I was going to comment upon the comment you made in another post about Gilbert Ling. I’d never heard of them or their ideas and had to go looking the other night. As a result I now know why I never did.
I am extremely tired right now and in the middle of preparing for my thesis committee meeting, so I cannot give this the attention it deserves right now. Come back in a day or three and I will either expand this reply or make another one.
For now:
*From what I’ve seen, Ling’s ideas seem to originally be based upon a few equivocal experiments from the sixties and seventies that have since been contradicted by just about all cellular electrophysiology, enzymology, and membrane biology known.
*All the odd results they point to about ion balance requiring too much energy to be accounted for via active-transport ion pumps in the membrane have been solved to my satisfaction by more recent work, especially in neurons, and all their talk about cells with compromised membranes maintaining ion balance seem like they can be explained by work on calcium-triggered membrane vesicle fusion from the nineties.
*This proposed role for ATP in unwinding proteins is experimentally unsupported by any good sources I can find, I fail to understand why it would be better for this purpose than just phosphate laying around the cell, and I use ATP nearly literally nearly every day to drive enzymes via hydrolysis. The ability of enzymes and channel proteins to couple its hydrolysis to all kinds of processes is not and never has been in doubt.
*Ion gradients established via membrane pumps are well-established as an energy source (or sink) in biology from the classic rotary ATPase that pumps protons across the bacterial membrane while breaking down ATP (with the proton gradient then driving flagellar motion in bacteria as it leaks back in through another rotary setup attached to the fiber and various transport systems), to the role of calcium as a signaling molecule that is sequestered out of the cytosol by active ion-specific pumps that can be poisoned, to the existence of channel proteins in our blood that form part of our innate immune system which exert their killing effect on bacteria by forming large nonspecific channels in their membranes that allow ions to leak across their gradient.
*Most of their writing that I did see in my hour of poking around contains incredulity about the very idea of powered channels being able to do what we know, experimentally, they do, along with some very elementary misunderstandings of molecular biology and evolution.
*The evidence for ordered water more extensive than hydration shells around proteins is not very good, and there is actually very interesting and productive research going on into protein hydration—I interviewed with and almost worked for a guy at Indiana University starting a project comparing the hydration shells around bacterial and eukaryotic proteins and finding that bacterial proteins had nice orderly hydration shells you can see in crystal structures while eukaryotic proteins are ‘sticky’ to other protein molecules because they are unable to hold onto water molecules well in the face of themal noise. You pointed to the work of Gerald Pollack supporting the idea of extensive ordered water and while this is a guy doing interesting materials science about the interaction of water and solutes with surfaces he is prone to making wild pronouncements on extremely small amounts of evidence and others have raised other possible explanations for his data that have been insufficiently followed up on.
There is a place for controversy in biology. At the university I am working at right now, I nearly worked with a guy who is busily basically trying to prove that the model of protein translocation across the ER membrane is wrong and far more complicated than the usual address-tag plus protein-channel for growing peptides setup in the textbooks. He even thinks that parts of it were crystallized in the public opinion of scientists before sufficient work had gone into proving it. But at a glance shoving out literally most of what is known pretty well about the way cellular metabolism and membranes work… color me sceptical to say the least.
If you have time to provide links, I would like to read the research you mention- especially on the thermodynamics of ATP and calcium-triggered membrane vesicle fusion. Ling’s work is all very old and doesn’t address any newer research, but Pollack addresses some of the issues you raise in his books. Pollack does love to speculate a lot, but he appears to be careful in distinguishing this speculation from things that have more evidence. Here is also a newer review paper that discusses this and some other ideas related to the role of entropy in biochemistry: Coherent Behavior and the Bound State of Water and K+ Imply Another Model of Bioenergetics: Negative Entropy Instead of High-energy Bonds
I’m gonna split up this reply, since I think part of it is important enough to be seen more and will go into a higher-level reply to the post itself. I will also preface this by saying that my primary areas of expertise are in energy metabolism (mostly glycolysis) and the cell division cycle, along with all the basic enzymology you need to know to do molecular biology.
