Peter L. Jørgensen

Purification and characterization of (Na+ + K+)-ATPase III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate

1. n1. A procedure is presented that allows preparation of (Na+ + K+)-ATPase with specific activities of 32–37 units · mg−1protein (units refer to μmoles ATP hydrolysed per min) by incubation of a microsomal fraction with sodium dodecylsulphate and ATP followed by a single zonal centrifugation. n n2. n2. In a technically simple version of this procedure preparations with specific activities of 20–26 units · mg−1 protein are obtained in quantities of 12–20 mg protein by a single centrifugation, in an angle rotor, of microsomal fractions from the outer medulla of rabbit, pig or sheep kidneys. n n3. n3. Binding of ATP and ADP partially protects (Na+ + K+)-ATPase against inactivation by sodium dodceylsulphate. In the incubation with sodium dodecylsulphate and ATP, 80% of the protein in the microsomal fraction is solubilized whereas the (Na+ + K+)-ATPase remains bound to membrane fragments. Tracer analysis shows that sodium dodecylsulphate forms less than 0.5% of the mass of the purified preparations. n n4. n4. Analysis of the protein composition by sodium dodecylsulphate gel electrophoresis and determination of the capacities for binding of ATP and ouabain and for sodium-dependent phosphorylation (Jorgensen, P. L. (1974) Biochim. Biophys. Acta 356, 53–67 show that the procedures lead to a true purification of the enzyme.

Structural basis for E1-E2 conformational transitions in Na,K-pump and Ca-pump proteins.

The primary active transport of Na +, K +, Ca 2T and H + in eukaryotic cells is driven by ATP-powered cation pumps, the Na,K pump, Ca pump and H,K pump. The intermediary steps of the pump reactions and their relationship to cation translocation have been examined in detail, particularly for the Na,K-pump and Ca-pump proteins. A common feature of these pumps is a protein of Mr close to 110,000 that binds ATP and accepts its y-phosphate in a covalent Asp-P bond. There is evidence that both phosphoand dephospho-forms of the protein exist in two major conformational states, E1 and E2, with different affinities and orientation of cation binding sites, and ATP-driven cation pumping is blocked by micromolar concentrations of vanadate [39, 68, 95, 96, 116, 132]. A wealth of structural information about the cation pump proteins appeared recently after identification of mRNA and sequencing of cloned cDNA. Several isoforms of the genes of o~-subunit of Na,K pump were identified in the human and rat genomes [147, 182] and the sequences ofa-subunit [110, 111,145, I46, 178,181] and B-subunit [22, 112, 140, 146, 179] were deduced from cDNA of mRNA from a variety of tissues in piscine and mammalian species. A similar intensity of work in adjacent fields provided the sequences of slow and fast twitch Ca pump from sarcoplasmic reticulum [19, 121] and H,K pump from stomach mucosa [180]. In addition the gene sequences are available for K pumps [82, 185] and H pumps [1, 177] from microorganisms. As paradigm for interpretation of this information the combined X-ray crystallographic structure at high resolution and

Purification and characterization of (Na+, K+)-ATPase. V. Conformational changes in the enzyme. Transitions between the Na-form and the K-form studied with tryptic digestion as a tool

1. Purified (Na+, K+)-ATPase consisting of membrane fragments was digested with trypsin. The time course of enzyme inactivation was related to the electrophoretic pattern of native and cleaved proteins remaining in the membrane. 2. Differences in both the inactivation kinetics and the cleavage of the large chain (mol. wt 98 000) allow distinction of two patterns of tryptic digestion of (Na+, K+)-ATPase seen with Na+ or K+ in the medium. 3. With K+, the inactivation of (Na+, K+)-ATPase is linear with time in semilogarithmic plots and the activity is lost in parallel with cleavage of the large chain to fragments with molecular weights 58 000 and 48 000. 4. With Na+, the inactivation curves are biphasic. In the initial phase of rapid inactivation, 50% of the activity is lost with minor changes in the composition of the large chain. In the final phase, the large chain is cleaved at a low rate to a fragment with a molecular weight of 78 000. 5. It is concluded that the regions of the large chain exposed in the presence of K+ are distinct from the regions exposed in presence of Na+ and that two conformations of (Na+, K+)-ATPase can be sensed with trypsin, a (t)K-form and a (t)Na-form. 6. Reaction of the (t)K-form with ATP cause transition to the (t)Na-form. Relatively high concentrations of ATP are required and Mg2+ is not necessary. Phosphorylation of (Na+, K+)-ATPase is accompanied by transition from the (t)Na-form to the (t)K-form. Previous kinetic data suggest that these conformational changes are accompanied by shifts in the affinities of the enzyme for Na+ and K+.

