First Man To The Pump
By Michael Schrøder
The story of the pump that led to the 1997 Nobel Prize in Chemistry is the story of an indefatigable research scientist who started out not wanting to be a researcher at all. It is also the story of a long series of coincidences which in the end led to an important scientific discovery. And the reward? That came 40 years later.
Early Days
To begin with Jens Christian Skou had no intention of becoming a research scientist, but instead dreamt of being a surgeon. This dream had started with an interest in anaesthesiology, but Skou's career took an unexpected twist when he chanced upon an interesting medical problem.In 1944 Skou graduated in medicine from the University of Copenhagen. He was 26. In that same year he found employment at Hjørring Hospital, where he then worked for the best part of two years. He then took up a position at the Orthopaedic Hospital in Aarhus, before moving yet again one year later to the Department of Physiology at the University of Aarhus. Here Skou felt that there was an opportunity for him to carry out research in what really interested him: the way in which local anaesthetics worked.
An Impecunious Environment
At that time - the mid-1940s - there were no professional anaesthesiologists as we know them today. Ether was much used for total anaesthesia, but it was an anaesthetic which involved much discomfort, both for the patient and for those administering the drug. The preference was therefore to use spinal anaesthesia or a local anaesthetic whenever this was at all possible.From his student years, Jens Christian Skou was aware that there was a link between the narcotic effect of a drug and its solubility in fatty substances (lipids). But did this also apply to those substances used as local anaesthetics, and what relevance did it have for the mechanisms that formed the underlying principles of anaesthesia? He decided that this was to be the topic of his doctoral thesis, which in turn would further his career as a surgeon.
Skou started his research project on local anaesthetics at the University's Department of Physiology. One might argue that a pharmacological department would have provided a more suitable base for such research, but at that time no such institution existed at the young University of Aarhus.
In 1947, just 19 years after its foundation, the University could not exactly boast a top-quality research environment for the various disciplines within the overall field of biology. And to make matters worse, with very few members of staff and 140 medical students, the teaching load was heavy.
A New Attempt
Skou quickly ascertained that the connection between anaesthetic substances and their solubility in lipids did not apply to local anaesthetics. Nevertheless, the weight of evidence appeared to suggest that lipids played some sort of role in local anaesthesia. But what exactly?In a book by N.K. Adam entitled The Physics and Chemistry of Surfaces, Jens Christian Skou learnt of Langmuir's investigation of the monomolecular layers of lipids on a water surface, and of how Schulman had shown that capillary active substances dissolved in a liquid with a lipid layer on top would actually be drawn up into the lipid layer. As the cell membrane comprises a bimolecular layer of lipids, Skou reasoned that it must be possible to use a monolayer of nerve membrane lipids placed on a water surface as a simple model of a nerve membrane, and that this model could then be used to investigate the effects of local anaesthetics on the nerve membrane.
His experiments showed that there was a connection between the ability of local anaesthetics to be drawn up from the liquid phase and into the fatty monolayer, and their ability to block neural conductance, i.e. to produce anaesthesia. It was already known that a nerve impulse occurs at the moment of a brief (1 millisecond) increased permeability of the cell membrane for sodium in a localised area, allowing sodium to run into the cell. As sodium carries a positive charge, this process leads to a reduction of the electric potential across the cell membrane. The next stage in the process involves the membrane opening up for potassium, which also carries a positive charge. Potassium now flows out of the cell, with the result that the membrane potential is re-established. What was not known was the nature of the structures in the membrane that mediated the transient increase in penetrability for sodium. Jens Christian Skou believed that it must be due to brief alterations in the structure of proteins in the membrane. In the light of his experiments with monolayers, Skou reasoned that local anaesthesia, by penetrating the lipids in the nerve membrane, brought about changes in the lipid part of the membrane, and in so doing indirectly obstructed the brief changes in the structure of proteins in the membrane which led to the increased permeability for sodium.
To be able to prove this hypothesis, it was necessary to make a monolayer comprising a mixture of lipids and proteins, and then to prove that local anaesthesia could affect the structure of the proteins by their penetration of the lipid element of the monolayer. Back in the early 1950s it was not possible to measure structural changes in proteins, but Skou supposed that if a protein with a high activity enzyme was used (i.e. one that could split a substance), any change in this enzyme's ability to split the substance could be used as a measurement of a change in the structure of the protein itself. For this purpose it was necessary to use an enzyme which not only was so active that it would be possible to measure the level of activity in the limited quantities present in a monomolecular layer, but which also originated from a cell membrane. Just such an enzyme, acetylcholinesterase, had recently been isolated from electric eels by Professor Nachmansohn at Columbia University, New York.