As for ATP thermodynamics, I looked deeper into Ling’s writings before replying and was more and more distressed by what I saw. They literally cannot correctly do thermodynamics and biochemistry that I learned in my senior year of high school. The end result of several extremely basic math and conceptual errors in their justification for their theories is that their calculated value for the free energy available from cellular metabolism to pump sodium and potassium across the membrane is approximately 1⁄12 the true value! Given that this is approximately the figure they give for the factor of insufficiency of cellular metabolism to provide enough energy to run the pumps (they give a factor of 15-30 [without stating the error bars] ), and that I have other reasons to distrust almost everything this person has ever done, it is safe to say that Ling’s objection to the sodium/potassium pump on thermodynamic grounds is quite simply unjustified.
I will be walking through this in more detail in my other top-level reply along with other reasons that you shouldn’t accept their work at face value.
I will only say more about the thermodynamics of the sodium potassium pump by pointing to a paper that I found after a few moments of google scholar searching indicating that hepatocytes under oxygen starvation conditions not dissimilar to those described in Ling’s experiments put about 75% of their cellular energy into maintaining the ion gradient, but that under normal circumstances they use a much more reasonable less than one quarter. This is not an unexplored area of research and insinuating that there is some sort of controversy here is simply false.
I will summarize the research on membrane formation and calcium-mediated membrane vesicle fusion before linking to a paper that you can see figures of without a paywall.
Membrane lipids and the contents of membrane-bound compartments move around between compartments via vesicles. Proteins are extruded into the interior of the endoplasmic reticulum compartment before being packaged through a series of other membrane-bound organelles before being secreted, and all the membrane-lipid-building enzymes are for the most part embedded in the ER membrane so the lipids have to get from there to all the other membranes somehow. This turns out to be done via extremely tiny submicroscopic vesicles. They are rather smaller than the wavelength of light because they are pinched off their parent membranes by molecular machines consisting of single-digit numbers of protein molecules and thus are invisible to a light microscope but you can see them with an electron microscope and in some places, like the growing tips of fungal threads, they are densely packed enough that they make the cytoplasm milky.
These vesicles are attached to their destination membranes by a complex of proteins called SNARES which require calcium to work. V-SNARES on the vesicles bind to T-SNARES on the destination membranes and as the complex forms they warp the membranes and cause them to fuse. I’ve seen very interesting molecular simulations from the Folding@Home project of this process, which if I recall correctly indicated that as the membranes get warped and pushed together by the binding together of the SNARES, the hydration shells of the hydrophilic head groups of the lipids clash and produce interesting ordered structures that suddenly exclude all solvent from between the two membranes, causing the membranes to rapidly fuse.
Calcium is ordinarily excluded from the cytoplasm almost entirely, being sequestered into the ER compartment and the extracellular fluid. As a result SNARE function is normally very slow, except near the surface of membranes that bear calcium channels. In neurons, calcium rushes in for a miniscule fraction of a second after the neuron fires and this is what mediates the fusion of neurotransmitter-carrying vesicles to the presynaptic membrane allowing the signal to be passed to another neuron.
Figure 1: something like an eigth of the membrane of a starfish egg is torn off and while there is an initial puff of cytoplasm that squirts out, a new membrane forms behind it and retains the cell contents within seconds.
Figure 2: same thing but in an egg that had been injected with a substance that only glows in the presence of calcium ions. Upon membrane tearing the calcium RUSHES in rapidly, and the area of very high concentration is where the new membrane forms. The remaining calcium that makes it past the new barrier then diffuses throughout the rest of the cell rather than being excluded from some kind of water matrix.
Figure 5: injecting fluorescent dye without calcium into a starfish egg lets the dye immediately diffuse throughout the cell, while injecting it with calcium ions causes a vesicle to form around the dye containing it and preventing it from getting into the cytosol.
Figure 7: the cytoplasm around a vesicle formed like in figure 5 is full of membrane vesicles of odd shapes and sizes.
