Chapter 5: Experimental Tests of the Alternate Theories
Is there enough metabolic energy for the Na+-pump? What about the other solute membrane and sub-membrane pumps?
As always, this is not medical advice, and reading this does not form a client relationship with me - your health is your responsibility.
Today’s Substack will continue with Chapter 5, Experimental Tests of the Alternate Theories, from Dr. Gilbert Ling’s “In Search of the Physical Basis of Life.”
Please feel free to skip to the parts you wish to read.
Starting with an overall summary of the chapter:
The primary supporting theories for the membrane pump theory include research showing - “full ionic dissociation of K+ salts in cell water; the high mobility of K+ in squid axon and frog muscle; the high K+ activity in squid axon; enzymes not normally found in bacteria; circumstantial evidence that the Na+, K+-ATPase is the Na+ pump; evidence that ATP provides the immediate source of energy for Na+ pumping; ATP-induced Na+ efflux in perfused or dialyzed squid axon and red blood cell ghosts; ATP-induced Na+ uptake by vesicles containing Na+, K+-ATPase.”
However, other studies showed Na+-pumping could occur without any metabolic energy source - there is always the possibility of another source (e.g., sunlight). Further experiments showed insufficient metabolic energy to run the Na+ pump, let alone the additional pumps needed to maintain a significant concentration difference intracellular to extracellular for many different solutes.
Red blood cell ghost studies showed that only 8% protein is needed to “accumulate K+ and extrude Na+, negating the assumption that these preparations are simply membranes.” Also, studies with open-ended cells (severed cells - so no pumps or membrane on one side) showed “selective K+ accumulation over Na+ that clearly is determined by cytoplasmic, not membrane, processes.”
5.1 Evidence Supporting the Membrane Pump Theory
Most supporting membrane pump research was published in the 1950s and 60s.
5.1.1. Full Ionic Dissociation of K+ Salts in Water at Ionic Strengths Similar to Those in Living Cells
1887: cellular concentrations of K+ and Na+ dissolved in water exist in the free state - not bound to anything else.
1940s and 50s: cellular K+ and Na+ believed to be in the free state
5.1.2. High Mobility of K+ in Living Cells
1950s and 60s: squid axon and frog muscle experiments gave support to K+, Na+, SO4-2, sorbitol, sucrose, and ATP-3 are in the free state, and Ca+2 is in the bound
5.1.3. High K+ Activity in Living Cells
The activity coefficient of K+ in cells was about equal to that found in a KCl solution. This further added to the belief that cellular K+ was free. The Na+ was shown to be ~76% free.
5.1.4. Genetic Control of Permeases or Sugar Pumps
Production of enzymes not generally produced by bacteria began by signaling DNA activity when exposed to the substrates that required enzyme usage. The inducer (substrate) binds to the repressor protein. This causes the repressor protein to unbind from the DNA leading to the operon (functioning DNA unit). - a precursor to epigenetics research. Also, why environmental changes have a marked effect on our health.
5.1.5. Na+, K+ - Activated ATPase as the Na+ Pump
1957: “if ATP provides the energy for the Na+ pump, the energy stored in the high-energy phosphate bonds of ATP could be made available only by a specific enzyme, i.e., an ATPase, that for maximal activity depends on the very same ions that are pumped, i.e., Na+ and K+.”
“the properties of the enzyme system, [ATPase], and the transport systems are qualitatively and quantitatively the same:
1. Both systems are present in the cell membrane.
2. Both systems utilize ATP but not inosine triphosphate.
3. Both systems require the presence of Na+ and K+.
4. Both systems require the same concentration of cations for half-maximal activity.
5. Both systems are inhibited by cardiac glycosides.”
“[T]his good correlation between enzyme activity and ion flux rate may not reflect a postulated pumping activity of this enzyme. It may merely reflect the fact that this large protein molecule in the cell surface may offer the seat of water polarization and that it is the total area of the cell surface occupied by this water that determines the rate of ion fluxes.”
5.1.6. “High Energy” Contained in the Phosphate Bonds of ATP Provides the Immediate Source of Energy for Na+ Pumping
Inhibiting cellular metabolism in muscle did not alter Na+-efflux. Creatine phosphate and arginine phosphate were thought to “step in” to provide the needed energy in the lower metabolic state -
Squid axons are sensitive to DNP, a popular weight loss aid that affects oxidative phosphorylation (metabolism). As such Na+-efflux did slow in the axon following DNP exposure and resumed to normal once the DNP was rinsed off.
Change in Na+-efflux rate and metabolism had no effect on action potentials - this is an important point that will be discussed in further chapters.
A reduction in external K+ concentration slowed the Na+-efflux.
“[T]here is a coupling of outward Na+ efflux with an inward K+ pumping. This coupled Na+ - K+ pump derives it energy from metabolism and is sensitive to cardiac glycosides, while a separate system of downhill Na+ and K+ movements is not sensitive to metabolic poisons or to cardiac glycosides. It was suggested that the pump is energized by the ionic gradients normally existing across the cell surface.”
Arginine phosphate and phosphopyruvate seem to act independently of ATP.
Na+-efflux rate and the level of cellular Na+ are not causally related.
“ATP does not even contain a package of energy that can be tapped for work performance in the way once widely believed.” - more on this in future chapters.
