A REMINISCENCE ABOUT SODIUM, POTASSIUM-ATPASE

Robert L. Post Department of Physiology, School of Medicine

Vanderbilt University Nashville, Tennessee 3 7232

The Discovery of Sodium, Potassium Adenosine Triphosphatase

Jens Skou discovered sodium, potassium adenosine triphosphatase. At Wood’s Hole in the summer of !953 my wife, Elizabeth, discovered Jens on the beach. Jens had come over t o the “New World” from Aarhus, Denmark in order t o learn about acetyl cholinesterase from Nachmansohn, and Nachmansohn had come t o the Marine Biological Laboratory at Wood’s Hole. There was not much for Jens t o do. He discovered me in Harry Grundfest’s laboratory where I was observing the injection of curare into the giant axon of the squid. A few such observations convinced Jens that he should spend his time on the beach or in the library. I was discovering a new world of bioelectricity, membrane potentials, oscilloscopes, and micropipettes, mostly by leaning over Eric Kao’s shoulder. Eric discovered that injection of anything into squid axons produced un- predictable effects.

It was in the library that Jens began t o read about lipoprotein ATPase’s.’ He wanted an enzyme that would help him make a model of a cell membrane. He had been putting cholinesterase into a lipid monolayer as a preliminary model, and now he thought that a lipoprotein enzyme would be more suitable. At the end of the summer he traveled with us t o the International Physiological Congress in Montreal. And so we became acquainted.

After the Congress we went home t o Nashville and Jens returned t o Aarhus. Jens now decided t o prepare an ATPase from nerve. Not having squid in Denmark, he chose crabs. He collected crabs by the basketful and his patient, conscientious, and good-looking laboratory assistant broke open the legs of the crabs and removed the nerve. A basketful of crabs could produce a few milliliters of pure nerve. The nerves he ground up in a sucrose solution and tested for ATPase activity. The crabs he boiled. His departmental chairman, grskov, repeatedly asked if the resulting stench was really necessary. Jens said it was. He knew that sodium ion was essential for the electrical impulses con- ducted by nerve axons and so he tested the action of sodium ion on the rate of splitting of adenosine triphosphate by his homogenate of crab nerves. Sodium ion increased the activity. The results were not reproducible precisely. Jens’ technician worried about this. Jens said later that it took him six months t o find out that the enzyme was also stimulated by potassium ions. Like other ATPase’s this enzyme also required magnesium ions.

What physiological function could the enzyme have? Sodium ions flow into axons during the rising phase of the action potential and are pumped out of the cell during the resting phase. This pump uses metabolic energy such as might be supplied by the splitting of ATP. Jens thought that his ATPase was involved in the active transport of sodium ions outward through the plasma membrane of nerve cells. The enzyme was in the particulate fraction of his homogenates, but at that time Jens did not know that it was embedded in the walls of tiny vesicles formed from pieces of broken plasma membrane.

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Post: Discovery of Na, K-ATPase

Jens had an unusual particulate ATPase from crab nerve which required sodium, potassium and magnesium ions simultaneously. He had carefully studied all the kinetics. What t o do? He presented his work at an international meeting in Copenhagen in 1956 and at an International Congress of Neuropharmacology in Aarhus in 1957. He visited McIlwain’s laboratory in London and gave a seminar. McIlwain was studying the effect of electrical stimulation on the metabolism of brain slices. He should have been interested. He said that the enzyme was very interesting. Jens concluded that he was not interested. In later years investigators in McIlwain’s laboratory worked extensively with the enzyme. One participant in the meeting in Copenhagen did examine homogenates of brain for the ATPase. With a collaborator he found it.2 After Jens’ paper3 came out in 1957, they did not carry their work further.

The Discovery by Jens Skou

In 1953 after the Congress in Montreal, Elizabeth and I returned to Nashville. I was beginning to set up again my apparatus for recording intracellular electrical potentials in the cells of frog heart when the Chairman of the Department, Rollo Park, called me into his office and said, “Post, I know your type. You are going to become a cynical old man. What you need is a problem. Furthermore, I am going to have a department centered around hormones and membranes and there will be no room for research on electrical potentials. Why don’t you discover the mechanism for the active transport of sodium ions across cell membranes?” I was shocked, but he was right. I gave up the celestial oscilloscope for the mundane pipet and marrow-chilling cold room. Rollo thought I should isolate the sodium carrier. Why not put a lipid extract of cell membranes into an oil film between solutions of sodium chloride and potassium chloride? Then one could detect the carrier by the appearance of a diffusion potential? Why not in- deed? Four months later I knew why not. I decided to approach the problem from the outside instead of from the inside and set out to observe sodium ion transport in human red blood cells. I might not know what I was doing, but at least the cells would be functioning. Human red blood cells had specific ad- vantages. They were easy to obtain, well-studied by other investigators, and most important of all, one could take aliquots. Nobody ever took an aliquot of a squid axon. Unfortunately transport was slow. This led to staying up late at night. Storage of the cells in the cold allowed sodium ions to leak in from the plasma, providing an increase in the quantity to be pumped out, but cold storage also ruined metabolism.

