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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
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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.]
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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.