Classic Experiment

15.1

Stumbling Upon Active Transport

n the mid-1950s, Jens Skou was a young physician researching the effects
of local anesthetics on isolated lipid bilayers. He needed an easily assayed

membrane-associated enzyme to use as a marker in his studies. What he discovered was an enzyme critical to the maintenance of membrane potential,
the Na!/K! 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 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 Skous 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 purposes, 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 proteins 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 reactionNa!, 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 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
15.1). 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
Skous 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
ATPase, he took no precautions to avoid the use of buffers

40

K 20 m M / I

Mg 6 mM / I

N a Cl 20 m M / I

20

30

M/I
K 350 m
K 3 mM/I

gP

gP

M/I
K 200 m

N a Cl 40 m M / I

30

K 120 m M / I

20

Mg 6 mM / I
N a Cl 10 m M / I

10

10

N a Cl 3 m M / I
K 0 mM/I

N a Cl 0 m M / I
0

20

40

60
K Cl m M / I

80

100

120

50

100
N a Cl m M / I

150

Fig ure 15.1

D e monstration of the dependence of the Na!/K! ATPase activity on the concentration of each ion. The graph on the left sho w s that
increasing K! leads to an inhibition of the ATPase activity. The graph on the right sho w s that with increasing Na!, the enzym e activity
increases up to a peak and then levels out. This graph also de monstrates the dependence of the activity on lo w levels of K!. [Adapted
from J. Skou, 1957, Bioche m. Biophys. Acta 23:394.]

200

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.