By now the reader has become thoroughly familiar with the subject of selective accumulation of K⁺ over Na⁺ in living cells, mostly in the context of the ideas of Moore and Roaf, Fischer, the LFCH and the PM theory.

The explanation offered by the membrane theory for this major cell physiological phenomenon has gone through several changes. First, the cell membrane was regarded as impermeable to both K⁺ and Na⁺ ions⁷⁶—it was this version of the membrane theory that provoked the criticism of Moore and Roaf ⁷⁷ cited in Chapter 7. Then the membrane was considered as permeable only to K⁺ but not to Na⁺ as the Mond-Amson-Boyle-Conway's version of the membrane theory suggested [4.3]. Finally, it was recognized that the cell membrane is also permeable to Na⁺.⁽³⁶⁾ When both K⁺ and Na⁺ can traverse the cell membrane, their intracellular concentrations should be the same as in the outside solution, contrary to facts.

It was in the emerging crisis thus created that the sodium pump hypothesis materialized. As mentioned above, the same or a broader category of metabolic pumps had been brought up from time to time throughout history.^{49; 333} In this specific version of the metabolic pump hypothesis, the low level of cell Na⁺ is maintained against its constant inward diffusion by the activity of a postulated outward pump located in the cell membrane. Like all pumps, ceaseless activity requires a ceaseless supply of energy.

Biochemical studies of energy metabolism in the 1930's led to the discovery that the final end-product of metabolism is adenosine triphosphate (also known as adenyl pyrophosphate) or ATP²⁷³ Meyerhofand Lohmann measured the heat of dephosphorylation of ATP (to ADP) and obtained a value of -12.0 Kcal/mole.³¹⁸ Based upon this and other relevant data, Lipmann then offered a new major cell physiological hypothesis,¹³² in which each one of the two terminal phosphate bonds in the ATP molecule contains an extra amount of free energy. This energy can be liberated to perform biological work with the aid of an ATP-splitting enzyme known as ATPase. These unusual phosphate bonds are given the name, "high energy phosphate bonds'" and represented as ~P.

Danish biochemist, Jens C. Skou found that the "membrane fraction" of a crab nerve homogenate contains a special kind of ATPase, which requires both K⁺ and Na⁺ for maximum activity—hence the name Na,K-ATPase. Skou then postulated that this and similar enzymes located in other cell membranes can liberate, and use the "high energy" in the high-energy-phosphate bonds of ATP to transport Na⁺ out of the cell, and K⁺ into the cell, both against concentration gradients. In other words, this membrane Na,K-activated ATPase is the postulated Na pump.⁵²

A vast amount of time, talents and resources have been spent in research related to Skou's hypothesis, culminating in the award of the 1997 Nobel Prize for Chemistry to Prof. J. C. Skou specifically for his work on the sodium pump. It seems to me that the decision-making majority of some committee laid an egg.²⁴⁷

My reason for this view is not hard to understand. Each piece of the experimental evidence supporting the LFCH [10.2] and the PM theory [11] is often sufficient (i) to reverse the evidence once considered to be in favor of the sodium pump hypothesis; (ii) and/or raise serious questions about the validity of the sodium pump hypothesis. However, to simplify the matter, I cite four additional sets of evidence focused specifically on questions that have direct bearing on the validity of the sodium pump hypothesis.

(1) The minimum energy need of the postulated sodium pump in frog muscle under rigorously controlled conditions has been shown to be at least 15 to 30 times the maximum available energy^{49, 98 pp 189-212} (Table 2). For more than ten years after its publication, none have disputed my conclusion that the sodium pump is energetically in violation of a basic law of physics, i.e., the Law of Conservation of Energy. Meanwhile, the essence of my finding has been twice confirmed.^{274; 275}

(2) The sodium pump is one of the pumps needed to keep the cell afloat. In an admittedly incomplete list. Ling, Miller and Ochsenfeld counted, in 1973, 18 pumps that had been proposed and published, some of which are not single pumps but each a multitude of pumps including the various sugar pumps and various free amino acid pumps.^{131 Table 2; 49 Table 1}

These are, however, only pumps at the cell membrane. Still other pumps are needed across the membrane of various subcellular particles. Due to their enormous surface area, the sodium pump at the sarcoplasmic reticulum (alone) in voluntary muscle cells, for example, requires 50 times more energy than a similar pump at the cell membrane.^{49 pp 130-133} Yet as shown in (1) Just one postulated pump alone at the cell membrane would demand for its operation more energy than what the cell has at its disposal.