Figure 9: starfish egg cytoplasm dripped out of a needle into non-calcium-containing media loses a fluorescent dye in it to the media, while when dripped into calcium-containing media it forms a membrane and holds it in.
Figure 10: cytoplasm centrifuged so that it no longer contains membrane-bound vesicles is unable to form a barrier in response to calcium while the centrifuged down membranous organelles and vesicles are able to.
Figure 11: a diagram of the proposed mechanism.
It appears that when the horrifically abnormally high levels of calcium that appear when a membrane is cut hit the small membrane vesicles present in the cytoplasm, they rapidly indiscriminately fuse until they manage to create a new membrane barrier from themselves and any other membranes they touch that holds the cytoplasm in and restores ion sequestration. Other papers both before and after this saw the resealing of membranes in normal body cells but were unable to closely examine it, the large egg cells made it possible to do all these interesting manipulations.
This immediately suggests an explanation for Ling’s experiment in which they sliced frog muscle cells in half and put the cut ends in an ion solution (“Ringer’s solution”) which they measured the ion flux in and out of and saw it was normal. I note that Ringer’s solution contains large amounts of calcium. They claim that they checked via electron microscopy that the cytoplasm did not reseal, but the insides of muscle fibers are horrifically dense complicated places and there’s no guarantee that it resealed RIGHT at the cut site – it could have resealed microns or even millimeters away.
Excellent post, thanks for putting so much work into a clear explanation. I will re-investigate Ling’s work more carefully, and also see if I can find the mistakes in his thermodynamics calculations you mention. I have been biased towards his work and not looking critically enough, because it seems to explain some surprising observations about drug activity I’ve found in my own research- but that’s no excuse.
I am interested in the possibility that Ling could be entirely wrong about membrane physiology, but this gel phase shift phenomena could still be important in the cell. If Ling and Pollack are wrong about long distance effects from protein surfaces, that might not destroy their arguments as the cytosol is very dense, and the distance between proteins is very short. Albert Szent-Györgyi also did some work on this idea that is very different from Ling’s.
One of my committee members works on physics simulations of protein hydration shells, and I am going to meet with him and see what he thinks about this. The simulations I have seen don’t show significant water structuring, as the water molecules have too much thermal energy.
I promise I’ll get my top level post made soon—I just finished my committee meeting a few hours ago.
The short and dirty version is that Ling seems to completely ignore the entropy contribution to the Gibbs free energy change associated with ATP hydrolysis and throws out about 3⁄4 of the enthalpy contribution on the grounds that it is the energy of solvation of the protons that come off the newly deprotonated middle phosphate rather than the potential energy of the phosphate-phosphate bond itself, when that simply doesn’t matter and you just can’t do that when considering equilibrium and reaction rates and the ability of one reaction coupling to another to drive it. It’s not as if that one bond alone charges up a battery or something, the whole reaction occurs.
I honestly don’t know what to make of the assertion that ATP unwinds proteins just by complexing with unwound backbone. I’ve never seen that claim anywhere else, and I use ATP all the time via standard active-site hydrolysis reactions to drive DNA-building and DNA-modifying reactions as I make the DNA I put into my modified cells.
The simulation I was speaking of about the SNARES was indicating small hydration shells just a molecule or two wide, not large ones. It was interesting though in that it found that when the two membranes were forced into odd geometries and very close proximity by the SNARES forming a tight ring, the hydration shells were forced together to form ordered structures just a few molecules wide between the membranes before suddenly emptying the space. It’s been a while since I’ve seen that paper though, and I’d encourage you to look at the folding@home website and find it if you are curious and you don’t trust my memory (which I do not entirely trust myself, that’s not exactly my field and it’s been a few years).
Odd drug metabolism stuff eh? Want to move that to a PM?
I was going to comment upon the comment you made in another post about Gilbert Ling. I’d never heard of them or their ideas and had to go looking the other night. As a result I now know why I never did.
