Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. XII. Evidence for interaction of the acyl phosphate at the active site with neighboring groups

The sodium-potassium adenosinetriphosphatase (NaK ATPase), partially purified from beef brain, has been phosphorylated with [γ-³²P]ATP in the presence of Na and Mg and digested with pronase. A single ³²P-labeled peptide spot has been identified on paper electrophoresis, accounting for 60% of the radioactivity in the ³²P-labeled enzyme, the remainder of the radioactivity being [³²P]-orthophosphate resulting from breakdown of the highly labile acyl phosphate during pronase digestion. The ³²P in the pronase peptide was released as [³²P]-orthophosphate by N-propylhydroxylamine—as to be expected of an acyl phosphate compound. The pH stability of the acyl phosphate in the denatured phosphorylated NaK ATPase, in the pronase peptide and in acetyl phosphate were quite different. The phosphorylated protein had the lowest stability of higher pHs, acetyl phosphate had the highest stability, and the pronase peptide had an intermediate stability. These results indicate that the neighboring groups in the polypeptide chain containing the acyl phosphate residue influence the stability of the acyl phosphate bond.     

Cerium-based cytochemical method for detection of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity at physiological pH

We have developed a new cytochemical method for detecting the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase (K-NPPase) activity of the sodium-potassium-activated adenosine triphosphatase (Na-K ATPase) complex. The incubation medium contains p-nitrophenylphosphate (p-NPP) as substrate, cerium chloride as capture agent, Tricine buffer, MgCl2, and KCl. Tricine buffer protected against the medium turbidity caused by non-enzymatic reaction at pH 7.5. Biochemically, the accumulation of p-nitrophenol and phosphate in the reaction precipitate was proportionally related to the enzyme concentration. Ultracytochemically, the reaction products of the K-NPPase activity were localized as fine and uniform electron-dense deposits in the cytoplasmic side of specialized basolateral plasma membranes of cells of kidney distal convoluted tubules, secretory cells of salt gland, and marginal cells of stria vascularis. This method has the advantage of being useful at physiological pH.     

The effect of (o-phenanthroline)2-cupric sulfate on several properties associated with (N-A+ + K+)-dependent adenosine triphosphatase.

Previous studies have shown that the large polypeptide of purified (Na⁺ + K⁺)-dependent adenosine triphosphatase (NaK ATPase) reacts to form a dimer and other higher oligomeric structures of the enzyme as a result of cross-linking with (o-phenanthroline)₂-cupric sulfate (CP). In the present communication, I show that both NaK ATPase activity and p-nitrophenylphosphatase (NPPase) activity decline rapidly and nearly in parallel when the enzyme is reacted with CP. Similarly, ATP binding is lost with kinetics close to those of ATPase activity and NPPase activity. The loss of ATPase activity, NPPase activity, and ATP binding occurs at a considerably faster rate than cross-linking of the large polypeptide, suggesting that CP may also be forming intrachain disulfide bonds. The binding of ouabain to NaK ATPase is also altered as a result of reacting the enzyme with CP. In marked contrast to ATP binding, however, ouabain binding is lost at a slower rate which closely parallels the rate of reaction of the large polypeptide to form cross-linked oligomeric structures.     

3-Phosphono-2-imino-4-oxoimidazoline--a competitive substrate of AMP-dephosphorylating enzymes from the cytosol fraction of the rat heart

