The intermediary complexes formed by (Na+ + K+)-dependent ATPase (original) (raw)
Related papers
Identification of intact ATP bound to (Na+ + K+)-ATPase
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1971
I. Native and ouabain-treated microsomes of turtle bladder epithelial cells incubated at o ° with [U-I*CIATP form a Mg*+-dependent, acid-stable complex with 1'C in a cooperative homotropic manner. 2. The bound a4C is readily identifiable as intact ATP by first cleaving the ~4C from the 1*C-labeled microsomal precipitate and by the subsequent chromatographic recovery of 14C-labeled ATP in the supernatant fluid. 3. The formation of E-ATP is ouabain inhibitable only in the presence of Mg2÷ + Na + + K+; and conversely the formation of E-ATP in the ouabain-treated enzyme in the presence of Mg 2+ is inhibitable by addition of Na t and K + together. This suggests that at least part of the E-ATP complex is an integral part of the (Na t + K+)-ATPase system. 4. The bond between the enzyme and ATP, probably a covalent one, resists increases in ionic strength and osmolality as well as increases in hydrogen bond dissolution. 5. The stoichiometric relations and turnover numbers of E-ATP and phosphoproteins are estimated with respect to their relative contributions to the overall catalyzed rate of hydrolysis of ATP.
Binding of ATP to and release from microsomal (Na+ + K+)-ATPase
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1973
I. "[ortle bladder micro~omes, which possess (Na* + K ~)-ATPase activity, form Mg2*-dependent acid-stable, radioactively labelled complexes during their incubation at pH 7.4 with o.oi mM concentrations of [U-t~C]ATP, ~y-32P~ATP, and !~-32P!ATP. 2. The formation of a 14C-labelled enzyme-ATP complex is demonstrated by the chromatographic isolation of intact [U-liC]ATP which had been cleaved (by 3 different chemical methods) from its binding sites on the liC-labelled microsomal precipitates; and this is confirmed by an analogous isolation of intact ~y-32P~ATP cleaved from ~,-a2P-labelled precipitate:,. 3. The pH-dependent patterns oi the formation of native complexes and of the breakdown of the acid-denatured complexes show that the ~4C-labelled microsomal protein is different from the Na+-dependent 7-32p-labelled protein. Tile pH dependency of JtC labelling of native microsomes incubated with ~U-14C]ATP is consistent with the behavior expected of a phosphoramido bond ; while that of ~,_3zp labelling of native microsomes incubated with 17-32p]ATP is consistent with the behavior expected of an acyl phosphate bond. 4. The enzyme-ATP complex (or complexes) possesses at least two bonds between the enzyme and ATP: one between the 7-phosphate of ATP and the protein; and the other between the adenosine of ATP and the protein. The presence of an adenosinyl-protein bond is suggested by the fact that the same reagent removes all of the ~_a2p label but only half of the U-14C label from paired sets of microsomal complexes incubated under identical conditions except for the radioactive label (~_32p vs U-x4C) on the substrate (ATP). 5. The pH-dependent pattern of 1'C labelling of microsomes by [U-I*C~ATP is nearly superimposable upon that of (Na-+ K+)-ATPase activity, while thgt of the Na+_stimulated ?,.a2p labelling of microsomes by [~,-32P]ATP is nearly superimposible upon the muco~al pH dependence of Na + transport in the intact tissue. 6. The addition of I.O mM ATP or ADP chases half of the ~*C label from native microsomes which had become labelled during their incubation with o.oi mM [UJ4C]ATP. Th~ significance of these and other data with respect to the reversibility of the formation of an enzyme-ATP complex and its role in ADP:ATP exchange is discussed. ATP-ATPase INTERACTION 209 7-The assignment of paralle! re~ctio,a paths to (Na + + K+I-ATPase (E) and to the sodium-stimulatable phospho-enzyme (E') is discussed in relation to the nature of the intermedialy complexes in the mictosomal system; and in relation to the energy transduction and ion translocation required of active Na:-transport in the intact system.
