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 10. Ling's Fixed Charge Hypothesis (LFCH) (p. 47-73) |
|
As mentioned earlier, I began my training
as a cell physiologist under Professor Ralph W. Gerard in the world-famous
Department of Physiology at the University of Chicago. Like virtually all my
peers, I was totally immersed in the belief that the membrane theory was the
one and only guiding light. Naturally my first full-length publications
(coauthored respectively with Prof. Gerard and W. Woodbury) are on the subject
of "membrane potential,"95 a name and concept taken straight
from the membrane theory. Following a presentation of the "Sodium Pump
Hypothesis" I gave at a departmental seminar49 p 124—based
solely on information gathered at the library—I carried out some simple
experiments of my own. My goal was to find out if the combined action of
metabolic poisons and low temperature (0°C) would promptly lower the
concentration of K+ in frog muscles as the hypothesis would have
predicted. The result was unforeseen, but also exciting. No change at all in
the K+ concentration at the
end of a five-hour-long experiment (see Table 8.4 and pp. 176-177 in Appendix 1
of Reference 49 for results of later more extensive confirmatory work). As I
became more and more drawn into the new direction of research thus started, my
doubts about the sodium pump hypothesis grew. In the years following, I spent much time trying to
dream up an alternative energy-saving mechanism to replace the hypothetical
sodium pump. It was tough going.
Years went by and I was getting absolutely nowhere. Then all of a sudden a
possible solution dawned on me while I was browsing and mulling in the
basement of the Welch Library of the Johns Hopkins Medical School in Baltimore.
This new seminal idea, the centerpiece
of what was later called Ling's Fixed Charge Hypothesis (LFCH),96
was also the first step toward the construction of the unifying theory of cell
physiology, called the association-induction hypothesis (Al Hypothesis;
AIH).98 The essence of this hypothesis will be presented here and in Chapters 11, 14 and 15. The idea that intracellular K+ could be selectively adsorbed
over Na+ was, to the best of my knowledge, first suggested by
Herbert Roafand Benjamin Moore in 1908. However, as pointed out once already
(and until the LFCH appeared), neither they nor anyone else had offered a
quantitative molecular mechanism for this striking discrimination between a
pair of ions so closely similar. Nor had anyone else suggested an explanation
why proteins—the most likely candidates inside the cells to offer the sites for
the adsorption of K+—can do the job in the living cell, but repeated
attempts in the past to reproduce the phenomenon in vitro had all
failed.99; 41 p 120 Nor had a molecular explanation been
offered why this ability of selective adsorption (if correct) is promptly lost
upon cell death. Ling's Fixed Charge Hypothesis represents the outcome of my
first attempt to answer these questions. 10.1 A theory of selective accumulation of K+ over Na+ Three new theoretical concepts were introduced in the
construction of a coherent theory of selective adsorption of K+ over Na+ and the
closely related phenomena just mentioned—a theory to be referred to later as
Ling's Fixed Charge Hypothesis (LFCH). However, only one of the theoretical
concepts described under (3) below represents what I mentioned above as the seminal
idea. (1)
Enhancement of counter-ion (or neutral molecules) association with site
fixation This is a major new concept introduced in 1952—not in the recognition of the existence of fixed charges, which had occurred long before100—but in the theory of full associations of these fixed charges with free counterions like K+ and Na+. The incorporation of this basic concept distinguishes LFCH (and the association-induction hypothesis) from other "fixed charge hypotheses" of the past and present.101 Indeed, without full counter-ion association, selective adsorption of K+ over Na+ by the mechanism proposed below under (3) or by others suggested elsewhere later [14.1]—would have been impossible. In the wake of the great theories of ionic
dissociation of Arrhenius102 and of Debye and Hückel,103
it has been widely believed that monovalent ions of one electric charge in a
dilute aqueous solution are fully dissociated from monovalent ions bearing the
opposite charge—regardless if one species is fixed in space. (For
illustrations of this deep-seated belief as late as 1961, see Figure 1 in
Reference 104, Figure 5 in Reference 100; Figure 1 in Reference 105.) In
harmony with this view of full ionic dissociation, the influential
protein-chemist, K. U. Linderstrøm-Lang of the Carlsberg Laboratory in
Stockholm described a protein molecule as an ellipsoid with electric charge
uniformly smeared over its surface. Counterions in number matching the excess
charges of the opposite polarity hover over the protein as a diffuse ion cloud.467
In his opinion, direct contact between proteins and counterions does not exist.
My view to the contrary, to be reviewed next, was definitely running against
the tide. For the enhancement of counterion association in
consequence of the spatial fixation of one species of ion,96 p 769
one of the two causes I gave in 1952 is the overlapping of the attractive
electric field of neighboring fixed charges—this field-overlapping is the
microscopic foundation of what is known as the Law of Macroscopic
Neutrality.97 pp 330-331 The result of this field-overlapping is
not merely an increase of the adsorption energy of the ion [14.2(1)],
enhancing its association with the fixed ion, but also the confinement of a
dissociated counterion to within a much smaller space surrounding the fixed
ions.96 p 769 This confinement reduces the entropy of
dissociation of the counterion and strengthens its association with the
fixed ion also. The second cause I gave in 1952 is kinetic in origin
(and nothing much more beyond that). Indeed, its specific detailed
mechanisms are given for the first time here and now: While the chance of
an anion and cation meeting and pairing may be assumed to be about the same
whether or not one species is fixed in space, the number of effective
collisions received from surrounding water molecules, which would tear apart
the associated pair, is at least halved if one species is fixed and thus
unmoved by the collision. The result is also an enhancement of association.