3-Phosphono-2-imino-1-methyl-4-oxoimidazolidine (PIMOI), AMP and p-nitrophenyl phosphate (pNPP) were dephosphorylated in the presence of rat heart cytosol at 37 degrees C pH 6.3 at the rates of 0.71, 0.45 and 1.07 mumol/mg X h, respectively. When mixed together, these compounds inhibited the hydrolysis of each other, which points to the participation of common enzyme(s) in this process. The inhibitor of 5'-nucleotidase (alpha,beta-methylene)-ADP, did not affect PIMOI cleavage and moderately inhibited AMP hydrolysis (by ADP, did not affect PIMOI cleavage and moderately inhibited AMP hydrolysis (by 30-50%), thus suggesting that acidic phosphatases are responsible for PIMOI and AMP hydrolysis under these conditions (pH 6.3). Phosphocreatine (PCr) and phosphocyclocreatine (PcCr) were stable to hydrolysis by the cytosolic fraction. However, addition of AMP to the medium containing PCr or PcCr resulted in AMP phosphorylation down to ATP due to the effects of these phosphagens and, probably, of microcontaminations of ATP. This was followed by gradual disappearance of PCr or PcCr and by accumulation of Pi as a result of the "ATPase" activity in the cytosol. The hydrolysis of AMP, PIMOI and p-NPP was sensitive to sulfhydryl reagents [5,5'-dithio-bis-(2-nitrobenzoate) and, in part, 2,4-dinitro-fluorobenzene] and fluoride ion. Thus, PIMOI is a competitive substrate of acidic phosphatases in heart cytosol with respect to AMP and p-NPP. This may partly explain the protective effect of PIMOI on ischemic myocardium.     

Study of the interaction of Na+ and K+-ATPase of erythrocytes with ouabain. Effect of acetyl phosphate and p-nitrophenyl phosphate]

Effects of ATP, acetyl phosphate (AcP) and p-nitrophenyl phosphate (p-NPP) on the inhibition of the Na+, K+-ATPase activity were studied. ATP, AcP and p-NPP were found to facilitate the ouabain-induced inhibition of the enzyme activity only after the injection of these phosphorylyzing agents into the erythrocyte ghosts. Inside the ghosts Na+ ions enhanced the effects of the phosphorylyzing agents. K+ ions in the environment removed the stimulating effects of ATP, AcP and p-NPP on the ouabain-induced inhibition of Na+, K+-ATPase activity. It is concluded that the sites of AcP and p-NPP hydrolysis as well as the active center for ATP are localized on the inner surface of the cell membrane.     

The effects of ATP, inorganic phosphate, protons, and lactate on isolated myofibrillar ATPase activity.

The purpose of this study was to examine the effects of lactate, protons, inorganic phosphate, and ATP on myofibrillar ATPase activity. Myofibrils were isolated from carp (Cyprinius carpio L.) fast-twitch white muscle, and myofibrillar ATPase activities were assessed under maximal activating calcium levels (pCa 4.0) at 10 degrees C in reaction media containing metabolic profiles similar to those seen in fatiguing muscles. The Ca(2+)-activated ATPase activity was assessed by an ATP regenerating assay that coupled the myofibrillar ATPase to pyruvate kinase and lactate dehydrogenase. This assay allowed the effects of ATP, inorganic phosphate, protons, and lactate on myofibrillar ATPase activity to be assessed. The coupled assay was found to give similar myofibrillar ATPase kinetics, with the exception of higher maximal activities, to those seen with a standard end-point assay. Myofibrillar ATPase activity was depressed by 35% when ATP concentrations were lowered to 2.5 mM. Lowering ATP levels to 0.5 mM reduced the myofibrillar ATPase activities by 85%. Lactate had no effect on myofibrillar ATPase activities. Inorganic phosphate levels up to about 20 mM significantly decreased the myofibrillar ATPase activities, after which further increases in inorganic phosphate content had minimal effects. The changes in ATPase activities were related to total inorganic phosphate, not to the content of diprotonated inorganic phosphate. Myofibrillar ATPase activity was highest at pH 7.5 and lowest at pH 6.0. The interactive effects of low ATP, decreased pH, and high inorganic phosphate levels were not additive, giving similar decreases in activity to those produced by increased inorganic phosphate levels alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone

Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.     

