JBC, Vol. 250, Issue 16, 6296-6303, Aug, 1975
Active potassium transport coupled to active sodium transport in vesicles reconstituted from purified sodium and potassium ion-activated adenosine triphosphatase from the rectal gland of Squalus acanthias
S. Hilden and L. E. Hokin
Vesicles containing a purified shark rectal gland (sodium +
potassium)-activated adenosine triphosphatase-(NaK ATPase) were prepared by
dialyzing for 2 days egg lecithin, cholate, and the NaK ATPase purified
from the rectal gland of Squalus acanthias. These vesicles were capable of
both Na+ and K+ transport. Studies of K+ transport were made by measuring
the ATP-stimulated transport outward of 42K+ or 86Rb+. Vesicles were
preloaded with isotope by equilibration at 4 degrees for 1 to 3 days.
Transport of 42K+ or 86Rb+ was initiated by addition of MgATP to the
vesicles. The ATP-dependent exit of either isotope was the same.
Experiments are presented which show that this loss of isotope was not due
to changes in ion binding but rather due to a loss in the amount of ion
trapped in the vesicular volume. The transport of K+ was dependent on
external Mg2+. CTP was almost as effective as ATP in stimulating K+
transport, while UTP was relatively ineffective. These effects of
nucleotides parallel their effects on Na+ accumulation and their
effectiveness as substrates for the enzyme. Potassium transport was
inhibited by ouabain and required the presence of Na+. The following
asymmetries were seen: (a) addition of external Mg2+ supported K+
transport; (b) ouabain inhibited K+ transport only if it was present inside
the vesicles; (c) addition of external Na+ to the vesicles stimulated K+
transport. External Li+ was ineffective as a Na+ substitute. The specific
requirement of external Na+ for K+ transport indicates that K+ exit is
coupled to Na+ entry. Changes in the internal vesicular ion concentrations
were studied with vesicles prepared in 20 mM NaCl and 50 mM KCl. After 1
hour of transport at 25 degrees, a typical Na+ concentration in the
vesicles in the presence of ATP was 72 mM. A typical K+ concentration in
the vesicles was 10 mM as measured with 42K+ or 6 mM as measured with
86Rb+. The following relationships have been calculated for Na+ transport,
K+ transport and ATP hydrolysis: Na+/ATP = 1.42, K+/ATP =1.04, and Na+/K+ =
1.43. The ratio of 2.8 Na+ transported in to 2 K+ transported out is very
close to the value reported for the red cell membrane. Potassium-potassium
exchange similar to that observed in the red cell membrane and attributed
to the Na+-K+ pump (stimulated by ATP and orthophosphate and inhibited by
ouabain) was observed when vesicles were prepared in the absence of Na+.
The results reported in this paper prove that the shark rectal gland NaK
ATPase, which is 90 to 95% pure, is the isolated pump for the coupled
transports of Na+ and K+.
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