Jens Christian Skou

The influence of some cations on an adenosine triphosphatase from peripheral nerves.

Leg nerves from the shore crab (Carcinus maenas) contain an adenosine triphosphatase which is located in the submicroscopic particles. The influence of sodium, potassium, magnesium and calcium ions on this enzyme has been investigated. The presence of magnesium ions is an obligatory requirement for the activity of the enzyme. Sodium ions increase the activity when magnesium ions are present. Potassium ions increase the activity when the system contains both magnesium and sodium ions. Potassium ions in high concentration inhibit that part of the activity which is due to Na+, while the activity due to Mg++ is not affected. Calcium ions inhibit the enzyme under all conditions. When Mg++ or Mg++ + Na+ are present in the system, the optimum magnesium concentration is equal to the concentration of ATP. If potassium ions are added, the optimum magnesium concentration is doubled. If calcium ions are also added, the optimum magnesium concentration becomes still higher, and it increases with the calcium concentration. A majority of these observations may be explained by assuming (a) that the substrate most readily attacked by the enzyme is sodium-magnesium-ATP, (b) that potassium ions stimulate the enzyme directly, and (c) that an increase in the concentration of potassium ions leads to a displacement of sodium ions from the substrate and accordingly to an inhibition of the reaction. If the system contains the four cations in concentrations roughly equal to those in the crab-nerve axoplasm, an increase in the sodium concentration as well as a decrease in the potassium concentration will lead to an intensification of the enzyme activity. This observation, as well as some other characteristics of the system, suggest that the adenosine triphosphatase studied here may be involved in the active extrusion of sodium from the nerve fibre.

Further investigations on a Mg++ + Na+-activated adenosintriphosphatase, possibly related to the active, linked transport of Na+ and K+ across the nerve membrane

The sodium activated ATPase prepared from crab nerve which was described in a previous paper1 has been subjected to a more detailed study of the relation between enzyme, substrate, and a number of monovalent and divalent cations. It has also been investigated whether the enzyme catalyses an ATP-Pi or an ATP-ADP exchange reaction, and whether these reactions are cation-dependent. Finally, the effect of g-strophanthin on the ATPase activity and the exchange reaction has been investigated. The simplest scheme by which the experimental results can be explained is as follows: (1)E + ATP + 2 Mg + nNa + mK ⇌ EnNamKMg2ATP (2)EnNamKMg2ATP ⇌ EnNamK ∼ 2 Mg + ADP (3)EnNamK ∼ P → E + Pi + nNa + mK where n and m are unknown numbers, and where EnNamKMg2ATP symbolises an enzyme-substrate-ion complex in which Mg++ is part of the enzyme-substrate complex and in which Na+ and K+ are attached to two different sites on the enzyme; these sites show high affinities for the respective ions. Phosphorylation of the enzyme, steps (1) + (2), will take place also when Mg++ is the only cation present in the system, but under these circumstances the release of inorganic phosphate, step (3), is very slow. The rate of step (3) is increased by the addition of Na++ to the system, but not by the addition of K+. In the presence of Mg++ + Na+, however, addition of K+ leads to a considerable increase of the rate of step (3). The binding of Na+ + K+ to the enzyme takes place during the attachment of ATP to the enzyme, i.e. during step (1). G-strophanthin has no effect on the phosphorylation of the enzyme, steps (1) + (2). But g-strophanthin inhibits the activating effect of Na+ and of Na+ + K+ on step (3), presumably by interfering with the binding of the cations to the enzyme during step (1). The observations lend further support to the suggestion made previously that this enzyme is involved in the active, linked transport of Na+ and K+ across the nerve membrane.

