrose, has (polarized and oriented) water-filled pores with average diameter five times that of the (virtually impermeant) sucrose molecule. This disproportion in size has once again eliminated a sieve mechanism for sucrose's near-impermeability but agrees with the polarized-water model following the size-rule.
Before closing this summary of the associative aspect of the AI Hypothesis on cell water, I shall fill in on some additional gains provided by the PM theory.
First, the PM theory has at long last offered the first (possible) explanation of why gelatin is different from most native proteins (by being, for structural reasons, permanently in the fully-extended conformation), and of what a colloid is (macromolecules or big aggregates of smaller entities polarizing water or other polar solvents in multilayers) or a coacervate (i.e., the stand-alone cooperative phase containing parallel-oriented colloids).
Second, based on results of early NMR investigations testing the PM theory, Raymond Damadian invented what was to be known as magnetic resonance imaging or MRI.
To be continued