I am extremely tired right now and in the middle of preparing for my thesis committee meeting, so I cannot give this the attention it deserves right now. Come back in a day or three and I will either expand this reply or make another one.
For now:
*From what I’ve seen, Ling’s ideas seem to originally be based upon a few equivocal experiments from the sixties and seventies that have since been contradicted by just about all cellular electrophysiology, enzymology, and membrane biology known.
*All the odd results they point to about ion balance requiring too much energy to be accounted for via active-transport ion pumps in the membrane have been solved to my satisfaction by more recent work, especially in neurons, and all their talk about cells with compromised membranes maintaining ion balance seem like they can be explained by work on calcium-triggered membrane vesicle fusion from the nineties.
*This proposed role for ATP in unwinding proteins is experimentally unsupported by any good sources I can find, I fail to understand why it would be better for this purpose than just phosphate laying around the cell, and I use ATP nearly literally nearly every day to drive enzymes via hydrolysis. The ability of enzymes and channel proteins to couple its hydrolysis to all kinds of processes is not and never has been in doubt.
*Ion gradients established via membrane pumps are well-established as an energy source (or sink) in biology from the classic rotary ATPase that pumps protons across the bacterial membrane while breaking down ATP (with the proton gradient then driving flagellar motion in bacteria as it leaks back in through another rotary setup attached to the fiber and various transport systems), to the role of calcium as a signaling molecule that is sequestered out of the cytosol by active ion-specific pumps that can be poisoned, to the existence of channel proteins in our blood that form part of our innate immune system which exert their killing effect on bacteria by forming large nonspecific channels in their membranes that allow ions to leak across their gradient.
*Most of their writing that I did see in my hour of poking around contains incredulity about the very idea of powered channels being able to do what we know, experimentally, they do, along with some very elementary misunderstandings of molecular biology and evolution.
*The evidence for ordered water more extensive than hydration shells around proteins is not very good, and there is actually very interesting and productive research going on into protein hydration—I interviewed with and almost worked for a guy at Indiana University starting a project comparing the hydration shells around bacterial and eukaryotic proteins and finding that bacterial proteins had nice orderly hydration shells you can see in crystal structures while eukaryotic proteins are ‘sticky’ to other protein molecules because they are unable to hold onto water molecules well in the face of themal noise. You pointed to the work of Gerald Pollack supporting the idea of extensive ordered water and while this is a guy doing interesting materials science about the interaction of water and solutes with surfaces he is prone to making wild pronouncements on extremely small amounts of evidence and others have raised other possible explanations for his data that have been insufficiently followed up on.
There is a place for controversy in biology. At the university I am working at right now, I nearly worked with a guy who is busily basically trying to prove that the model of protein translocation across the ER membrane is wrong and far more complicated than the usual address-tag plus protein-channel for growing peptides setup in the textbooks. He even thinks that parts of it were crystallized in the public opinion of scientists before sufficient work had gone into proving it. But at a glance shoving out literally most of what is known pretty well about the way cellular metabolism and membranes work… color me sceptical to say the least.
If you have time to provide links, I would like to read the research you mention- especially on the thermodynamics of ATP and calcium-triggered membrane vesicle fusion. Ling’s work is all very old and doesn’t address any newer research, but Pollack addresses some of the issues you raise in his books. Pollack does love to speculate a lot, but he appears to be careful in distinguishing this speculation from things that have more evidence. Here is also a newer review paper that discusses this and some other ideas related to the role of entropy in biochemistry: Coherent Behavior and the Bound State of Water and K+ Imply Another Model of Bioenergetics: Negative Entropy Instead of High-energy Bonds
I’m gonna split up this reply, since I think part of it is important enough to be seen more and will go into a higher-level reply to the post itself. I will also preface this by saying that my primary areas of expertise are in energy metabolism (mostly glycolysis) and the cell division cycle, along with all the basic enzymology you need to know to do molecular biology.
