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.

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+.     

Ouabain binding to phospholipid-dependent adenosine triphosphatase.

The role of phospholipid in the binding of ouabain to the (Na+ + K+)-dependent adenosine triphosphatase was studied. Enzyme preparations obtained from rabbit kidney were treated with Lubrol WX to remove the phospholipid component essential for ATPase activity. Reconstituted enzyme samples were prepared by the addition of phosphatidylserine and sedimentation of an enzymically active lipid-protein complex. The binding of ouabain to both kinds of preparations was measured under equilibrium conditions with the use of 3H-labelled ouabain and initial ouabain concentrations in the range 0.01-1 micrometer. The main findings were: (i) (Mg2+ + Pi) promoted binding of significant quantities of ouabain only to the reconstituted enzyme; (ii) the absence of added Na+, (Mg2+ + ATP) similarly promoted binding only to the reconstituted samples; (iii) the addition of Na+ in the presence of (Mg2+ + ATP) increased the amount of ouabain bound to the reconstituted enzyme when the ouabain concentration was below about 0.1 micrometer, but it had no effect when the ouabain concentration was about 1 micrometer; (iv) (Mg2+ + ATP) induced ouabain binding to the depleted enzyme only when Na+ was also added; (v) the amount of ouabain bound to both depleted and reconstituted enzymes was the same in the presence of (Mg2+ + ATP + Na+); (vi) the reconstituted enzyme appeared to have a greater affinity for Na+ than did the depleted enzyme.     

Organ secificity of rat sodium- and potassium-activated adenosine triphosphatase.

We compared several Na,K-ATPase preparations from various organs of the rat. The brain Na,K-ATPase differed from the enzymes of other organs in its pH dependence and responses to ouabain and N-ethylmaleimide in spite of similarities in the kinetic parameters of activation by Na+, K+, Mg2+, and ATP. The optimum pH of the brain MaI-enzyme was at 7.4 to 7.5 at 37 degrees D. The Lubrol extract of this brain enzyme preparation showed a lower optimum oH of 6.6. When the Lubrol extract of the brain was fractionated wtih (NH4)2SO4, the activity of the precipitate in the neutral pH region was restored. On the other hand, the optimum pH of the kidney NaI-enzyme was slightly affected by Lubrol and ammonium sulfate treatments (pH 7.5 leads to 7.3). The brain enzyme (K 1/2 = 0.9 microM) showed about 100-fold higher sensitivity to ouabain than the enzymes from other organs (I 1/2 = 100 microM) in the presence of 120 mM Na+ and 10 mM K+. In a Hill plot of the ouabain inhibition, the former failed to give a linear relationship, while the latter gave a straight line with a Hill coefficient of 1.0. The effect of K4 on the brain enzyme-ouabain interaction led us to consider that the brain enzyme might have two components as regards ouabain affinity, high and low affinity components. The time course of N-ethylmaleimide inhibition of the brain enzyme was rapid and biphasic, while the kidney enzyme showed only a slow phase following pseudo-first order kinetics. ATP protected the kidney enzyme activity completely agai,st N-ethylmaleimide inhibition, but the protection of the brain enzyme activity by ATP was only partial. We divided rat Na,K-ATPases into two groups, the brain type, which is restricted to the central nervous system, and the kidney type, which is found in most organs.     

Formation of adenosine triphosphate from Pi and adenosine diphosphate by purified Ca-2+-adenosine triphosphatase.

Ca-2+-ATPase purified from sarcoplasmic reticulum of rabbit muscle forms a phsophoeznyme when exposed to inorganic phosphate in the presence of Mg-2+. On addition of ADP and Ca-2+ virtually all of the phosphate bound to the enzyme is transferred to form ATP. It has been shown previously and confirmed by us that (a) the purified ATPase contains one major polypeptide and about 30% phospholipids; (b) on removal of residual detergent by passage through Sephadex the enzyme forms vesicular membranes; and (c) these vesicles are leaky and incapable of accumulating Ca-2+. Our findings therefore indicate that we have observed ATP generation from ADP and P-i without the formation of an ion gradient across a membrane. We propose that the energy derived from ion-protein interaction drives the formation of ATP.     

Binding of divalent cation to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase.