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1988
1. Sea bass kidney microsomal preparations contain two Mg 2+ dependent ATPase activities: the ouabain-sensitive (Na + + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na + + K+)-ATPase under the optimal conditions ofpH 7.0, 100 mM Na ÷, 25 mM K ÷, 10 mM Mg 2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 #mol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH6.0, 40mM Na +, 1.5mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 #tool Pi/mg protein per hr.
Mg2+-dependent (Na+ + K+)- and Na+-ATPases in the kidneys of the gilthead bream (Sparus auratus L.)
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1990
1. The (Na + + K÷)-and Na+-ATPases, both present in kidney microsomes of Sparus auratus L., have different activities and optimal assay conditions as, in the first of the two stocks of fish used (A), the spec. act. ofthe former is 51.7 #tool P~ mg prot -) hr -) at pH 7.5, 100mM Na +, 10raM K +, 17.5 mM Mg 2+, 7.5 mM ATP and that of the latter is 6.5 #mol P~ mg prot -~ hr -~ at pH 6.5, 40 mM Na +, 4.0 mM Mg 2+, 2.5 mM ATP.
FTIR Study of ATP-Induced Changes in Na+/K+-ATPase from Duck Supraorbital Glands
Biophysical Journal, 2003
The Na 1 /K 1-ATPase uses energy from the hydrolysis of ATP to pump Na 1 ions out of and K 1 ions into the cell. ATP-induced conformational changes in the protein have been examined in the Na 1 /K 1-ATPase isolated from duck supraorbital salt glands using Fourier transform infrared spectroscopy. Both standard transmission and attenuated total internal reflection sample geometries have been employed. Under transmission conditions, enzyme at 75 mg/ml was incubated with dimethoxybenzoin-caged ATP. ATP was released by flashing with a UV laser pulse at 355 nm, which resulted in a large change in the amide I band. The absorbance at 1659 cm ÿ1 decreased with a concomitant increase in the absorbance at 1620 cm ÿ1. These changes are consistent with a partial conversion of protein secondary structure from a-helix to b-sheet. The changes were ;8% of the total absorbance, much larger than those seen with other P-type ATPases. Using attenuated total internal reflection Fourier transform infrared spectroscopy, the decrease in absorbance at ;1650 cm ÿ1 was titrated with ATP, and the titration midpoint K 0.5 was determined under different ionic conditions. In the presence of metal ions (Na 1 , Na 1 and K 1 , or Mg 21), K 0.5 was on the order of a few mM. In the absence of these ions, K 0.5 was an order of magnitude lower (0.1 mM), indicating a higher apparent affinity. This effect suggests that the equilibrium for the ATP-induced conformational changes is dependent on the presence of metal ions.
Revealing of proteins interacting with Na,K-ATPase
Biochemistry. Biokhimii͡a, 2003
Proteins interacting with alpha 1 beta 1-type of Na,K-ATPase were revealed in pig kidney outer medulla and duck salt glands using three different methods (immunoprecipitation, protein overlay, and chemical cross-linking). Immunoprecipitation was performed after solubilization of protein homogenate with Triton X-100 so that both membrane and cytosol proteins bound to Na,K-ATPase could be revealed. Two other methods were used to study the interaction of cytosol proteins with purified Na,K-ATPase. The sets of proteins revealed by each method in outer medulla of pig kidney were different. Proteins interacting with Na,K-ATPase that have molecular masses 10, 15, 70, 75, 105, 120, and 190 kD were found using the immunoprecipitation method. The chemical cross-linking method revealed proteins with molecular masses 25, 35, 40, 58, 68-70, and 86-88 kD. The protein overlay method revealed in the same tissue proteins with molecular masses 38, 42, 43, 60, 62, 66, 70, and 94 kD.
Restoration of phosphorylation capacity to the dormant half of the α-subunits of Na+, K+-ATPase
FEBS Letters, 1996
Purified kidney Na+,K+-ATPase whose a-subunit is cleaved by chymotrypsin at Leu~66-Ala 267, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of s-cleavage in the course of proteolysis, total EP increased with time before it declined. The magnitude of this rise indicated doubling of the number of phosphorylation sites after cleavage. Together with previous findings, these data establish that half of the ~-subunits of oligomeric membrane-bound enzyme are dormant and that interaction of the N-terminal domain of c¢-subunit with its phosphorylation domain causes this half-site reactivity. Evidently, disruption of this interaction by proteolysis abolishes overall activity while it opens access to phosphorylation sites of all c¢-subunits.
