Lp6b Non-existent "crucial experiment" and
other fiascoes to resurrect the sodium pump

In 1962 I published in detail the experimental evidence that the sodium pump hypothesis is
untenable because under carefully controlled condition, the minimum energy need for
pumping sodium ion from frog muscle cells is from 15 to 30 times that of the maximum
energy available. The evidence and conclusion deduced had remained unchallenged until a
reporter from the Science magazine, Dr. Gina Kolata published in 1976 a review entitled
"Water Structure and Ion Binding: A Role in Cell Physiology?" (Sience, vol. 192, p.1220,
1976). In this article she claimed that two scientists, A and B had produced "crucial
experiments and calculations ....that provide strong evidence for the existence of pumps".

This public report was surprising to me on two accounts.

1. Two surprises, discovered twenty years later
Firstly, I and other scientists, including Dr. Raymond Damadian, Dr. Freeman Cope, Dr.
Carlton Hazlewood, had each individually received and responded to Kolata's article in the
Letters to the Editor Column of a later issue of Science (193: 528, 1976). Yet neither my
response, nor my recollection of the content of Kolata's article indicate that she had in her
article a section mentioned the so-called "crucial experiments and calculations". The
question arose: Did Kolata send us an earlier version of the article (and we responded to it)
but put in print later a different version containing this "crucial experiment and
calculations"(and other related and unrelated statements)?
I raised this issue in my letter to Dr. Kolata dated June 21, 1996. She answered my letter on
another issue (see below) but not on this point specifically. On November 5, 1996 I wrote
her another letter addressing this issue once more and exclusively. Again I got no response.
On February 5, I repeated my request once more, asking for a yes-or-no answer, this time in
a letter with a return slip (which was duly acknowledged and returned to me ). But still no
answer.Whatever the reason for her unwillingness to respond to my simple question, her
statement in her article could very well have produced---unintentionally, I am sure--- the
spurious impression that I was not able to rebut the "crucial experiments and calculations"--
-utterly contrary to the truth (see below).
Feeling at the time that I had already answered all the relevant questions she raised in the
manuscript she made available to us, I went on to other work and was not to learn about
this "crucial experiment and calculations" claim until twenty years later--- when a friend, Dr.
Gerald H. Pollack brought it to my attention. Since Drs. B and A were once my graduate
students, the fact that I did not respond promptly to these alleged "crucial experiments and
calculations" might have been misconstrued as evidence that the en masse departure of my
former graduate students from my laboratory (see home-page under "Absolute Power
Corrupts Absolutely) was for purely scientific reasons----a misconception that could be made
into a powerful weapon in the hands of those who would prefer that my NIH-supported
work be stopped, as it was (see home-page).
Surprise number two from Kolata's article was B and A's alleged "crucial experiments and
calculations "--- which had, in Gina Kolata's opinion, reversed my earlier refutation of the
sodium pump theory. What are these experiments and calculations?
Neither B nor A had conducted any experimental studies on the energy need of the sodium
pump while they were working in my laboratory. Could they have gone into this new field
after they left my laboratory? To find an answer, I went to the Citation Index and did a
thorough search for all B's and A's scientific publications. I could not find a trace of what
could be conceivably regarded as the source of Kolata's "crucial experiments and
calculations. I then wrote to all three: Kolata and my two former students.
B never answered my letter or another follow-up letter I sent later. Kolata did answer and
here is what she wrote me in her letter dated July 16, 1996:
"I am sorry to say that I can no longer remember where I got that information about Dr. B
and Dr. A's (real names here and below withheld by G.L.) conclusions. I'm especially
chagrined because Dr. A and Dr. B never published their results and Dr. A says I never even
interviewed them. In retrospect, that seems hard to believe, but I was very young then and
maybe I really was so inexperienced that I included that statement about their work without
calling either Dr. A or Dr. B ..."

