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8945d_017-020 6/18/03 11:07 AM Page 17 mac85 Mac 85:1st shift: 1268_tm:8945d: Classic Experiment 7.1 STUMBLING UPON ACTIVE TRANSPORT n the mid-1950s Jens Skou was a young physician researching the effects of B local anesthetics on isolated lipid bilayers. He needed an easily assayed mem- brane-associated enzyme to use as a marker in his studies. What he discovered was an enzyme critical to the maintenance of membrane potential, the Na1/K1 ATPase, a molecular pump that catalyzes active transport. Background During the 1950s many researchers around the world were actively investigating the physiology of the cell membrane, which plays a role in a number of biological processes. It was well known that the concentration of many ions differs inside and outside the cell. For example, the cell maintains a lower intracellular sodium (Na) concentration and higher intracellular potassium (K) concentration than is found outside the cell. Somehow the membrane can regulate intracellular salt concentrations. Additionally, movement of ions across cell membranes had been observed, suggesting that some sort of transport is system is present. To maintain normal intracellular Na and K concentrations, the transport system could not rely on passive diffusion because both ions must move across the membrane against their concentration gradients. This energy-requiring process was termed active transport. At the time of Skou’s experiments, the mechanism of active transport was still unclear. Surprisingly, Skou had no intention of helping to clarify the field. He found the Na/K ATPase completely by accident in his search for an abundant, easily measured enzyme activity associated with lipid membranes. A recent study had shown that membranes derived from squid axons contained a membrane-associated enzyme that could hydrolyze ATP. Thinking that this would be an ideal enzyme for his pur- poses, Skou set out to isolate such an ATPase from a more readily available source, crab leg neurons. It was during his characterization of this enzyme that he discovered the protein’s function. The Experiment Since the original goal of his study was to characterize the ATPase for use in subsequent studies, Skou wanted to know under what experimental condition its activity was both robust and reproducible. As often is the case with the characterization of a new enzyme, this requires careful titration of the various components of the reaction. Before this can be done, one must be sure the system is free from outside sources of contamination. In order to study the influence of various cations, including three that are critical for the reaction—Na, K, and Mg2 —Skou had to make sure that no contaminating ions were brought into the reaction from another source. Therefore, all buffers used in the purification of the enzyme were prepared from salts that did not contain these cations. An additional source of contaminating cations was the ATP substrate, which contains three phosphate groups, giving it an overall negative charge. Because stock solutions of ATP often included a cation to balance the charge, Skou converted the ATP used in his reactions 8945d_017-020 6/18/03 11:07 AM Page 18 mac85 Mac 85:1st shift: 1268_tm:8945d: to the acid form, so that balancing cations would not affect the experiments. Once he had a well-controlled environment, he could characterize the enzyme activity. These precautions were fundamental to his discovery. Skou first showed that his enzyme could indeed catalyze the cleavage of ATP into ADP and inorganic phosphate. He then moved on to look for the optimal conditions for this activity by varying the pH of the reaction, and the concentrations of salts and other cofactors, which bring cations into the reaction. He could easily determine a pH optimum as well as an optimal concentration of Mg2, but optimizing Na and K proved to be more difficult. Regardless of the amount of K added to the reaction, the enzyme was inactive without Na. Similarly, without K, Skou observed only a low-level ATPase activity that did not increase with increasing amounts of Na. These results suggested that the enzyme required both Na and K for optimal activity. To demonstrate that this was the case, Skou performed a series of experiments in which he measured the enzyme activity as he varied both the Na and K concentrations in the reaction (see Figure). Although both cations clearly were required for significant activity, something interesting occurred at high concentrations of each cation. At the optimal concentration of Na and K, the ATPase activity reached a peak. Once at that peak, further increasing the concentration did not affect the ATPase activity. Na thus behaved like a classic enzyme substrate, with increasing input leading to increased activity until a saturating concentration was achieved, at which the activity plateaued. K, on the other hand, behaved differently. When the K concentration was increased beyond the optimum, ATPase activity declined. Thus, while K was required for optimal activity, at high concentrations it inhibited the enzyme. Skou reasoned that the enzyme must have separate binding sites for Na and K. For optimal ATPase activity, both must be filled. However, at high concentrations K could compete for the Na-binding site, leading to enzyme inhibition. He hypothesized that this enzyme was involved in active transport, that is, the pumping of Na out of the cell, coupled to the import of K into the cell. Later studies would prove that this enzyme was indeed the pump that catalyzed active transport. This finding was so exciting that Skou devoted his subsequent research to studying the enzyme, never using it as a marker, as he initially intended. (a) (b) 40 Discussion Skou’s finding that a membrane ATPase used both Na and K as substrates was the first step in understanding active transport on a molecular level. How did Skou know to test both Na and K? In his Nobel lecture in 1997, he explained that in his first attempts at characterizing the 40 K 20 mM / I Mg 6 mM / I K 120 mM/ I K 200 m NaCl 40 mM / I 30 30 K 350 m M/ I M/ I 20 µgP µgP K 3 mM / I NaCl 20 mM / I 20 Mg 6 mM / I NaCl 10 mM / I 10 10 NaCl 3 mM / I K 0 mM / I NaCl 0 mM / I 0 0 20 40 60 KCl mM / I 80 100 120 0 0 50 100 NaCl mM / I 150 200 Demonstration of the dependence of the Na/K ATPase activity on the concentration of each ion. The graph on the left shows that increasing K leads to an inhibition of the ATPase activity. The graph on the right shows that with increasing Na, the enzyme activity increases up to a peak and then levels out. This graph also demonstrates the dependence of the activity on low levels of K. [Adapted from J. Skou, 1957, Biochem. Biophys. Acta 23:394.] 8945d_017-020 6/18/03 11:07 AM Page 19 mac85 Mac 85:1st shift: 1268_tm:8945d: ATPase, he took no precautions to avoid the use of buffers and ATP stock solutions that contained Na and K. Pondering the puzzling and unreproducible results that he obtained led to the realization that contaminating salts might be influencing the reaction. When he repeated the experiments, this time avoiding contamination by Na and K at all stages, he obtained clear-cut reproducible results. The discovery of the Na/K ATPase had an enormous impact on membrane biology, leading to a better understanding of the membrane potential. The generation and disruption of membrane potential forms the basis of many biological processes including neurotransmission and the coupling of chemical and electrical energy. For this fundamental discovery, Skou was awarded the Nobel Prize for Chemistry in 1997.