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase: Nuclear magnetic resonance studies of 23Na binding to the purified and partially purified enzyme

²³Sodium binding to a partially purified beef brain and purified dogfish rectal gland (sodium + potassium)-activated adenosinetriphosphatase (NaK ATPase) has been studied by pulsed nmr. In both preparations addition of ATP (in the absence of Mg) increased the amount of Na bound to the enzyme protein. In the less-pure brain preparation there was some binding of Na to the protein in the absence of ATP but in the purer preparation from the rectal gland there was little or no binding without ATP. With the dogfish enzyme, potassium readily displaced bound sodium. The K_D for sodium determined by nmr agreed closely with that determined kinetically. This, coupled with the fact that the dogfish enzyme required ATP for sodium binding suggests that the sodium detected by nmr in this preparation is due to binding at its specific site(s).     

Evidence for nonbridged coordination of p-nitrophenyl phosphate to the dinuclear Fe(III)-M(II) center in bovine spleen purple acid phosphatase during enzymatic turnover.

The pH dependence of the catalytic parameters k(cat) and K(M) has been determined for the Fe(III)Fe(II)- and Fe(III)Zn(II)-forms of bovine spleen purple acid phosphatase (BSPAP). The parameter k(cat) was found to be maximal at pH 6.3, and a pK(a) of 5.4-5.5 was obtained for the acidic limb of the k(cat) vs pH profile. Two different EPR spectra were detected for the phosphate complex of the mixed-valent diiron enzyme; their relative amounts depended on the pH, with an apparent pK(a) of 6. The EPR spectra of Fe(III)Fe(II)-BSPAP.PO(4) and Fe(III)Zn(II)-BSPAP.PO(4) at pH 5.0 are similar to those previously reported for Fe(III)Fe(II)-Uf.PO(4) and Fe(III)Zn(II)-Uf.PO(4) complexes at pH 5.0. At higher pH, a new Fe(III)Fe(II)-BSPAP.PO(4) species is formed, with apparent g-values of 1.94, 1.71, and 1.50. The EPR spectrum of Fe(III)Zn(II)-BSPAP does not show significant changes upon addition of phosphate up to 30 mM at pH 6.5, suggesting that phosphate binds only to the spectroscopically silent Zn(II). To determine whether the phosphate complexes were good structural models for the enzyme substrate complexes, these complexes were studied using rapid-freeze EPR and stopped-flow optical spectroscopy. The stopped-flow studies showed the absence of burst kinetics at pH 7.0, which indicates that substrate hydrolysis is rate limiting, rather than phosphate release. The EPR spectrum of Fe(III)Fe(II)-BSPAP.p-NPP is similar, but not identical, to that of the corresponding phosphate complex, both at pH 5 and pH 6.5. We propose that both phosphate and p-NPP bridge the two metal ions at low pH. At higher pH where the enzyme is optimally active, we propose that hydroxide competes with phosphate and p-NPP for coordination to Fe(III) and that both phosphate and p-NPP coordinate only to the divalent metal ion.     

Identification of the phosphorylated intermediate of the Neurospora plasma membrane H+-ATPase as beta-aspartyl phosphate

The chemical nature of the phosphoryl enzyme linkage of the electrogenic proton-translocating ATPase (ATP phosphohydrolase, EC 3.6.1.3) in the plasma membrane of Neurospora has been identified as a mixed anhydride between phosphate and the beta-carboxyl group of an aspartic acid residue in the polypeptide chain. Incubation of isolated Neurospora plasma membrane vesicles containing 32P-labeled ATPase in buffers of increasing pH followed by analysis of the hydrolysis products yielded a pH versus hydrolysis profile characteristic of an acyl phosphate linkage. Reaction of labeled membranes with hydroxylamine at pH 5.3 also released [32P]i from the ATPase. Amino acid analyses of the Na[3H]BH4 reduction products obtained from membranes containing phosphorylated and dephosphorylated ATPase identified [3H]homoserine, the expected reduction product of beta-aspartyl phosphate, as the only additional tritiated reduction product in the samples from phosphorylated membranes. Tritium was not found in alpha-amino-delta-hydroxyvaleric acid, the reduction product of gamma-glutamyl phosphate, nor in proline, the degradation product of alpha-amino-delta-hydroxyvaleric acid. These results indicate that the phosphorylated intermediate of the Neurospora plasma membrane ATPase is a beta-aspartyl phosphate identical with that already known to exist in the Na+:K+- and Ca2+-translocating ATPases of animal cell origin. A common model for the mechanisms of all 3 ion-translocating ATPases is presented.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, K_D = 1–2 μM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (K_I = 240–300 μM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This “tightly bound” ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetic studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, P_i can maintain the active form of the enzyme.     