This notwithstanding, what she wrote as Dr. A and Dr. B's "crucial experiments and
calculations" could be seen as the center piece of her whole article. Dr. A's response in his
letter to me dated June 28, 1996, corroborated the fact that Kolata did not interview Dr. A
or B. However, Dr. A too could not provide me with a copy of what might have been the
source of Kolata's "crucial experiments and calculations ". This is the main portion of what
Dr. A wrote:

"When I was a postdoc at Cornell and Dr. B was one at Yale, the two of us, still fresh from
the tumultuous experiences in your lab, wrote a manuscript on analyzing the Na+ efflux
data in muscle--a sort of literature review--taking into account compartmentation effects. It
was an attempt to show, among other things, that your apparently fast efflux data could be
reconciled with a multicompartment membrane theory. (Remember, though, that I also
considered the rates measured in your zero-degree experiments too high, as I discussed at
length in my thesis.) Dr. B and I had fun writing the paper, and we sent it to J. Membrane
Biology, I believe, where it was immediately rejected...We didn't try to publish it after this
rejection. So I suppose that's what Gina Kolata was talking about there. But the big puzzle is:
how did she know about it? I don't think that we sent the manuscript to Science, and I'm
certain that we never published it. But we may have circulated it around to friends, etc. So
maybe she heard about it in the grapevine. That's just conjecture, but it seems to be a
plausible scenario. As for citing it, that's impossible: never having passed through the fire of
peer-review, it doesn't exist, and so it isn't part of the literature--nothing for you to argue
with. I don't have a copy of the paper, having thrown out the manuscript as useless junk
over a decade ago..."

So the much touted "crucial experiments" were never performed.

2.Why it was really "useless junk"
Even though now Dr. A does not regard what he and Dr. B wrote to have value beyond that
of "useless junk", he did mention in the letter cited above that he had already rejected the
association-induction hypothesis while still in my laboratory, and that he has given "the
reason for this rejection at length in his Ph. D. Thesis"---a thesis I had never read before
because by the time he started writing his thesis, he was no longer my graduate student.
On my request, Dr. A sent me the relevant portion of his Ph.D. thesis. From this thesis, one
gets a closer look at the materials which had led Dr. A to reject the AI Hypothesis and to
launch him in his new-found faith in the membrane pump theory. Before going into the
details of what he had to say in his Ph. D. thesis, let us first construct a bird-eye view of the
whole problem of the hypothetical membrane pumps.

The sodium-pump hypothesis does not offer a basic molecular mechanism for the
postulated pump. It is therefore nothing much more than just a name, a rephrasing of one
arbitrarily chosen observation. I have clearly pointed out as far back as 1955 (J. Phys. Med.,
34: 89, 1955, p.94, paragraph 2) and repeated many times afterward ("A Physical Theory of
the Living State: the Association-Induction Hypothesis", Blaisdell, 1962, p. 216; "A
Revolution in the Physiology of the Living Cell", Krieger, 1992, p. 17) that if a membrane
pump is a mechanism offered in ernest to explain the asymmetrical distribution of a
permeant ion, it cannot be limited to just one arbitrarily chosen ion (i.e., the sodium ion).
Instead, "we must have 'pumps' for all these ions" which, like sodium ion, are also permeant
and do not follow the prediction of the Donnan ratio according to the membrane pump
theory ". Nor can one postulate pumps only for ions. Pumps must be postulated for
nonelectrolytes, chemicals like sugars bearing no net electric charges; many of them also do
not follow the equal distribution pattern predicted by the membrane theory--- from its basic
tenet that a living cell represents a membrane-enclosed dilute water solution.

Thus while it is legitimate and meaningful to choose just one pump to disprove the
membrane pump theory on energy ground--- as I did in 1962. To argue that the membrane
pump theory is tenable energetically, one must take into account all the pumps which must
be postulated. And then show that the total energy needs of all pumps added together is
within the limit of the energy available. Doing anything less would have created the false
impression that the membrane-pump theory is all right, while it is not.

In his hopeless and pathetic attempt to resurrect the membrane pump theory, Dr. A did just
that. Instead of dealing with all the postulated pumps together, he limited himself arbitrarily
to one pump, the sodium pump. At the time when he was preparing his Ph.D. thesis, there
were at least 20 pumps already postulated and published (some of which are not single
pumps but long lists of pumps like the various sugar pumps and various free-amino-acid
pumps). Worse, this list of pumps was collected from the literature by nobody else than A
himself. Later this list was printed and published in a review A co-authored with Ochsenfeld
and myself ( Ann. NY Acad. Sci. 204: 6, 1973, Table 2 on page 9).

