Abstract
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+.
Highlights
ATPase purified from the rectal gland of Squalus acanthias
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+
Potassium-potassium exchange similar to that observed in the red cell membrane and attributed to the Na+-K+ pump was observed when vesicles were prepared in the absence of Na+
Summary
BBRb+ were purchased from New England Nuclear Corp. Ouabain and cholic acid were purchased from Aldrich Chemical Co.; egg lecithin (“highly purified”), from Schwa&Mann; valinomycin, Tris-ATP, Na,CTP, and choline chloride, from Sigma; Na,ATP and NaUTP, from P-L Biochemicals. Five-tenths or 1 ml of vesicles prepared in the usual dialysis buffer was preincubated at 25” for 30 min with added 5 mM MgCl,, 10 FM CaCl,, and 2 x lo-’ M ouabain. Correction for nonenzymatic breakdown of ATP was made with two different controls: (a) radioactive ATP in the usual incubation medium without vesicles and (b) vesicles suspended in the dialysis buffer with added nonradioactive ATP. Both controls were preincubated and incubated as described above. After correction for nonenzymatic hydrolysis of ATP, this percentage was used to calculate ATPase activity and expressed as micromoles of P, per ml of vesicles per 10 min of incubation at 25”
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