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. XI. Comparison of kinetic properties of the purified with the impure membrane-bound enzyme from Squalus acanthias

A variety of kinetic parameters have been compared in the membrane-bound and purified forms of the (sodium + potassium)-activated adenosinetriphosphatase (NaK ATPase) from the rectal gland of the spiny dogfish, Squalus acanthias. The kinetic parameters which have been studied have been temperature optima, pH optima, Mg-activation curves, optimum ATP/Mg ratios, K_m for ATP, ouabain-inhibition curves, and Na and K-activation curves. All kinetic parameters were remarkably similar for both forms of the enzyme. This encourages us to believe that information obtained from the pure enzyme can be extrapolated to the enzyme in its native membrane environment and should throw light on the molecular mechanism of Na and K transport.     

Deficiency of uncoupler-stimulated adenosine triphosphatase activity in yeast mitochondria

Oligomycin-sensitive ATPase activity was studied in isolated yeast mitochondria. The protonophore CCCP, at a concentration which completely inhibited ATP synthesis, induced only a low rate of hydrolysis of externally added ATP, and the extent of hydrolysis was dependent upon phosphate (Pi) concentration. CCCP promoted hydrolysis of intramitochondrial ATP. However, hydrolysis of externally added ATP was total in a medium containing potassium phosphate plus valinomycin. Without ionophores, ATPase activity was only observed at high external pH or with detergent-treated mitochondria. Under state 4 conditions, external ATP had access to the catalytic nucleotide site of ATPase as shown by 32Pi-ATP exchange experiments. These results are discussed in terms of a limitation of the translocase-mediated ATP/ADP exchange in uncoupled mitochondria.     

Effect of Na+, K+, Mg2+ and ATP on the Relative Electrophoretic Mobility of Sugar Beet Membrane Particles with NaK ATPase Activity

Electrophoretic measurements on membrane coated particles were performed with a Zytopherometer. Tris-HCl buffer 0.2 M pH 7.0 at 37°C with addition of different combinations of Na⁺, K⁺, Mg²⁺ and ATP was used as test medium. The membranes were of two types, an untreated preparation with low NaK ATPase activity and a deoxycholate treated preparation with high NaK ATPase activity. There was no marked difference in reaction between the two types of membranes. To both types of membranes Mg²⁺ gave a strong positive and ATP a slight negative addition to the membrane charge. In the presence of ATP Na⁺ gave a higher charge contribution than did K⁺ or a combination of Na⁺ and K⁺. This implies that K⁺ gives a higher affinity for ATP than Na⁺ does and or that ATP mediates a higher affinity for Na⁺ than for K⁺.     

Interaction of Na,K-ATPase with modifying ATP analogs and chloromethylphosphonic acid

The interaction of synthetic ATP analogs, containing active groups in the triphosphate moiety and in the 8-position of the nucleotide molecule, with highly purified Na, K-ATPase from the medullar layer of porcine kidney was studied. It was found that 11 out of 17 ATP analogs studied irreversibly inhibit the ATPase activity of the enzyme. The pH optimum of the enzyme inactivation by adenosine-5'-(beta-chloroethylphosphate) and adenosine-5'-(p-fluorosulfonylphenylphosphate) beside the pronounced protective effect of ATP suggests possible covalent blocking of histidine and dicarboxylic amino acid residues in the enzyme active center. The irreversible inhibition of the enzyme by "oxo-ATP" containing aldehyde groups in the modified ribose residue in the presence of sodium borohydride suggests a possible presence of the lysine residue epsilon-amino group in the ATP binding site of the enzyme. Na, K-ATPase was found to possess an inorganic phosphate binding site, which is specifically blocked by chloromethylphosphonic acid. the accessibility of this site for modification depends on ATP, NA+ and K+.     