5.2. Evidence against the Pump Hypothesis
5.2.1. There Is Not Enough Energy to Operate the Na+ Pump
Living cells have a different concentration of ions and nonelectrolytes than their surrounding medium.
To stop diffusion from occurring, which would lead to the concentrations equilibrating, the following three would need to happen:
An energy barrier - what’s in the cell stays in, and what is out stays out.
Pumps - continually running and the only to require a constant energy source.
A difference in the “physiochemical environments in the two spaces.”
Radioactive tracer experiments disproved 1 - decay of the radioisotopes showed they move in and out of the cells, so the ions and non electrolytes are not fixed
One of the central presuppositions of physics is the law of energy conservation: energy in = energy out - this can be used to test the other two possibilities. There are energy requirements for membrane pumps and submembrane, like the mitochondria.
Respiration is inhibited by nitrogen.
Glycolysis is inhibited by insole-3-acetic acid, a naturally occurring plant hormone.
Ling and follow on experiments showed: “The minimum energy need of the postulated Na+ pump is from 1500% to 3000% of the maximum energy available. Before accepting these findings, the possibility of other energy sources must be examined.” - prior experiments only looked at ATP, ADP, and Creatine phosphate.
Experiments measuring the total heat output from metabolism matched the energy output from ATP, CrP, and lactate - “This indicates that there is no unknown energy source.”
“I concluded that the Na+ pump alone could consume far more energy than the cell has at its command. There is, therefore, logically no need to consider the many other postulated plasma membrane pumps and subcellular particle membrane pumps.”
5.2.2. Reassessment of the High Energy of the “High-Energy Phosphate Bond”
“Podolsky and Kitzinger (1955) and Podolsky and Morales (1956) in very carefully conducted calorimetric experiments showed that the enthalpy of hydrolysis of ATP is not -12 kcal/mole as was once believed, but is only -4.75 kcal/mole. This finding removed the experimental basis for the high-energy phosphate bond theory.”
“These different approaches all led to a serious doubt that one can really separate classes of phosphate bonds as being “high-energy” or “low-energy” in character. The key role of ATP in biological work performance is not the capture and storage of energy within a certain bond and its subsequent release to drive pumps or contractile machines or other types of biological work. Rather, ATP must function in a different manner.” - low energy phosphate bonds were believed to play a role in ribopolynucelotides.
5.2.3. Failure to Demonstrate Selective K+ Accumulation and Na+ Exclusion by a Cytoplasm-Free Squid Axon Membrane Sac
Squid axon membrane sheaths that had the cytoplasms removed continued to retain their electrical properties - bringing into question the delayed K+ outward current seen with the polarization change from the membrane potential by Hodgkin and Keynes compared to the physiologic state. Also, added ATP did not lead to a net exclusion of Na+.
5.2.4. Failure to Prove Selective Ion Pumping in Membrane Vesicles
“[T]he vesicles contain as much or more solids than the original intact cells and therefore cannot possibly be pure membrane vesicles.”
5.2.5. Studies of the Red Cell Ghost
“[E]xposure of red blood cells to a hypotonic solution causes hemolysis.”
“The remains of the hemolyzed red cells are called ghost or stroma.”
‘It is probable that ... hemoglobin is bound in some way to the cell structure. One reason for this belief is that purely mechanical agencies will not liberate the pigment. The cell may be torn into the finest shreds, yet each minute particle still retains its hold upon the hemoglobin.’
“[R]ed cell ghosts are solid, [not hollow].”
Ghosts were believed to be cytoplasm-free and proof that ATP led to Na+ and K+ pumping. However, follow on research showed that the ghosts “do not consist of hollow membranes… [and] they do transport K+ and Na+ against concentration gradients.” Other ghost experiments did show hollow membranes but no pumping.
“Since the salt-free dry solid is virtually all protein, these data clearly show that it is the cytoplasmic proteins that determine both the selective K + uptake and the Na+ exclusion. Red cell ghosts prepared and treated by the Freedman procedure can neither accumulate K+ nor exclude Na + when their water content rises above 92%. In no instance have we observed K+ uptake in and Na+ extrusion from ghosts with a 95% water content.”
“In summary, red cell ghosts do not produce evidence proving the membrane pump theory, contrary to widely held belief.”
5.2.6. Ouabain-Sensitive Selective Accumulation of K+ over Na+ in an Effectively Membrane (Pump)-less Open-Ended Muscle Cell (EMOC) Preparation
“A basic requirement of a membrane pump is that it must have both a sink and a source. Thus, to pump K+ inward there must be an external source of K+; to pump Na+ outward there must be an external sink into which the Na+ pumped out is taken up. Without the source and sink, the pump cannot function.”
Experiments were done that involved cutting the end of a sartorius muscle cell. These showed the cut end maintained its “membrane” and did not pump “22Na+ (isotope) into the extracellular space.” No membrane “regenerated for as long as 28 hr at 25C.” The “pumping” of Na+ and K+ was shown to “reflect primarily the properties of the muscle cytoplasm… The only major difference is the slow but spreading decline of the selectively accumulated K+ and rise of Na+ near the cut end, an observation that is attributed to damage there and to a decline of ATP.” - there was an “abrupt drop of total K+ and a [rise] Na+ at the cut end.”