The Na,K-ATPase

The energy dependent exchange of cytoplasmic Na+ for extracellular K+ in mammalian cells is due to a membrane bound enzyme system, the Na,K-ATPase. The exchange sustains a gradient for Na+ into and for K+ out of the cell, and this is used as an energy source for creation of the membrane potential, for its de- and repolarisation, for regulation of cytoplasmic ionic composition and for transepithelial transport. The Na,K-ATPase consists of two membrane spanning polypeptides, an α-subunit of 112-kD and a β-subunit, which is a glycoprotein of 35-kD. The catalytic properties are associated with the α-subunit, which has the binding domain for ATP and the cations. In the review, attention will be given to the biochemical characterization of the reaction mechanism underlying the coupling between hydrolysis of the substate ATP and transport of Na+ and K+.

Preparation from mammallian brain and kidney of the enzyme system involved in active transport of Na+ and K+

Abstract A procedure is described for the preparation from mammalian brain and kidney of an enzyme system which hydrolyses ATP to ADP and P 1 , and which requires both Mg 2+ , Na + and K + for full activity. The enzyme system prepared behaves qualitatively in its relations to cations and to g-strophanthin as the Mg 2+ + Na + + K + -activated, ATP-hydrolysing enzyme system previously isolated from peripheral crab nerves and red blood cells. The finding of this enzyme system in tissues from brain and kidney in which there is an active, linked transport of Na + and K + lends support to the assumption made on basis of the experiments with the enzyme system from peripheral nerves and from red blood-cell membranes that this enzyme system is involved in the active, linked transport of Na + and K + across the cell membrane.

The (Na++K+) activated enzyme system and its relationship to transport of sodium and potassium.

It seems to be the membrane bound (Na + +K + )-activated enzyme system which transforms the energy from a hydrolysis of ATP into a vectorial movement of sodium out and potassium into the cell against electrochemical gradients, i.e. this systems seems to be the transport system for sodium and potassium (see, for example, review by Skou, 1972; Hokin & Dahl, 1972).

Purification and characterization of (Na+ + K+)-ATPase. I. The influence of detergents on the activity of (Na+ + K+)-ATPase in preparations from the outer medulla of rabbit kidney

1. 1. Incubation of a microsomal fraction from the outer medulla of rabbit kidney with deoxycholate rapidly increases the specific activity of (Na+ + K+)-ATPase (ouabain sensitive; ATP phosphohydrolase, EC 3.6.1.3) from 45 to 270 μmoles Pi per mg protein per h if the conditions for incubation are optimal with respect to temperature, pH and concentrations of protein and detergent. A procedure for evaluation of the conditions for maximum activation by deoxycholate is described. 2. 2. Measurements of the surface tension show that the marked influence of changes in the pH on the activation by deoxycholate is due to changes in the capillary activity of deoxycholate. The optimum concentrations of deoxycholate, sodium dodecyl sulfate, and Lubrol-14 for activation of (Na+ + K+)-ATPase are different, but it is common for the three detergents that maximal activation is obtained when the critical micelle concentration is reached. 3. 3. Fractionation by zonal centrifugation shows that the (Na+ + K+)-ATPase remains associated with membranes after activation by deoxycholate, whereas inactive protein is removed and solubilized by the detergent. The treatment with deoxycholate reduces the content of Mg2+-ATPase (ouabain insensitive; ATP phosphohydrolase, EC 3.6.1.3) in the fractions which contain the (Na+ + K+)-ATPase. 4. 4. Tracer studies show that the activation of (Na+ + K+)-ATPase is not associated with binding of significant amounts of deoxycholate to the membranes. The activation does not change the molecular activity of (Na+ + K+)ATPase. 5. 5. The data suggest that the activation of (Na+ + K+)-ATPase is due to exposure of latent enzyme sites in the preparation. The removal of protein may lead to opening of vesicular structures resulting in free access of substrate and activators to their respective sites on the membrane.