In 1954 I found an old bottle of adenosine in a dusty cabinet. I sprinkled some on my erthrocytes and they pumped sodium well for the first time. I was so excited that I tried everything else in the cabinet; nothing else worked. It was hardly any extra trouble with the flame photometer to measure potassium ion also and so I measured both. Potassium was transported in when sodium was transported out. The two transports appeared to be linked together. In 1953 Schatzmann had published his classic discovery of the inhibition of the active transport of sodium and potassium ions by cardioactive steroids, such as ouabain. This was terrific. It provided pharmacological agents that would distinguish pump transport from transport through other pathways. I was working on the right system. I had a message, stoichiometric linkage of sodium and potassium transport through the pump, and I had an experimental system to demonstrate it. In the fall of 1955 and spring of 1956, I set about to make the most convincing

8 Annals New York Academy of Sciences

demonstration that I could. MaizelsS had already discovered some sort of linkage and E. J. Harris6 had already proposed stoichiometry (2 Na+ outward per 1 K+ inward), but I was fortunately not reading the literature and was able t o think that I was on t o something. Actually my only original contribution was the ratio of 3 sodium ions moving outward per 2 potassium ions moving inward. As it turned out, Harris had not invested much midnight oil in stoichiometric linkage and abandoned it a few years later. In late 1956 Ian Glynn’ published a paper supporting stoichiometric linkage (1 Na+ per 1 K+). I could hardly bear t o read it, In 1957 my linkage paper8 came out. I was firmly convinced that the pump had t o have both sodium and potassium ions in order t o work.

Red blood cells do not consume any of the oxygen that they carry. They convert one molecule of glucose into t w o molecules of lactic acid and in the process synthesize two high-energy phosphate bonds of ATP. What could be more reasonable than that the pump should consume ATP? If so, should it not be possible t o demonstrate an ATPase activity in a preparation of broken membranes that would require sodium, potassium, and magnesium simul- taneously for activity and that would be inhibited by ouabain? That such a thing could be true seemed an impossible piece of luck at the time. I only suggested the search for such an ATPase t o a medical student who was de- termined not t o work on any student project on which another student was working. Unfortunately, he did not learn even how t o d o an analysis for in- organic phosphate. About this time I did try an experiment t o see if red cells that were pumping sodium and potassium without glucose would lose the ability t o take u p added glucose faster than those that were not pumping. They did. I used the ability t o take up glucose as a measure of the presence of intra- cellular ATP. So I thought that pumping cells would use u p ATP. Ned D ~ n h a m ~ . ’ ~ and Ron Whittam” were having similar ideas about the same time. When I read Jens’ paper in 1957, my first reaction was that he had got i t ; my second reaction was that he had not got all of i t ; and my third reaction was that there was really too strong an inhibition by potassium ion at certain concentra- tions for it t o really be right. And so I put it aside. My mind was on lithium ion as a substitute for potassium ion in inward transport.

The next development was at the International Biochemical Congress in Vienna at the end of the summer of 1958. Jens and I met again. Almost a t once he began t o tell me about his ATPase, which required sodium, potassium, and magnesium ions together, which was found in particles from crab nerve, and which he thought was involved in the active transport of sodium ions outward across the cell membrane. As I listened t o him tell me about it in person, face t o face, it became crystal clear that he had it, and I told him so. This was the first solid encouragement he had received. At that time the world was full of ATPase’s without visible physiological significance and a new one, slightly fancier than the others, had failed t o trigger interest. I asked if his enzyme was inhibited by ouabain. “Ouabain, what’s that?”, he replied. Linkage was new t o him also. 1 began t o tell Jens about linkage of active transport in red blood cells and of its inhibition by ouabain. Jens immediately invited me t o Aarhus t o give a seminar on transport in red blood cells. He also arranged by telephone t o test the effect of ouabain on his ATPase. After the Vienna meeting, when I gave my talk in Aarhus, Jens told me that ouabain did inhibit his ATPase, but that the effective concentrations were much higher than those in human red blood cells. After his talk we agreed that I could look for his ATPase in the membranes of red blood cells when I got home.

