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Vol. 26 No. 2
March-April 2004

Role Models in Chemistry: Jens Christian Skou

by Balazs Hargittai and István Hargittai

Jens Christian Skou
Photo by I. Hargittai, Aarhus, 2003.

Jens Christian Skou (b. 1918 in Lemvig, Denmark) is professor emeritus at the Department of Biophysics of the University of Aarhus, Denmark. He received half of the Nobel Prize in Chemistry in 1997 "for the first discovery of an ion-transporting enzyme, Na+,K+-ATPase." His story tells how scientific curiosity, persistent work, and international interactions bring a medical doctor working in a rather isolated place to a major discovery in biochemistry. Skou—who was training as a surgeon—wanted to understand the action mechanism of local anesthetics. His studies eventually led to the problems of transport of ions across cell membranes. This is an area of research, which is at the interface of chemistry and biology and has implications for the medical sciences.

Jens Christian Skou received his M.D. degree from the University of Copenhagen in 1944. He studied in the medical school when Denmark was under German occupation in the Second World War. That period had a lasting impact on his life. Members of his medical student group knew that one of the students was an informer for the Germans; unknown people liquidated him. Because of fear of revenge from the Gestapo against the group, teaching was cancelled. Many of Skou’s teachers took part in the resistance against the Germans and they went underground or escaped to Sweden.

After graduation, Skou started his internship at a hospital in the northern part of Denmark. In the surgical ward the head of the department had escaped from the Gestapo to Sweden. The next in line in the department was anxious to teach him how to operate, which was unusual for someone who had just started his internship. Eventually Skou realized that the reason for this was that his superior surgeon was involved in receiving weapons from England delivered by plane at night. When they were on night duty together and the surgeon had to leave to receive weapons he wanted to be sure that Skou could take over. Skou also received his Doctor of Medical Sciences degree from the University of Copenhagen in 1954.

In order to understand the importance of professor Skou’s discovery, we have to go back a little in science history. In the 1870s it was shown that there is a difference in the concentration of sodium and potassium inside and outside the cell. The potassium concentration is higher in the cell than outside while for the sodium concentration the reverse is the case. This was explained around 1913 in the following way. If the cell contains proteins, which cannot pass through the cell membrane and there is potassium chloride, which can pass through the cell membrane, the product of potassium and chloride concentrations in the cell will be equal to the product of potassium and chloride concentrations on the outside at equilibrium. But as there must be electro-neutrality on the inside, the concentration of potassium must be higher than the chloride concentration, because part of the potassium concentration is used to neutralize the protein negative charges. On the outside, the product consists of equal components. If there are two components in a product with unequal size, which is equal to a product of two components of equal size, then the sum of the two unequal components is higher than the sum of the two equal components. This means that there is a higher concentration of potassium + chloride inside than outside, which also means a higher osmotic pressure.

Jens Christian Skou standing next to the street sign that bears his name in Aarhus, Denmark.
Photo by I. Hargittai, Aarhus, 2003.

Furthermore, there are the proteins in the cell, which also adds to the osmotic pressure. Equilibrium can only be obtained if water is prevented from flowing in. As the cell membrane is permeable to water, the only way to establish the equilibrium is by adding some ions on the outside to compensate for the high osmotic pressure inside the cell, and that ion is sodium. That is why the sodium concentration must be higher outside than inside. But why is sodium not distributed like potassium. The answer was that this is because the membrane is impermeable to sodium. But in l939, it was shown that the muscle membrane was permeable to sodium, and it was suggested that there are pumps in the membrane that pump sodium out (i.e., the distribution of sodium is not an equilibrium distribution but a steady state distribution that requires energy). In the following years it was shown that cell membrane permeability to sodium was a general phenomena.

Skou started his related research in the beginning of the 1950s. By then the existence of the ion pump in the cell membrane had been accepted, but it was not known what the nature of the pump was. While Skou was in the surgical ward, he became interested in the action mechanism of local anesthetics. He knew that for narcotics, which are neutral substances, there is a correlation between lipid solubility and narcotic potency. The local anesthetics are weak bases, which in aqueous solution dissociate into a water-soluble ionized part and a non-ionized part, which is lipid-soluble. Did a similar correlation between lipid solubility and anaesthetic potency exist for local anesthetics? Experiments showed that it did not and neither was there a correlation between the capillary activity and the potency of the local anesthetics.