Note that the first (field-overlapping) cause is restricted to particles
carrying net electric charges (i.e., ions), the second (kinetic) cause does not
suffer such a restriction and may therefore underlie all localized adsorption
including the adsorption of ions and water. The theory of enhanced counterion (and neutral
adsorbent) association has been reiterated and discussed in my publications in
years following.98 pp 17-28; 106 pp 152-155; 107 pp 39-41 importance
in compelling close-contact association in living phenomena is put
center-stage by the title of the general theory, association-induction hypothesis. Experimental
confirmations on inanimate models of the predictions of this theory as applied
to ions469; 98 pp 17-22; 107 p 40 and to water470 had
been already in the literature—before the (only) theory for its explanation
known to me (i.e., LFCH just reviewed) was published. One of these experimental
studies will be summarized here. Kern showed that the activity coefficient of Na+
in 0.0125 to 0.2 M Na salts of isobutyric acid (СН3СНСООНСН3) ranged from 0.90 to 1.00, indicating that 90% or
more of the Na+ is free. When these isobutyric-acid
monomers are joined end-to-end into the linear polymer, polyacrylic acid (-СН2СНСООНСН2-)n,
and the carboxylic groups thus (not completely but substantially) fixed in space, the activity
coefficient of Na+ at the same concentration range, fell to 0.168 to
0.315, indicating that from 68% to 83% of the Na+ is now associated
with the carboxyl groups.469 (2)
The salt-linkage hypothesis and a critical role for ATP According to the LFCH, the negatively-charged β-
and γ-carboxyl groups of isolated native proteins are largely engaged in salt-linkages
with fixed cations (e.g., positively-charged ε-amino groups and guanidyl
groups belonging respectively to lysine and arginine residues of intracellular
proteins)108 and thus not free to adsorb cations like K+.
In the LFCH, these β- and γ-carboxyl groups can be made available for
adsorbing K+
ions by adenosinetriphosphate or ATP,
when ATP occupies key controlling cardinal
sites [14.3(3)]. In this early model, ATP serves its role by the same
mechanism proposed earlier by Riseman and Kirkwood for keeping the contractile
proteins like myosin from collapsing upon themselves and shortening, i.e.,
long-range electrostatic repulsion (transmitted through space).109
Later, in the association-induction hypothesis, I introduced a new (though
related) mechanism (see [14.3] below). ATP is, of course, the end product of energy
metabolism. Its critical role in maintaining selective K+ adsorption
explains the loss of this ion upon cell death, when metabolism ceases and ATP
regeneration comes to an end. 3)
The 1952 electrostatic model for the selective accumulation of K+
over Na+ in living cells Taking into account dielectric
saturation110 (rapidly declining dielectric constant as one
approaches an electrically charged site or ion, as illustrated in the inset of
Figure 6) in the electrostatic interaction between a fixed anion and a free monovalent
counter-cation, one can calculate the statistical probability of finding the
counter-cation at different distances from the center of the negatively charged
oxygen atom (of a β- and γ-carboxyl group) (Curve 2 in Figure 6).
Figure 6. A theoretical model for the selective adsorption of K+
over Na+ on a fixed oxyacid site presented in 1952 as part of Ling's
Fixed Charge Hypothesis (LFCH) Computation takes into account the decrease of
the dielectric constant of water (referred to in the Inset as "radial
differential dielectric coefficient") when approaching an ion as
illustrated in Inset. Theoretical curve (2) shows the probability of finding a
monovalent cation (e.g., K+, Na+) associated with the
fixed oxyacid anion—partially represented at extreme left of bottom section of
the figure—at distance (away from the center of the oxygen atom of the oxyacid
group) indicated on the abscissa in Angstrom units. Note that only the hydrated K+
with its smaller radius shown in the bottom figure can enter the "shell of
high probability of association" around the oxygen atom of the
negatively-charged oxyacid group and becomes selectively adsorbed over the
larger hydrated Na+ (the center of which stays largely out of the
shell of high probability) also shown in the bottom part of the figure. (Ling,96 reproduced from Phosphorus Metabolism by permission of The Johns Hopkins University
Press)
The bottom part of Figure 6 also shows the diameters
of the smaller hydrated K+
and the larger hydrated Na+ ion.98 p 548;
111 Note that only the center of the smaller hydrated
K+ can enter the
"shell of high probability of association" around the fixed anion and
become selectively adsorbed, but the hydrated Na+ is too large to do
the same and is thus largely left out. What is illustrated in Figure 6 is the first-of-its-kind quantitative molecular mechanism for the selective adsorption of K+ over Na+. Anticipating new developments to be described below in [14.1], I recast this mechanism in an adulterated but easier-to-parley lingo: Due to the different distances separating (the center of) the fixed anion and (the center of) the smaller hydrated K+, the electrostatic field strength experienced by a K+ is stronger than that by the larger hydrated Na+ The resulting preferential adsorption of K+ over Na+ on the β- and γ-carboxyl groups provides an energy-conserving, quantitative molecular mechanism for the selective accumulation of K+ over Na+ in living cells. (For a more detailed presentation of this theoretical model, see pp. 54-57 in Reference 98.) In summary, proteins like myosin in muscle cells carry many β- and γ-carboxyl groups. Adsorption of ATP on the controlling cardinal site(s) {[14.3(3)]} on myosin enables these β- and γ-carboxyl groups to associate with (or adsorb) either K+ or Na+ Since the hydrated K+ is smaller than hydrated Na+, it is energetically more favorable for K+ to be adsorbed. Accordingly, K+ is selectively accumulated in the cell over Na+. That said, I must add that this was how the
LFCH (and the association-induction hypothesis) began in 1952. The proposed
mechanisms are as valid today as then, as will be made clear in the next
Section. To be continued |
|
|
Разделы книги
Contents (PDF
218 Kb) |
|
|
На страницу
книги "Life at the Cell and Below-Cell Level..." |
|
