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." |
Chapter 16. Summary Plus (p. 233-271) |
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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 |
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