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. n1. 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. n n2. n2. 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. n n3. n3. 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. n n4. n4. 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. n n5. n5. 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.

Purification and characterization of (Na+ + K+)-ATPase IV. Estimation of the purity and of the molecular weight and polypeptide content per enzyme unit in preparations from the outer medulla of rabbit kidney

Abstract 1. 1. The purpose has been to examine the purity of the highly active preparation of ( Na + + K + )- ATPase described recently (Jorgensen, P. L. (1974) Biochim. Biophys. Acta, 356, 36–52) and to determine the molecular weight and the polypeptide content per enzyme unit. 2. 2. The concentration of sites for binding of ATP and ouabain and for sodium-dependent phosphorylation is proportional to the activity of ( Na + + K + )- ATPase in a range from 15 to 37 μmoles Pi · min−1 · mg−1 protein. The maximum content of the large phosphorylated polypeptide M r = 96 00 ) is 72% of the total protein and the content of small polypeptide ( M r = 35 000–57 000 ) is close to 1 mole per mole large chain. 3. 3. The receptor capacities (pmoles · (μmoles Pi · min−1)−1) are 105 ± 13 for ATP, 112 ± 5 for ouabain and 221 ± 12 for sodium-dependent phosphorylation. The maximum weight per site is 250 000 g protein per mole ATP bound, 278 000 g per mole ouabain bound and 132 000 g per mole phosphate incorporated. 4. 4. The results suggest that the enzyme unit binding one molecule of ATP or ouabain contains two chains of large polypeptide and that both chains are phosphorylation from ATP in the presence of Mg2+ and Na+. 5. 5. The purity of the preparation with respect to protein is close to the maximum if the enzyme contains one mole of small chain per mole of large chain. The capacities for binding of ATP or ouabain are higher chain per mole of large chain. The capacities for binding of ATP or ouabain are higher than or equal to the highest values reported before. The catalytic functions of ( Na + + K + )- ATPase are well preserved since the molar activity is high and all large chains in the preparation can be phosphorylated from ATP.

Structure–function relationships of Na+, K+, ATP, or Mg2+ binding and energy transduction in Na,K-ATPase

Abstract The focus of this article is on progress in establishing structure–function relationships through site-directed mutagenesis and direct binding assay of Tl + , Rb + , K + , Na + , Mg 2+ or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E 2 [2K] or E 1 P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu 327 in transmembrane segment M4, Glu 779 in M5, Asp 804 and Asp 808 in M6 are essential for tight binding of K + and Na + . Asn 324 and Glu 327 in M4, Thr 774 , Asn 776 , and Glu 779 in 771-YTLTSNIPEITP of M5 contribute to Na + /K + selectivity. Free ATP binding identifies Arg 544 as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp 710 or Asn 713 do not interfere with free ATP binding. Asp 710 is essential and Asn 713 is important for coordination of Mg 2+ in the E 1 P[3Na] complex, but they do not contribute to Mg 2+ binding in the E 2 P-ouabain complex. Transition to the E 2 P form involves a shift of Mg 2+ coordination away from Asp 710 and Asn 713 and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp 369 .

Regulation of the (Na+ + K+)-activated ATP hydrolyzing enzyme system in rat kidney. I. The effect of adrenalectomy and the supply of sodium on the enzyme system

Abstract 1. 1.Activity of the (Na+ + K+)-activated ATP hydrolyzing enzyme was measured in the subcellular fractions from normal and adrenalectomized rat kidneys before and after incubation with sodium deoxycholate. 2. 2.By incubation with deoxycholate + EDTA activity of the (Na+ + K+)-activated enzyme increased about 3-fold, while the activity with Mg2+ alone remained unchanged. Using this procedure total activity of the (Na+ + K+)-activated enzyme could be measured in a reproducible way. 3. 3.A 35 to 46% reduction in activity of the (Na+ + K+)-activated enzyme was observed after adrenalectomy in preparations treated with deoxycholate + EDTA, while the changes in the fresh preparations were small and located in the microsomal fractions. Thus part of the decrease in activity could be ascribed to a change in response to deoxycholate of the enzyme in kidneys from adrenalectomized rats. 4. 4.The decrease in amount of the (Na+ + K+)-activated enzyme during developing adrenal insufficiency corresponded to the rate of change of plasma concentrations of sodium and potassium. 5. 5.A high sodium diet could postpone and partly prevent the decrease in activity of the enzyme during developing adrenal insufficiency. This finding suggests that the enzyme is not primarily under the control of the adrenals, and might indicate an effect of sodium on activity levels of the (Na+ + K+-activated enzyme in rat kidney.

Soluble and active renal Na, K-ATPase with maximum protein molecular mass 170,000 +/- 9,000 daltons; formation of larger units by secondary aggregation.