Post: Discovery of Na, K-ATPase 9

On the way home I stopped in Cambridge, England t o visit Ian Glynn and Ned Dunham. Ned was starting two years of postdoctoral work in Ian’s laboratory and they were considering the possibility of relating Jens’ observa- tions on the ATPase in crab nerve t o sodium-potassium transport in red blood cells, specifically t o look for a sodium and potassium ATPase in red blood cell membranes. I told them about my visit t o Jens’ laboratory and that ouabain in- hibited his enzyme. We all thought that it was a promising line of experimenta- tion but neither I nor they were ready t o say that we would surely work on it without further consideration. As far as I was concerned, there were still prob- lems left over from my 1957 paper that needed clarification. As it turned out , they did not get clarified until nine years later, when they were n o longer of much interest.

While still on the return journey home I attended a meeting of the Red Cell Group in Bethesda, and discussed the relationship of linkage of sodium and potassium transport in red blood cells t o ouabain-sensitive sodium- and potassium-dependent ATPase activity. Joe Hoffman and Dan Tosteson in particular encouraged me t o try this line of experimentation and said they would be interested t o know if it worked. And so in October 1958 I set about learning how to estimate ATPase activity by the Fiske Subbarow method. I also learned how t o make preparations of red blood cell membranes. The ATPase activity of the membrane had been described, but had not been tested for sensitivity t o sodium and potassium or ouabain. There was not much activity so that most of the reaction volume was occupied by the membrane suspension. By December the experiments were working reliably, just in time for an abstract t o be sent t o the Federation Meeting.I2 Years later I learned that Ned Dunham’s similar efforts were not yet successful, apparently because of traces of impurities in his distilled water. My only precaution against impurities was t o keep a little EDTA with the membranes all the time since I had been raised t o believe that traces of heavy metals were bad for enzymes and i t was desirable t o have a little EDTA in the reaction mixture on general principle. At about this time a graduate student, Richard Kinsolving, began t o help me. We were having trouble with “lumpy enzyme”; that is, duplicate samples from a single suspension of membranes did not give identical results. Later o n we learned t o filter out small clots. In the summer of 1959 two medical students also helped with the experi- ments. They were Cullen Merritt and Charlie Albright. I tried t o pull in every consideration I could think of to strengthen the case for the participation of the ATPase in active transport. About a year after 1 started the experiments, I began t o write the manuscript that came out in 1960,13 which was about the same time that Jens published a further studyI4 of the kinetics of his ATPase together with a demonstration of its inhibition by ouabain. He also proposed a phosphoenzyme as an intermediate.

Th e Ph osph oenzy rn e

In December 1958 Victor Najjar told me, “Post, you will never have t o d o anything else in your life.” This was not really true. I did have t o remain visible. When Rollo Park heard Lowell Hokin propose that phospholipids were the sodium carriers,I5 he told me that I should get a piece of that action. 1 did not much want t o trail behind Lowell, but 1 agreed that I should d o something about it. Hermann Bader was undertaking chromatography of phospholipids in the next laboratory and so I made some radioactive ATP with spinach chloroplasts, put it on red cell membranes, and Hermann chromatographed the phospholipids for

10 Annals New York Academy of Sciences

me. There was a beautiful peak of radioactivity coming off the column before any of the ordinary phospholipids. This was quite exciting until we discovered that addition of radioactive inorganic phosphate to membranes already denatured with acid would produce the same result.I6 That was the end of that.