 

Table 2. Comparison of the minimal energy need of the postulated sodium pump in frog muscle cells with the maximum available energy after both respiration and glycolysis were stopped. Metabolic inhibition was achieved by a combination of low temperature (0^oC), pure nitrogen (99.99% pure nitrogen further purified by activated copper at high temperature). 1 mM NaCN and 1 mM sodium iodoacetate. Under these conditions, the only energy available^{131 pp 11-12} to the muscle cells were from the store of ATP, ADP and creatine phosphate at the time when the poisons had begun to exercise their full effect and a minute trickle of glycolysis, which had escaped the iodoacetate blockage (but was accurately determined and taken into account in the energy balance calculation from analysis of the total lactate formed and analyzed). Rate of sodium pumping (according to the sodium pump hypothesis) was determined from small muscle-fiber bundles isolated from semitendinosus muscles of North America leopard frogs (Ranu pipiens pipiens, Schreber). Pumping was against both an electric potential gradient (i.e., the resting potential measured with Gerard-Graham-Ling microelectrodes) and a concentration gradient (computed from the labeled Na⁺ in the cells and in the bathing solution) under similar experimental conditions described above. For more details and supportive evidence for the experimental procedures adopted, see a recent review.⁴⁹ (From Ling⁹⁸ with permission)

---

(3) One year before Skou presented his sodium pump hypothesis, Podolsky and Morales demonstrated that the high-energy-phosphate-bond concept was in serious trouble.¹³³ To wit, the so-called "high energy phosphate bond" does not contain high energy. Podolsky and Morales's conclusion was based on the more precise measurement of the heat of hydrolysis of ATP and a (judicious) correction for the heat of neutralization of the acid liberated during the hydrolysis (Table 3). Thermodynamic analyses led George and Rutman¹³⁴ to a similar conclusion: that there is no high energy in the so-called high-energy phosphate bond.

Table 3. Heat content (enthalpy) change in myosin-catalyzed hydrolysis of ATP in phosphate buffer. Purified myosin labeled Sample 48 was dissolved in 0.60 M KC1. Total orthophosphate concentration was 0.05 M; pH, 8.00. n_ATP represents the number of moles of ATP hydrolyzed; n_HCl, the number of moles of HCl in the 0.6 M KC1 neutralized. The last column represents heat content change with hydrolysis of one mole of ATP after correction for the heat of neutralization of the H⁺ produced. Parallel studies with other buffers yielded similar results. σ is the standard deviation. (Podolsky and Morales,¹³³ by permission of Journal of Biological Chemistry)

With the truth about the "high energy phosphate bond" concept brought to light, even the 1500% to 3000% energy disparity figures for the postulated sodium pump in frog muscle cited under (1) appear to be serious underestimations.⁴⁹

---

 

Since there is no utilizable free energy stored in the phosphate bonds of ATP, the Na,K-activated ATPase or any other ATPase cannot by hydrolyzing ATP provide energy for pumping Na⁺ and K⁺ ions.

(4) The technique of removing the cytoplasm or axoplasm of squid nerve axon was perfected in two laboratories in the year 1961.¹³⁵ The healthy condition of the "axoplasm-free" squid-axon membrane thus obtained has been established by its normal electric activities. When the open ends of such a hollow membrane sheath are tied, after sea water containing essential nutrients have been introduced into the sac, it provides an ideal preparation to test the membrane pump theory. There is every reason to expect it to pump Na⁺ out and K⁺ in against concentration gradients—that is, if the membrane-pump theory is fundamentally sound. In fact, active transport of K⁺ or Na⁺ against concentration gradients could not be demonstrated in these ideal preparations by some of the most skilled workers in the field.^{136 p 95; 137; 138}