Inhibition of the purified sodium-potassium activated adenosinetriphosphatase from the rectal gland of Squalus acanthias by antibody against the glycoprotein subunit.

Rabbits have been immunized with purified shark rectal gland NaK ATPase and its glycoprotein component. Serum, globulin from this serum, and a purified antibody fraction from rabbits immunized either with holoenzyme or with glycoprotein inhibited NaK ATPase activity. These antibodies also inhibited a purified NaK ATPase from the electric organ of the electric eel, but to a lesser extent, suggesting some cross reactivity. Ouchterlony double diffusion showed precipitation bands between shark NaK ATPase and serum or globulin containing antibodies against the holoenzyme or the glycoprotein. The inhibition of the NaK ATPase by antibody directed against the purified glycoprotein provides some direct evidence that the glycoprotein is a subunit of the NaK ATPase.     

Dicyclohexylcarbodiimide-sensitive ATPase in Halobacterium saccharovorum

Membranes from Halobacterium saccharovorum contained a cryptic ATPase which required Mg2+ or Mn2+ and was activated by Triton X-100. The optimal pH for ATP hydrolysis was 9-10. ATP or GTP were hydrolyzed at the same rate while ITP, CTP, and UTP were hydrolyzed at about half that rate. The products of ATP hydrolysis were ADP and phosphate. The ATPase required high concentrations (3.5 M) of NaCl for maximum activity. ADP was a competitive inhibitor of the activity, with an apparent Ki of 50 microM. Dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis. The inhibition was marginal at the optimum pH of the enzyme. When the ATPase was preincubated with DCCD at varying pH values, but assayed at the optimal pH for activity, DCCD inhibition was observed to increase with increasing acidity of the preincubation medium. DCCD inhibition was also dependent on time of preincubation, and protein and DCCD concentrations. When preincubated at pH 6.0 for 4 h at a protein:DCCD ratio of 40 (w/w), ATPase activity was inhibited 90%.     

Steady state kinetics of soluble and membrane-bound mitochondrial ATPase

Steady state kinetic measurements of the rate of hydrolysis of ATP to ADP and inorganic phosphate by beef heart mitochondrial ATPase have been performed with both the solubilized enzyme and with the enzyme attached to a mitochondrial membrane fraction at 25° in 0.1 M NaCl with Mg²⁺ as the metal ion activator. These studies indicate the ATP Michaelis constants are somewhat larger for the soluble enzyme and the turnover numbers are considerably larger. In addition, the steady state parameters are essentially independent of pH over the range 7–9 for the membrane-bound enzyme, while the turnover number for the soluble enzyme varies considerably with pH. The product, ADP, is a competitive inhibitor of ATP and inhibits the soluble enzyme much more strongly than the membrane-bound enzyme. Oligomycin inhibits the membrane-bound enzyme very strongly, but has no effect on the activity of the soluble enzyme. The oligomycin inhibition is noncompetitive in nature.     

Steady state kinetics of soluble and membrane-bound mitochondrial ATPase

Steady state kinetic measurements of the rate of hydrolysis of ATP to ADP and inorganic phosphate by beef heart mitochondrial ATPase have been performed with both the solubilized enzyme and with the enzyme attached to a mitochondrial membrane fraction at 25° in 0.1 M NaCl with Mg²⁺ as the metal ion activator. These studies indicate the ATP Michaelis constants are somewhat larger for the soluble enzyme and the turnover numbers are considerably larger. In addition, the steady state parameters are essentially independent of pH over the range 7–9 for the membrane-bound enzyme, while the turnover number for the soluble enzyme varies considerably with pH. The product, ADP, is a competitive inhibitor of ATP and inhibits the soluble enzyme much more strongly than the membrane-bound enzyme. Oligomycin inhibits the membrane-bound enzyme very strongly, but has no effect on the activity of the soluble enzyme. The oligomycin inhibition is noncompetitive in nature.