Electric Eels And Squids
A Danish colleague and friend of Nachmansohn, Professor E. Lundsgaard from the University of Copenhagen, introduced Skou to Nachmansohn, and Jens Christian Skou took the opportunity to ask whether he might visit Columbia University in August and September of 1953. Professor Nachmansohn was not in New York in August, but at the marine biology station at Woods Hole on the Atlantic coast near Boston. He proposed that Skou should join him there instead, and then in September accompany him to New York where they could purify the enzyme.Jens Christian Skou accepted the invitation despite the fact that he had no idea what he should do at Woods Hole, there being no access to electric eels. But Woods Hole proved to be a veritable eldorado for scientists. Every summer researchers from all over the world flocked to the station to work. The attraction? Access to squids caught by the station's fishing boat - squids with a 'big nerve' containing nerve fibres with a diameter of anything between 0.5 and 1 mm. This allowed scientists to carry out tests that were simply impossible on other nerves. Jens Christian Skou was thrown into a level of scientific activity and a scientific environment vastly different from that which he had experienced in Aarhus, and the visit made a great impression on him.
During his stay at Woods Hole, Skou, quite by chance, read an article about an ATP-splitting enzyme, an ATPase found in the membrane of the giant nerve of the squid. He already knew that ATP is the fuel for body cells. Splitting ATP releases the energy used to power many cellular processes, and he therefore wondered what function it might have in a nerve membrane. An enzyme is a protein, and as it was to be found in the membrane it must be a protein that could exist alongside lipids. And that, of course, was exactly the type of protein Jens Christian Skou was interested in using in his monolayer experiments. He decided to take a closer look at this on his return to Aarhus.
Crabs In Aarhus
Skou spent the September of 1953 at Columbia University purifying acetylcholinesterase from electric eels.On returning to the Department of Physiology at the University of Aarhus he renewed his work with the monomolecular layer, but now using acetylcholinesterase. In the autumn of 1954, Skou began to search for a suitable organism from which he could isolate the ATP-splitting enzyme in the nerve fibres. As squid was not available, his choice fell on crabs. Crab nerves are similar to those of squid, inasmuch as they are not myelinated. However, there is one disadvantage: while the large nerve fibres of the squid are between a half and one millimetre thick, crab nerve fibres are the size of the thinnest of sewing thread. So what was needed was an awful lot of crabs.
Jens Christian Skou got in touch with a fisherman from Norsminde, to the south of Aarhus, who agreed to supply him with crabs, and during the ensuing years finding the
Department of Physiology at the University of Aarhus was the easiest of tasks: you could smell it a mile away. Many a crab was sacrificed in the name of science during that period.
The experiments showed that the ATP-splitting enzyme also existed in the membrane of crab nerve fibres. Magnesium was required to achieve the splitting process. Sodium combined with magnesium resulted in a slightly higher level of activity, while potassium did not appear to have any effect. However, the many experiments related to enzyme activity produced variable results; sometimes the activity was high, sometimes low, without there being any obvious explanation. Uncertain of what step to take next, Jens Christian Skou decided to leave matters as they were for a while, and left his laboratory to enjoy a much-needed Christmas holiday.
The experiments were resumed in the new year, but still with the fluctuating results, and still without Skou being able to pinpoint the reason for the inconsistencies. By now he was close to abandoning this work, so he temporarily dropped everything and left on his summer vacation.
If At First ...
After the summer break he observed that potassium, which in the earlier experiments had not had any effect, now increased enzyme activity. Closer investigation of the experimental conditions showed that this was because there was sodium present in the medium. In other words, it seemed that to activate the enzyme it was necessary to use a combination of sodium and potassium. This explained the fluctuations that had proved so frustrating. ATP was purchased in the form of a barium salt, but when it was used, it was converted into either a sodium or potassium salt. This was because when the nerve membranes were cut up, this was done sometimes in a sodium chloride solution, and sometimes in a sucrose solution, which had led to the varying amounts of potassium and sodium in the various solutions. As it was known that potassium alone played no role, and that sodium only produced a minimal effect, the fact that a combination of the two might have a crucial effect had not been considered. This then, appeared to be the explanation for the variations in the level of enzyme activity.A systematic investigation of the combined effect of the two ions showed that not only was the presence of sodium necessary for potassium to activate the enzyme, but also that potassium actually affected the process in two different ways in its own right. When present at low concentrations, potassium activated the enzyme; but if the concentration was increased, potassium not only inhibited its own activating effect, but also the low-level activation produced by sodium. This latest round of results showed that the ions affected the enzyme in two places. One of these sites was where sodium is normally to be found. When the sodium was in position, potassium produced increased activity at the other site; but when the concentration of potassium was increased, potassium displaced sodium from its site. And with potassium at both sites there was no activity at all.