At some point Skou realized that a phospholipid monolayer is similar to one half of the cell membrane, and that is why it could be used as a model of the water lipid interface of the cell membrane; the cell membrane is a double layer of phospholipids. He then used this model to test the effect of the local anesthetics. He observed that the local anesthetics when added to the waterphase underneath a monolayer of lipids extracted from peripheral nerves increased the pressure in the monolayer at a given area, indicating that they penetrated up into the monolayer. There was a correlation between the ability to increase the pressure in the monolayer and the blocking potency of the local anesthetics.

These results sufficed for Skou to write up his Danish thesis and publish six papers in English. At this point, he could have stopped research, but his curiosity did not let him stop. He wanted to determine what the connection was between the penetration and pressure increase in the monolayer and the blocking of the nerve impulse. The local anaesthetic in blocking concentrations had no effect on the membrane potential. Skou thought it was possible that the local anaesthetic—by the pressure increase—blocked the opening of the membrane for sodium, thereby stopping the influx of sodium and, consequently, the initiation of the nerve impulse.

Jens Skou and his wife skiiing.

Skou thought that a protein—through a change in its conformation—was responsible for opening the nerve membrane for sodium and that the local anaesthetic, by penetrating into the membrane, blocked the conformational change. In order to show this, he thought that the protein should be incorporated into a monolayer of lipids and then a local anaesthetic should be added to the water phase so it could be determined whether the penetration into the monolayer had an influence on the conformation.

A problem was that the protein responsible for the opening of the nerve membrane for sodium was unknown, and that no methods existed for measuring conformations of proteins in a monolayer. Instead, he decided to use a protein with enzymatic activity, incorporate this in a lipid monolayer, add local anesthetics to the water phase, and see if penetration up into the monolayer had an influence on the enzymatic activity, and take this as an indication of an effect on the conformation. He had to find an enzyme with a high turnover number, as the amount of protein he could incorporate into a monolayer was very limited, and if possible an enzyme that was membrane bound. One of his candidates was acetylcholinesterase. It is a membrane bound protein, which is prepared from the electric organ of the electric eel. Skou had no access to electric eels, but he knew that David Nachmansohn, a former refugee from Nazi Germany, now at Columbia University, prepared the enzyme from electric eels. Skou spent two months with Nachmansohn in 1953, the first month in Woods Hole and the second in New York City.

"Woods Hole . . . was where he realized for the first time that science was a serious affair and not just a hobby."

Woods Hole turned out to be a great experience for Skou. It was where he realized for the first time that science was a serious affair and not just a hobby. In contrast to Woods Hole, Aarhus was a 19-year old university then, and Skou’s department consisted of three young doctors with no scientific background who were trying to do research. At Woods Hole, he encountered such giants of science as Albert Szent-Györgyi, Severo Ochoa, George Wald, and others. Scientists interested in neurophysiology came from all over the world to work there in the summer time, because there was access to squids and their giant axons, nerve fibers with a diameter of 0.5–1 mm, which were very useful for neurophysiological experiments.

Skou read in a book by Nachmansohn that in l948 Libet had shown that there is an ATP hydrolyzing enzyme in the membranes of the giant axons, an ATPase. He knew that ATP is the energy source in cells and wondered what was the function of an ATP hydrolyzing enzyme in the nerve membrane. Furthermore, being in the membrane it had to be a lipoprotein, and that was what he needed for his monolayer experiments. He decided to take a look at the enzyme when he returned to Aarhus. In September, in New York, he prepared acetylcholinesterase.

Upon his return home he prepared monolayers of the acetylcholinesterase and measured the enzymatic activity in the monolayer, and observed that the enzymatic activity of the monolayer was pressure dependent. The next step was to prepare a phospholipid monolayer containing the protein and to see if the addition of local anesthetics to the water phase could influence the enzymatic activity. But before doing this he wanted to take a look at the nerve membrane ATPase.

He had no access to giant axons, but used membranes from crab leg nerves instead. A fisherman sent him some 200 small crabs every week and he used several thousand crabs before the study was over. Skou had two laboratory assistants pull the nerves out of the legs. A problem was how to kill the crabs. They started with a hammer, but this led to crab pieces all over the room. At the end, they cut the legs with a sharp pair of scissors above a big pot with boiling water and dumped the crabs into the pot, which killed them immediately. The procedure gave them another problem—the smell of boiled crabs. One could easily locate the Institute of Medical Physiology on the campus of Aarhus University based on its smell alone. Today such experiments might not be tolerated.