In the summer of 1960 Alan Rosenthal came t o work with me as a summer student before he entered medical school. He was an intense young man deter- mined t o discover something important and impatient with halfway measures. He was subject t o wide variations in mood from elation on one day t o despair on the next. But he kept me moving. Next year, during his time in the medical physiology course, I arranged that he should d o a student project with Janey Park. Janey was labelling an active site in triose phosphate dehydrogenase with radioactive acetate at that time and she taught Alan all that was necessary to get that system t o work. During the next summer, we started out on something trivial, but in the middle of June I visited Arthur Solomon’s laboratory at Harvard Medical School and heard Manfred Karnovsky give a lecture t o an audience, which included Lowell and Mabel Hokin, on the role of phospholipids in the active transport of sodium and potassium. That did it. When I got back home, I turned Alan loose on the phosphoenzyme. We made radioactive ATP with spinach chloroplasts and sodium, potassium ATPase from beef kidney from the grocery store. The kidney membranes had much more ATPase activity in them than the red cell membranes did. From the specific activity of the ATP and guessing at the turnover number of the enzyme, I calculated that we should just be able t o detect a phosphoenzyme if one was present. We put both sodium and potassium in the reaction system so that the enzyme would surely be turning over and added ouabain as an inhibitor t o change its level. About half the time ouabain produced a barely detectable increase in the level of phosphorylation. Finally after two months we changed all the variables separately and it was obvious that sodium and magnesium produced the highest level of phosphoryla- tion and that potassium and magnesium produced the lowest level.” Alan got a few more experiments t o work, one of which we eventually published, but the system remained erratic. At the end of the summer he went back t o medical school. In 1962 John Charnock came, and I gave him Alan’s problem. After six months the experiments began t o work in a useful way and John sent in an abstract t o the Federation Meetings for the spring of 1963.’’ Unfortunately for me he got a nice job in Australia starting in January of that year and now I had to repeat and confirm the experiments, because I would be presenting the paper for him. I really wasn’t doing it right and asked Rollo if I should drop the whole business. He said not to get married t o a system that doesn’t work. Fortunately, on the Saturday before the meeting it did work again, and I presented it as if it were real. I wrote t o John that he could write up the material for publication, which fortunately he did promptly,’’? 2o since it turned out that Wayne Albers” was working on the same problem at the same time. In May, Alan visited me t o find out why I wasn’t moving. “Perhaps AMP would control the erratic back- ground?” No, it didn’t. In the summer of 1963, Amar Sen came back from India t o work with me again and took up the problem. The results of his first experiment were as erratic as any, but in the second experiment we added un- labeled ATP to the acid, which stopped the reaction. This was exactly what was needed and for the next few months he was doing a publishable experiment almost every day. The problem was t o show that the phosphorylated material was functionally related t o the sodium and potassium ATPase activity. Just when we thought we were finished, Dan Tosteson pointed out a lack of direct

Post: Discovery of Na, K-ATPase 11

evidence for acceleration of dephosphorylation by potassium. Fortunately the necessary transient experiments worked. That paper came out in 1965” along with independent support from Elwood and Ahmed and J ~ d a h . * ~ Jens was skeptical (and still is) that the form of the phosphoenzyme detected in the presence of sodium ion alone is necessarily the active form when sodium and potassium ions are present together in the system. The active site is another story.

References

1. LIBET, B. 1948. Fed. Proc. 7: 72. 2. HESS, H. H. & A. POPE. 1957. Fed. Proc. 16: 196. 3. SKOU, J. C. 1957. Biochim. Biophys. Acta 23: 394-401. 4. SCHATZMANN, H.-J. 1953. Helv. Physiol. Acta 11: 346-354. 5. MAIZELS, M. 1954. Symp. SOC. Exp. Biol. 8: 202-227. 6. HARRIS, E. J. 1954. Symp. SOC. Exp. Biol. 8: 228-241. 7. GLYNN, 1.M. 1956. J. Physiol. 134: 278-310. 8. POST, R. L. & P. C. JOLLY. 1957. Biochim. Biophys. Acta 25: 118-128. 9. DUNHAM, E. T. 1957. Fed.Proc. 16: 33.

10. DUNHAM, E. T. 1957. Physiologist 1: 23. 11. WHITTAM, R. 1958. J. Physiol. 140: 479-497. 12. POST,R. L. 1959. Fed.Proc. 18: 121. 13. POST, R. L., C. R. MERRITT, C. R. KINSOLVING & C. D. ALBRIGHT. 1960. J. Biol.

14. SKOU, J. C. 1960. Biochim. Biophys. Acta 42: 6-23. 15. HOKIN, L. E. & M. R. HOKIN. 1960. J . Gen. Physiol. 44: 61-85. 16. BADER, H., H. E. MORGAN, R. L. POST & C. R. PARK. 1961. Pflugers Arch.

17. POST, R. L. & A. S. ROSENTHAL. 1962. J. Gen. Physiol. 45: 614A. 18. CHARNOCK, J. S., A. S. ROSENTHAL & R. L. POST. 1963. Fed. Proc. 22: 212. 19. CHARNOCK, J . S. & R. L. POST. 1963. Nature 199: 910-911. 20. CHARNOCK, J . S., A. S. ROSENTHAL & R. L. POST. 1963. Australian J . Exp. Biol.

21. ALBERS, R. W., S. FAHN & G. J . KOVAL. 1963. Proc. Nat. Acad. Sci. USA

22. POST, R. L., A. K. SEN & A . S. ROSENTHAL. 1965. J. Biol. Chem. 240: 1437-1445. 23. GIBBS, R., P. M. RODDY 6t E. TITUS. 1965. J. Biol. Chem. 240: 2181-2187. 24. AHMED, K. & J. D. JUDAH. 1965. Biochim. Biophys. Acta 104: 112-120.

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