The effect of sulphydryl-blocking reagents and of urea on the (Na+ + K+)-activated enzyme system

Summary Incubation of the (Na + + K + )-activated ATP-hydrolysing enzyme system with SH-blocking reagents decreases its activity. With SH-blocking reagents such as maleimide, N -ethylmaleimide and 1 fluoro-2,4-dinitrobenzene the activity with Mg 2+ decreases more than the activity with Mg 2+ + Na + + K + , i.e. the (Mg 2+ + Na + + K + )/Mg 2+ activity ratio is increased. ATP in the incubation medium protects against the decrease in activity with Mg 2+ + Na + + K + but not against the decrease in activity with Mg 2+ ; incubation with ATP therefore gives a further increase in activity ratio. The same effect is obtained by incubation with urea and ATP. Na + in the incubation medium gives a further decrease in activity with Mg 2+ + Na + + K + , whereas K + has less effect. With ATP, K + decreases the activity more than Na + , and the concentrations of the cations which give half the maximum decrease are lower than without ATP. When G-strophanthin is present together with ATP, Na + gives a further decrease in activity, whereas there is no change in the effect with K + . The experiments suggest (a) that ATP has an effect on the structure of the enzyme system and on the affinities of the enzyme system for Na + and K + ; and (b) that the activity with Mg 2+ and with Mg 2+ + Na + + K + is due to the same enzyme. When activated by Mg 2+ the enzyme system is “locked” in a structural configuration in which ATP cannot induce the change in affinities which leads to the activation by Na + + K + .

Preparation of highly active (Na+ + K+)-ATPase from the outer medulla of rabbit kidney

Abstract It is described how (Na + + K + )-ATPase from the outer medulla of rabbit kidney was purified to a specific activity of 881 ± 25 moles Pi/mg protein per hr at 37 C. The procedure consists of treatment of a microsomal fraction with deoxycholate and subsequent fractionation by differential centrifugation and sucrose gradient centrifugation. The “purity” of the preparation was estimated to be 31–61%.

Preparation of membrane-bound and of solubilized (Na+ + K+)-ATPase from rectal glands of Squalus acanthias. The effect of preparative procedures on purity, specific and molar activity.

Abstract A simple method is described for the routine preparation of larger quantities of purified (Na + + K + )-ATPase from the rectal glands from Squalus acanthias and for solubilization of the purified enzyme in a highly active form. Microsomes are prepared by homogenization of the glands in a Waring Blendor followed by differential centrifugation. They keep their activity for years when stored at −70°C. Based on the earlier method (Jorgensen, P.L. and Skou, J.C. (1971) Biochim. Biophys. Acta 233, 366–380), enzyme with a specific activity of 1500 μmol Pi · mg−1 protein · h−1 was prepared by treating the microsomes with low concentrations of deoxycholate followed by differential centrifugation, and with a yield of 70% of the activity in the deoxycholate-treated microsomes. The purified enzyme can be dissolved in deoxycholate in the presence of cholesterol, and after a single centrifugation to remove undissolved enzyme, the specific activity of the solubilized enzyme is increased to 2400–2600 μmol Pi · mg−1 protein · h−1. Precipitation of the solubilized enzyme leads to a decrease in specific activity to 1500 μmol Pi · mg−1 protein · h−1 and to a decrease in molar activity.

Eosin, a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase

Abstract (1) Eosin bound to the ( Na + + K + )- ATPase in the presence of K+ has practically the same fluorescence as eosin without enzyme while in the presence of Na+ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K+ concentration but with a low affinity. With equal concentrations of Na+ and K+ more is bound in the presence of Na+, and the difference between 150 mM Na+ and 150 mM K+ shows one high-affinity eosin binding site per 32P-labelling site ( K D 0.45 μM). With lower concentrations of the cations there are between one and two Na+-dependent high-affinity eosin binding sites per 32P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na+-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na+ (+Mg2+). (4) Eosin, like ATP, increases the Na+ relative to K+ affinity ( Na + + K + = 150 mM ) for Na+ activation of hydrolysis and for Na+ protection against inactivation by N- ethylmaleimide . (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na+ opens a high-affinity site for ATP while K+ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na+ relative to K+ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of Na + + K + the system reacts with two different ATP molecules.