As for ATP thermodynamics, I looked deeper into Ling’s writings before replying and was more and more distressed by what I saw. They literally cannot correctly do thermodynamics and biochemistry that I learned in my senior year of high school. The end result of several extremely basic math and conceptual errors in their justification for their theories is that their calculated value for the free energy available from cellular metabolism to pump sodium and potassium across the membrane is approximately 1⁄12 the true value! Given that this is approximately the figure they give for the factor of insufficiency of cellular metabolism to provide enough energy to run the pumps (they give a factor of 15-30 [without stating the error bars] ), and that I have other reasons to distrust almost everything this person has ever done, it is safe to say that Ling’s objection to the sodium/potassium pump on thermodynamic grounds is quite simply unjustified.
I will be walking through this in more detail in my other top-level reply along with other reasons that you shouldn’t accept their work at face value.
I will only say more about the thermodynamics of the sodium potassium pump by pointing to a paper that I found after a few moments of google scholar searching indicating that hepatocytes under oxygen starvation conditions not dissimilar to those described in Ling’s experiments put about 75% of their cellular energy into maintaining the ion gradient, but that under normal circumstances they use a much more reasonable less than one quarter. This is not an unexplored area of research and insinuating that there is some sort of controversy here is simply false.
I will summarize the research on membrane formation and calcium-mediated membrane vesicle fusion before linking to a paper that you can see figures of without a paywall.
Membrane lipids and the contents of membrane-bound compartments move around between compartments via vesicles. Proteins are extruded into the interior of the endoplasmic reticulum compartment before being packaged through a series of other membrane-bound organelles before being secreted, and all the membrane-lipid-building enzymes are for the most part embedded in the ER membrane so the lipids have to get from there to all the other membranes somehow. This turns out to be done via extremely tiny submicroscopic vesicles. They are rather smaller than the wavelength of light because they are pinched off their parent membranes by molecular machines consisting of single-digit numbers of protein molecules and thus are invisible to a light microscope but you can see them with an electron microscope and in some places, like the growing tips of fungal threads, they are densely packed enough that they make the cytoplasm milky.
These vesicles are attached to their destination membranes by a complex of proteins called SNARES which require calcium to work. V-SNARES on the vesicles bind to T-SNARES on the destination membranes and as the complex forms they warp the membranes and cause them to fuse. I’ve seen very interesting molecular simulations from the Folding@Home project of this process, which if I recall correctly indicated that as the membranes get warped and pushed together by the binding together of the SNARES, the hydration shells of the hydrophilic head groups of the lipids clash and produce interesting ordered structures that suddenly exclude all solvent from between the two membranes, causing the membranes to rapidly fuse.
Calcium is ordinarily excluded from the cytoplasm almost entirely, being sequestered into the ER compartment and the extracellular fluid. As a result SNARE function is normally very slow, except near the surface of membranes that bear calcium channels. In neurons, calcium rushes in for a miniscule fraction of a second after the neuron fires and this is what mediates the fusion of neurotransmitter-carrying vesicles to the presynaptic membrane allowing the signal to be passed to another neuron.
Cells DO get rips and tears in their membranes but often manage to repair them before losing undue cell contents. The main research on how this happens was done in starfish and echinoderm egg cells and embryos because they are cheap ways of getting lots of cytoplasm. A representative paper can be seen here: “Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca 2+ dependent Vesicle–Vesicle Fusion Events”.
Figure 1: something like an eigth of the membrane of a starfish egg is torn off and while there is an initial puff of cytoplasm that squirts out, a new membrane forms behind it and retains the cell contents within seconds.
Figure 2: same thing but in an egg that had been injected with a substance that only glows in the presence of calcium ions. Upon membrane tearing the calcium RUSHES in rapidly, and the area of very high concentration is where the new membrane forms. The remaining calcium that makes it past the new barrier then diffuses throughout the rest of the cell rather than being excluded from some kind of water matrix.
Figure 5: injecting fluorescent dye without calcium into a starfish egg lets the dye immediately diffuse throughout the cell, while injecting it with calcium ions causes a vesicle to form around the dye containing it and preventing it from getting into the cytosol.