All these postulated pumps A had gathered from the literature are confined to pumps at the
cell membrane or plasma membrane. As mentioned in linked page, lp6a(1), pumps are also
needed at the membranes of the subcellular particles because the ion and nonelectrolyte
distribution across their surfaces are also as a rule asymmetrical. One kind of such
subcellular particles is the sarcoplasmic reticulum, (SR). In frog muscle cells, the SR has a
total surface 50 times larger than the plasma membrane. Since the energy need of the
membrane pump (otherwise the same), varies directly with the size of the surface area of
the particle, a similar pump at the surface of the SR would consume 50 times more energy
than that at the cell membrane (see Ling, "A Revolution in the Physiology of the Living Cell",
Krieger, 1992, Malabar, Fl., p.19).

Not only do sodium ion, magnesium ion, sugars, free amino acids etc. etc., which are found
in the natural environment of the living cells, require pumps; exotic solutes, including those
synthesized and thus created for the first time by organic chemists in chemistry laboratories
also as a rule distribute asymmetrically across cell membranes and thus need pumps. This
need of pumps for man-made chemicals poses two difficult if not insurmountable problems
for the membrane-pump theory: First, since the living cell's genome had never been
exposed to these new molecules in past history, how could the cell evolve pumps in
anticipation of their future synthesis by humans? Second, since there is no limit to the
number of new chemicals organic chemists can create, how is the limited space of the
plasma membrane and subcellular particle membrane accommodate an infinity of pumps?
In my computation of the maximum energy available to operate the sodium pump of
poisoned frog muscle given in detail in 1962, the energy source was to a large extent limited
to the energy in the so-called "high energy phosphate bonds" of ATP and phosphocreatine
present initially in the muscle cells. I then used values of these energies available in the
literature. However, Podolsky and Morales (J. Biol. Chem. 218: 945, 1956) and George and
Rutman (Prog. Biophys. Biophys. Chem. 10: 1, 1960) have conclusively demonstrated that
the high-energy-phosphate-bond concept itself is a mistake. There is no such high-energy to
speak of.

The downfall of the high-energy phosphate bond concept was a tremendously important
landmark event in the history of cell physiology and it has not been challenged since its
publication. Among its many major impacts, it demands that the maximum energy available
for the postulated sodium pump I estimated must be revised. As a result, the figure of 22,95
cal/kg/hr for the total available free energy of poisoned frog muscle as given in my original
publications (Table 8.5, Ling "A Physical Theory of the Living State", 1962, pp. 202-203)--- as
the sum of the contributions from the decomposition (and presumed usage to pump the
sodium ion) of creatine phosphate (21.57 cal/kg/hr), of ATP (0.64 cal/kg/hr), of ADP (0.18
cal/kg/hr) and from the residual glycolysis producing lactate (0.56 cal/kg/hr)--- must now be
replaced by the free energy from the residual lactate production alone. This, of course,
amounts to a mere 0.56 cal/kg/hr. A reduction of the maximum available energy by a factor
of a little over forty-fold (40 times) is the result.

With this corrected maximum available energy, the disparity between minimum energy
need and maximum energy available would rise sharply from 30.6 times, 15.4 times and
18.0 times from the three sets of experiments performed in 1956 (Table 8.9, ibid) to 1224
times, 616 times, and 720 times respectively ( " A Revolution in the Physiology of the Living
Cell, Krieger, 1992, pp. 12-16) (see below, however, for a two-fold reduction of these figures
due to partial adsorption of intracellular sodium ion). All of these new facts ( and much
more ) are what a scientist wishing in earnest to resurrect the membrane-pump theory must
deal with. Sadly, A could not and did not do it. Therefore, what he did was not a serious
scientific inquiry. To call it "useless junk" as he himself did, is not an exaggeration.
Nonetheless, it is of at least record-keeping interest, to look deeper into what A himself has
to say in his Ph.D. thesis, which was not thrown away as trash and remains a part of the
existing literature.

But before that, I must review some other works I have published which are directly
relevant to understanding my original computation of the excessive energy and which A set
out to challenge in a way that has excited one reader at least (Kolata) to (unearned)
ebullience.