Figure 33. Reuptake of K⁺ and extrusion of Na⁺ from red-blood-cell "ghosts" prepared from washed human red blood cells. The study followed rigorously a procedure described in Reference 471. Freshly drawn blood was obtained (mostly) from different donors. When blood from the same donors was used, it was drawn at at least 6-weeks intervals. Each data point represents the difference of K⁺ or Na⁺ concentration in samples of the ghosts at the beginning of incubations and after 18 hours of incubation in the presence of ATP (37°C). Straight lines corresponding to the two equations shown in the graph were obtained by the method of least squares. Total protein content represented as P in the equations was obtained by subtracting the sum of the weights of lipids, phospholipids, salt ions, and sucrose from the dry weights of the ghosts. (Ling, Zodda and Sellers¹⁴⁰)

---

Similarly, resealed red blood cell ghosts also freed of all cytoplasmic proteins and supplied with ATP do not transport K⁺ or Na⁺ against concentration gradients.^{139, 140, 107 pp 25-27} In contrast, similarly-treated, resealed red cell ghosts supplied with ATP do re-accumulate K⁺ and extrude Na⁺ if and only if, a measurable amount of the cytoplasmic proteins remains in the ghosts.^{107 pp 25-27} Indeed, the levels of K⁺ re-accumulated is directly proportional to the level of proteins remaining in the ghosts (mostly hemoglobin), as shown in the upper plot of Figure 33. And the level to which Na⁺ is brought down is inversely related to the concentration of residual intracellular proteins also¹⁴⁰ as shown in the lower plot of Figure 33.

Since Nature's own membrane—when freed of cytoplasm—cannot do what the membrane pump theory has predicted, it is not surprising that claims of success in demonstrating K⁺ and Na⁺ transport against concentration gradients by man-made "membrane pumps" of isolated Na,K-ATPase are themselves mistakes. The mistakes appear to have come from an over­looked isotope leakage from the isotope-loaded vesicles while passing through a Sephadex column—in order to separate the vesicles from the isotope-loading solution—as pointed out in some detail by Ling and Negendank in 1980.^{138 p 224; 107 pp 22-25}

Parenthetically, Ling and Negendank also pointed out^{138 pp 234-235} that the data perhaps prematurely interpreted as indicating ATP-energized pumping (in the wrong direction) are not without value. For they suggest that water in the vesicular membrane (see Figure 5D for an EM picture of similar vesicles) might have become polarized and oriented by the Na,K-activated ATPase, which assumes at least in part the fully-extended conformation under the influence of ATP as a cardinal adsorbent [15.1]. The reduced rate of permeation of labeled Na⁺ through the polarized-oriented water may then slow down the loss of labeled Na⁺ from the vesicles during their passage through the Sephadex column, and account for the higher level of labeled Na⁺ found in these vesicles than in the otherwise similarly-treated vesicles but without AT P.

When all the above evidence is taken into account, I have reached the conclusion that the selective accumulation of K⁺ over Na⁺ in nerve, muscle, erythrocytes and other cells with a uniform cell membrane, and called unifacial cells, is not at all likely the consequence of the ceaseless activity of a postulated pump in their cell membranes. The sodium pump hypothesis is thus no longer a useful model, and it is high time for its retirement.

 

In 1976, two of my former graduate students and an inexperienced fledgling reporter for Science magazine attempted to convince readers that the sodium pump hypothesis was still viable. A careful analysis of their output showed that there is little merit in it as it was based on some non-existent "crucial experiments" and on not telling the whole truth—as was made clear in an article entitled "Debunking the Alleged Resurrection of the Sodium Pump Hypothesis" I published in 1997.⁴⁹

  

As was made clear in [4.3], the "pump" was installed as the last resort to keep the membrane theory afloat. The retirement of the sodium pump hypothesis, therefore, also spells the end of the tenure of the paradigm of living cells as membrane-enclosed dilute solutions, a paradigm that has dominated the field of cell physiology throughout its entire history. However, not everything introduced in the membrane theory has been proven wrong. Thus, there is strong evidence that living cells are covered with a diffusion barrier of some sort, as originally postulated in the membrane theory. This barrier variously called the protoplasmic skin, plasma membrane or cell membrane is the subject matter of the following chapter.