In order to study the action of the divalent cation which is essential for phosphorylation of sodium- and potassium-transport adenosine triphosphatase, magnesium ion, the normal ligand, was replaced with calcium ion, which had properties diffeerent from those of Mg2+, Mn2+, Fe2+, Co2+, Ni2+, or Zn2+. Phosphorylation of the enzyme from ATP at pH 7.4 in the presence of Na+ and Ca2+ yielded a Ca.phosphoenzyme (60% of the maximal level) with a normal rate of dephosphorylation following a chase with unlabeled Ca.ATP (PK = 0.092S-1 at 0 degrees C). In contrast, after a chase by a chelator, namely ethylenediaminetetraacetic acid, 1,2-cyclohexylenedinitrilotetraacetic acid, or ethylene glycol bis-(beta-aminoethyl ether)N,N'-tetraacetic acid, dephosphorylation slowed within 5 s and half of the initial phosphoenzyme remained with a stability about 5-fold greater than normal. Three states of the phosphoenzyme were distinguished according to their relative sensitivity to ADP or to K+ added during a chase. Normally prepared Mg.phosphoenzyme was sensitive to K+ but not to ADP; Ca.phosphoenzyme was sensitive either to ADP or to K+; and the stabilized phosphoenzyme prepared from Ca.phosphoenzyme by addition of a chelator was sensitive neither to ADP nor to K+ nor to both together. Addition of Ca2+ to the stabilized phosphoenzyme restored the reactivity to that of Ca.phosphoenzyme. Addition of Mg2+ to the stabilized phosphoenzyme changed the reactivity to that of Mg.phosphoenzyme. Therefore, this unreactive, stabilized state of the phosphoenzyme appeared to be a divalent cation-free phosphoenzyme. With respect to sensitivity to ouabain, Ca.phosphoenzyme was as sensitive as Mg.phosphoenzyme but calcium-free phosphoenzyme was much less sensitive. It was concluded that the divalent cation required for phosphorylation normally remains tightly bound to the phosphoenzyme and is required for normal reactivity. Calcium ion was almost unique in dissociating relatively easily from the phosphoenzyme. Strontium ion appeared to act similarly to Ca2+.     

Inhibition of chloroplast adenosine triphosphatase activity by adenosine triphosphatase inhibitor from beef heart mitochondria.

A new amino-acid sequence is proposed for silk fibroin peptide Cp, after automatic Edman degradation studies. The proposed sequence is: Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Gly-(Ser-Gly-(Ala-Gly)_n)₈ Tyr, where n is usually 2.     

Transient state in the phosphorylation of sodium- and potassium- transport adenosine triphosphatase by adenosine triphosphate

The rate of phosphorylation of sodium and potassium ion-transport adenosine triphosphatase by 10 microM [gamma-32P]ATP was much slower with Ca2+ than with Mg2+ (0.13-10 mM) in the presence of 16 to 960 mM Na+ at 0 degrees C and pH 7.4. In the presence of a fixed concentration of Mg2+ or Ca2+, the rate became slower with increasing Na+ concentration. When the Na+ concentration was fixed, the rate became slower with decreasing divalent cation concentration. Sodium ions appear to antagonize the divalent cation in the phosphorylation to slow its rate. In the presence of 1 mM Ca2+ and 126 or 270 mM Na+, the rate was slow enough to permit the manual addition of a chasing solution at various times before the phosphorylation reached the steady state. Therefore, we studied the time-dependent change of the sensitivity to ADP or to K+ of the phosphoenzyme by a chase with unlabeled ATP containing ADP or K+ during the time range from the transient to the steady state of the phosphorylation. The ADP sensitivity decreased and the K+ sensitivity increased with the progress of the phosphorylation. With 270 mM Na+, the phosphoenzyme found at 1 s, when its amount was 5.5% of the maximum level, was virtually completely sensitive to ADP. Under these conditions, it was concluded that the form of the phosphoenzyme initially produced from the enzyme.ATP complex has ADP sensitivity and that the phosphoenzyme acquires K+ sensitivity later. The initially produced ADP-sensitive phosphoenzyme partially lost its normal instability and sensitivity upon adding a chelating agent, probably because of dissociation of a divalent cation from the phosphoenzyme.