The Journal of Membrane Biology, 2014
We characterize the kinetic properties of a gill (Na ? , K ? )-ATPase from the pelagic marine seabob Xiphopenaeus kroyeri. Sucrose density gradient centrifugation revealed membrane fractions distributed mainly into a heavy fraction showing considerable (Na ? , K ? )-ATPase activity, but also containing mitochondrial F 0 F 1 -and Na ?and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na ? , K ? )-ATPase a-subunit with an M r of &110 kDa. The a-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na ? , K ? )-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with V M = 109.5 ± 3.2 nmol Pi min -1 mg -1 and K M = 0.03 ± 0.003 mmol L -1 . Mg 2? (V M = 109.8 ± 2.1 nmol Pi min -1 mg -1 , K 0.5 = 0.60 ± 0.03 mmol L -1 ), Na ? (V M = 117.6 ± 3.5 nmol Pi min -1 mg -1 , K 0.5 = 5.36 ± 0.14 mmol L -1 ), K ? (V M = 112.9 ± 1.4 nmol Pi min -1 mg -1 , K 0.5 = 1.32 ± 0.08 mmol L -1 ), and NH 4 ? (V M = 200.8 ± 7.1 nmol Pi min -1 mg -1 , K 0.5 = 2.70 ± 0.04 mmol L -1 ) stimulated (Na ? , K ? )-ATPase activity following site-site interactions. K ? plus NH 4 ? does not synergistically stimulate (Na ? , K ? )-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K ? binding that can be occupied by NH 4 ? , stimulating the enzyme. Ouabain (K I = 84.0 ± 2.1 lmol L -1 ) and orthovanadate (K I = 0.157 ± 0.001 lmol L -1 ) inhibited total ATPase activity by &50 and &44 %, respectively. Ouabain inhibition increases &80 % in the presence of NH 4
STUDIES ON THE TWO PHOSPHOENZYME CONFORMATIONS OF Na++ K+-ATPase
Annals of the New York Academy of Sciences, 1974
Many proposed reaction mechanisms for Na+ t K+-ATPase postulate the existence of a phosphoenzyme that reacts readily with ADP and poorly with K+, generally called the E l Q P form of the phosphoenzyme.' The direct identification of this postulated intermediate in native Na+ + K+-ATPase has proved difficult, although it is relatively easy t o demonstrate such an intermediate in enzymes irreversibly inhibited with NEM (N-ethylmaleimide).2 In this communication we present evidence for the direct identification of this intermediate in native Na+ + K+-ATPase and its role in the reaction mechanisms of this enzyme. Methods Rat-brain Na+ + K+-ATPase prepared by the method of Akera and Brody3 or the guinea pig kidney enzyme prepared as described by Post and Sen4 were used throughout. Phosphorylation, [ HI ouabain binding, and Na+ t K+-ATPase activity were measured as described by Tobin and coworkers.' Because of the ease with which El % P may be demonstrated in rat-brain Na+ + K+-ATPase this preparation was used in the phosphoenzyme experiments. In experiments involving [ HI ouabain binding equilibria the guinea pig kidney Na+ + K+-ATPase was used because of the rapidity with which glycoside binding t o this particular enzyme equilibrates. Results One of the ligands of Na++K+-ATPase used to monitor changes in the reactivity of the enzyme in these experiments is [ HI ouabain, and the initial series of experiments deal with the effects of bound ATP on the interaction of this enzyme with [ 3H] ouabain. FIGURE 1 shows the actions of Na+ and ATP on the rate of dissociation of [ HI ouabain from rat-brain Na+ t K+-ATPase. For this experiment [ 3H] ouabain was bound t o the enzyme in the presence of Mg++ and Pi, and the enzyme then washed free of ligands and unbound [ 3 H ]ouabain. The preformed enzyme-ouabain complex was then allowed t o dissociate into a buffered solution containing 1 mM EDTA and unlabeled ouabain. As shown in FIGURE 1 , the rate of dissociation of [3H] ouabain was slow under all