"Looking for the answer, You hunt it, you catch it, you fool yourself; the answer is always a step ahead."
—Jens C. Skou's opening Nobel Lecture on 8 December 1997

Skou at that point entered a frustrating period in his research. The membrane pieces from the crab nerves contained—as described for the giant axon—an enzyme that hydrolyzed ATP in the presence of magnesium, but the activity was low; and the addition of potassium had no effect, while sodium gave a slight increase. Further experiments gave, however, varying results, with some times higher and some times lower activity. This went on for a year and a half. He needed enormous stamina not to give up. Opportunities came up for clinical jobs and at one point Skou decided to take up such an appointment, but his wife knew him better and suggested that he stay at the university.

As it turned out, eventually Skou discovered that the enzyme required a combined effect of sodium and potassium for activity. This was an unexpected surprise, and it explained the varying results, which were due to varying concentrations of the two cations in the test tube, which came from buffers and the solutions used for the preparation of the membranes. As the first experiments had shown no effect from potassium and little effect from sodium, the concentration of these cations in the medium had not been controlled.

Skou was interested in the effect of local anesthetics on the nerve conduction. As the nerve conduction is related to the opening and closing of the nerve membrane for sodium, followed by an opening for potassium, his first reaction was that the enzyme, which was influenced by a combined effect of sodium and potassium, was related to this change in permeability for sodium and potassium, and that it was a channel in the nerve membrane. But as the change in permeability of the nerve membrane was voltage dependent, and the enzyme activity was ATP dependent, he came to the conclusion that the enzyme was part of or the sodium pump.

He published his findings in a paper titled “The Influence of Some Cations on an Adenosine Triphosphatase from Peripheral Nerves” [Skou, Biochimica et Biophysica 1957, 23, 394]. He ended the paper by saying that the crab nerve ATPase seems to fulfill a number of the conditions that must be imposed on an enzyme, which is thought to be involved in the active extrusion of sodium from the nerve fiber. This was the paper that was the principal basis for his Nobel Prize. However, the paper had only limited immediate impact.

It was again the international interactions that gave a decisive push to his career. In 1958, he took part in an international biochemistry conference in Vienna, Austria. There he renewed his contacts forged some years before in Woods Hole. One of the discussions gave him a clue to a chemical test that would unambiguously demonstrate that the ATPase he worked with was the sodium pump. He also found out that the crucial observation was the combined effect of the potassium and sodium cations.

Gradually Skou’s discovery spread and when he visited the United States in 1963, the editor of Physiological Review asked him to prepare a review article on the ATPase, which was published in 1965 [Skou, Physiol. Rev. 1965, 45, 596]. The article provided all the evidence that this enzyme had all the characteristics needed for it to be the sodium pump. It was named the Na+,K+-ATPase.

"From his seminal 1957 paper, the Nobel recognition took 40 years."

From his seminal 1957 paper, the Nobel recognition took 40 years. During that time, Skou diligently worked on the enzyme. He was mostly working alone and most of his papers carry his name as the sole author. After his 1965 paper, however, he had more visibility and more co-workers. Although tremendous progress has been made, Skou stressed in his Nobel lecture that the molecular basis of the action mechanism of the ATPase pump is not yet fully understood.

Professor Skou never thought of the Nobel Prize and it came to him as a surprise. He was happy that his field was selected, but was disappointed that the two other major figures, I. M. Glynn of Cambridge, England, who had made important contributions to the development of the concept of active transport, and Robert Post of Nashville, Tennessee, who had done very important work for the understanding of the involvement of the Na+,K+-ATPase in active transport, were not included. The Nobel recognition came late in Skou’s life and did not disrupt the creative period of his activities. It took him two full years to deal with its consequences, the celebrations, lectures, interviews, answering fan mail, etc. Of course, there was tremendous press coverage of the prize in Denmark and the University of Aarhus also benefited from the recognition.

Today, Skou is deeply concerned about the funding system in science. He thinks that it hinders the development of young, independent scientists who would have original ideas to pursue. The funding system favors those who are already in secure position and it leaves little room for new thinking. The present funding system may lead to the loss of new initiatives and almost ensures mediocrity. In Denmark, Skou has been very vocal about the need for a better funding system to support basic research, especially to provide the opportunity for young people to work on their ideas. He has become sort of a politician, in a way following in the footsteps of his wife, Ellen-Margrethe, who has been active in county politics for a long time.

Dr. Balazs Hargittai is at St. Francis University in Loretto, Pennsylvania, and Dr. István Hargittai is at the Budapest University of Technology and Economics.

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