Figure 7: the cytoplasm around a vesicle formed like in figure 5 is full of membrane vesicles of odd shapes and sizes.
Figure 9: starfish egg cytoplasm dripped out of a needle into non-calcium-containing media loses a fluorescent dye in it to the media, while when dripped into calcium-containing media it forms a membrane and holds it in.
Figure 10: cytoplasm centrifuged so that it no longer contains membrane-bound vesicles is unable to form a barrier in response to calcium while the centrifuged down membranous organelles and vesicles are able to.
Figure 11: a diagram of the proposed mechanism.
It appears that when the horrifically abnormally high levels of calcium that appear when a membrane is cut hit the small membrane vesicles present in the cytoplasm, they rapidly indiscriminately fuse until they manage to create a new membrane barrier from themselves and any other membranes they touch that holds the cytoplasm in and restores ion sequestration. Other papers both before and after this saw the resealing of membranes in normal body cells but were unable to closely examine it, the large egg cells made it possible to do all these interesting manipulations.
This immediately suggests an explanation for Ling’s experiment in which they sliced frog muscle cells in half and put the cut ends in an ion solution (“Ringer’s solution”) which they measured the ion flux in and out of and saw it was normal. I note that Ringer’s solution contains large amounts of calcium. They claim that they checked via electron microscopy that the cytoplasm did not reseal, but the insides of muscle fibers are horrifically dense complicated places and there’s no guarantee that it resealed RIGHT at the cut site – it could have resealed microns or even millimeters away.
Excellent post, thanks for putting so much work into a clear explanation. I will re-investigate Ling’s work more carefully, and also see if I can find the mistakes in his thermodynamics calculations you mention. I have been biased towards his work and not looking critically enough, because it seems to explain some surprising observations about drug activity I’ve found in my own research- but that’s no excuse.
I am interested in the possibility that Ling could be entirely wrong about membrane physiology, but this gel phase shift phenomena could still be important in the cell. If Ling and Pollack are wrong about long distance effects from protein surfaces, that might not destroy their arguments as the cytosol is very dense, and the distance between proteins is very short. Albert Szent-Györgyi also did some work on this idea that is very different from Ling’s.
One of my committee members works on physics simulations of protein hydration shells, and I am going to meet with him and see what he thinks about this. The simulations I have seen don’t show significant water structuring, as the water molecules have too much thermal energy.
I promise I’ll get my top level post made soon—I just finished my committee meeting a few hours ago.
The short and dirty version is that Ling seems to completely ignore the entropy contribution to the Gibbs free energy change associated with ATP hydrolysis and throws out about 3⁄4 of the enthalpy contribution on the grounds that it is the energy of solvation of the protons that come off the newly deprotonated middle phosphate rather than the potential energy of the phosphate-phosphate bond itself, when that simply doesn’t matter and you just can’t do that when considering equilibrium and reaction rates and the ability of one reaction coupling to another to drive it. It’s not as if that one bond alone charges up a battery or something, the whole reaction occurs.
I honestly don’t know what to make of the assertion that ATP unwinds proteins just by complexing with unwound backbone. I’ve never seen that claim anywhere else, and I use ATP all the time via standard active-site hydrolysis reactions to drive DNA-building and DNA-modifying reactions as I make the DNA I put into my modified cells.
The simulation I was speaking of about the SNARES was indicating small hydration shells just a molecule or two wide, not large ones. It was interesting though in that it found that when the two membranes were forced into odd geometries and very close proximity by the SNARES forming a tight ring, the hydration shells were forced together to form ordered structures just a few molecules wide between the membranes before suddenly emptying the space. It’s been a while since I’ve seen that paper though, and I’d encourage you to look at the folding@home website and find it if you are curious and you don’t trust my memory (which I do not entirely trust myself, that’s not exactly my field and it’s been a few years).
Odd drug metabolism stuff eh? Want to move that to a PM?