Articles
But what possible role could an enzyme that splits ATP play in a nerve membrane? And why did this split occur as a result of the combined effects of sodium and potassium?Had Skou known more about active transport through the cell membrane, the answer might well have been easier to find. In the early 1950s it was in fact known that the transport of sodium out of the cell was linked to the transport of potassium in the opposite direction, and that it was therefore probable that the activation of the pump mechanism was due to the combined effects of the two ions. But Skou was not aware of this. Local anaesthesia was still his main interest. His first reaction was therefore to presume that this ATP splitting enzyme might be the protein in the cell membrane responsible for opening and closing the flow of sodium and potassium, and by so doing provoke the breakdown and reestablishment of the membrane potential, i.e. the nerve impulse - the nerve impulse which was blocked by local anaesthesia. This theory was, however, quickly rejected, since it was known that the opening and closing for the ion stream is dependent on the membrane potential and not on the splitting of ATP. His conclusion, therefore, was that the enzyme must have something to do with the active, energy-demanding transport of ions through the membrane: the sodium pump.
It was at this stage that Skou began to concentrate his reading in the field of active transport. He found that others had already discovered that the sources of energy for this active transport are organic substances combined with what are known as high-energy phosphate bonds. As ATP is such a substance, this new knowledge supported his supposition that the enzyme might be involved in the active transport process.
In 1956 Jens Christian Skou wrote the article describing his experiments with crab nerves, and put forward the idea that the ATP-splitting enzyme was a part of the sodium pump, or even constituted the sodium pump in its entirety. The theory that cell membranes contain pumps that regulate the sodium-potassium balance in cells was first put forward in 1941, and it was widely agreed that such a pump must exist. But so far no research had identified the pump. Skou's article was published in 1957 with the title 'The Influence of Some Cations on the Activity of an Adenosine Triphosphatase in Peripheral Nerves'. He considered using the word 'pump' in the title, but felt that this would be too daring - hence the somewhat obscure wording. The result was that very few people read the article, and that it certainly did not receive much attention among researchers working with active transport.
The Conclusive Experiment
It was over a year later, in 1958, when Jens Christian Skou participated in the 4th International Congress for Biochemistry in Vienna, that international researchers began to take note of the results being produced in Aarhus. During the congress Skou told his American colleague, Robert L. Post, whom he had first met five years previously at Woods Hole, that he believed he had identified the pump as a sodium-potassium ATPase in the cell membrane. Judging by the look on Post's face, this was news indeed. Suddenly Skou began to grasp the importance of his many years of research.Post asked whether the activity of the system was inhibited by the digitalis glycoside ouabain. To which Skou could only answer with a question of his own: "What is ouabain?" Post explained that ouabain is the most water-soluble of the digitalis glycosides, and that Schatzmann, a Swiss scientist, had demonstrated in 1954 that digitalis glycosides, which are found in the digitalis plant, are specific inhibitors of active transport in blood corpuscles. Skou telephoned straight away to his laboratory technicians in Aarhus and got them to set up an experiment with ouabain.
Robert Post, who was more than interested, returned to Aarhus with Jens Christian Skou after the congress. The experiment proved that ouabain did in fact inhibit the activity, and this was sufficient to convince Post that Skou had indeed identified the pump.
By this time Skou was beginning to look for the enzyme in red blood corpuscles (erythro-
cytes), because he had learned that erythrocytes are the classic test object for investigating the active transport of sodium and potassium. Post asked whether he could continue work on these experiments when he got back to the States. Unlike Jens Christian Skou,
Robert Post had experience of working with erythrocytes, so Skou accepted the proposal and concentrated his own efforts on investigating whether the enzyme was present in other tissue with active transport.
It Is The Pump!
In 1959 Skou participated in the 21st International Physiology Congress, which was held in Buenos Aires. It was here that he gave his first actual presentation on the crab nerve experiments. A year later Robert Post published the article in which he proved that the enzyme was also present in erythrocytes, and that it was responsible for the active transport of sodium and potassium. Post's article had a better title than Skou's, namely 'Membrane Adenosine Triphosphatase as a Participant in the Active Transport of Sodium and Potassium in Human Erythrocytes', and, since Post was already a highly respected researcher in the field, his article was widely read. The result was a huge upsurge of interest in the field. Within the space of just a few years a considerable number of articles appeared discussing the connection between the enzyme and active transport in many different tissues.In the light of this wide-ranging activity, Skou was able to produce (in 1965) a survey of the work done to date. The article was entitled 'Enzymatic Basis for the Active Transport of Na+ and K+ across the Cell Membrane', and Skou used it to demonstrate that the enzyme met the necessary requirements for a system that could transport sodium and potassium across the membrane while using energy, and that this therefore must be 'the pump'.
Over the years Jens Christian Skou has been the recipient of many awards, but the highest award of them all, the Nobel Prize, came 40 years after his initial discovery. But no one disputes the fact that Skou was the first man to the pump.