Summary Purified membrane-bound Na,K-ATPase from pig kidney was solubilized with nonionic detergent, dodecyloctaethylenglycol monoether (C 12 E 8 ) as 70–90% active protein units with S 20,w 7.4 ± 0.2 and maximum molecular mass 170,000 ± 9,000 daltons indicating that the soluble complex predominantly consisted of protomeric αβ-units. Inactivation of Na,K-ATPase by excess C 12 E 8 was not related to the aggregation state of the protein. On storage both the soluble Na,K-ATPase and soluble Ca-ATPase (115,000 daltons) from sarcoplasmic reticulum underwent secondary aggregation which may account for previous reports of higher molecular weights.

Purification and characterization of (Na+ + K+)-ATPase. VI. Differential tryptic modification of catalytic functions of the purified enzyme in presence of NaCl and KCl

1. Two distinct patterns of tryptic modification of the catalytic functions of purified (Na+ + K+)-ATPase can be related to the two previously described patterns of enzyme inactivation and cleavage of the large chain seen with NaCl and KCl (Jorgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415). 2. With NaCl, in phase A, the rapid inactivation of 50-55% of the (Na+ + K+)-ATPase activity is associated with loss of 85% of the K+-phosphatase activity and an increase in Na+-ADP-ATP exchange activity to 150% of control. ATP binding and phosphorylation are unchanged and the inactivation may result from cleavage of bonds within the large chain which are involved in dephosphorylation reactions. In phase B with NaCl, ATP binding and phosphorylation are lost slowly in parallel to inactivation of (Na+ + K+)-ATPase and cleavage of the large chain to a fragment with Mr=78 000. 3. With KCl, cleavage of the large chain to almost equal fragments abolish ATP binding and phosphorylation in parallel to the inactivation of (Na+ + K+)-ATPase. An additional split seems required for inactivation of the K+-pNPPase activity. 4. After completion of the digestion in phase A with NaCl a stable preparation can be isolated in which the activity of (Na+ + K+)-ATPase is 40%. ATP binding and phosphorylation are 90%, K+-phosphatase is 15%, and Na+-ADP-ATP exchange is 150% of control. We currently examine if these levels are related to changes in phosphorylation kinetics. 5. The ATP binding area is much more stable to trypsin with NaCl than with KCl, but loss of the binding capacity is in both cases correlated to a distinct cleavage of the large chain. The relationship between the fractional loss of ATP binding and cleavage of the large chain suggests that the nucleotide binding area is confined to one of the two large chains in the protein complex with Mr=270 000 which binds one molecule of ATP. 6. The data also suggest that the phosphatase site is remote from the ATP binding area. It is proposed that the protein complex with Mr=270 000 contains two large chains with different catalytic functions and that each chain forms a cation channel.

Soluble and enzymatically stable (Na++K+)-ATPase from mammalian kidney consisting predominantly of protomer αβ-units: Preparation, assay and reconstitution of active Na+, K+transport

Soluble (Na++K+)-ATPase consisting predominantly of αβ-units with Mr below 170 000 was prepared by incubating pure membrane-bound (Na++K+)-ATPase (35–48 μmol Pi/min per mg protein) from the outer renal medulla with the non-ionic detergent dodecyloctaethyleneglycol monoether (C12E8). (Na++K+)-ATPase and potassium phosphatase remained fully active in the detergent solution at C12E8/protein ratios of 2.5–3, at which 50–70% of the membrane protein was solubilized. The soluble protomeric (Na++K+)-ATPase was reconstituted to Na+, K+ pumps in phospholipid vesicles by the freeze-thaw sonication procedure. Protein solubilization was complete at C12E8/protein ratios of 5–6, at the expense of partial inactivation, but (Na++K+)-ATPase and potassium phosphatase could be reactivated after binding of C12E8 to Bio-Beads SM2. At C12E8/protein ratios higher than 6 the activities were irreversibly lost. Inactivation could be explained by delipidation. It was not due to subunit dissociation since only small changes in sedimentation velocities were seen when the C12E8/protein ratio was increased from 2.9 to 46. As determined immediately after solubilization, S20,w was 7.4 S for the fully active (Na++K+)-ATPase, 7.3 S for the partially active particle, and 6.5 S for the inactive particle at high C12E8/protein ratios. The maximum molecular masses determined by analytical ultracentrifugation were 141 000–170 000 dalton for these protein particles. Secondary aggregation occurred during column chromatography, with formation of enzymatically active (αβ)2-dimers or (αβ)3-trimers with S20,w=10–12 S and apparent molecular masses in the range 273 000–386 000 daltons. This may reflect non-specific time-dependent aggregation of the detergent micelles.