STUDIES ON THE TWO PHOSPHOENZYME CONFORMATIONS OF Na++ K+-ATPase (original) (raw)
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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.
Kinetics studies on the interaction between ouabain and (Na+,K+)-ATPase
Biochimica et biophysica acta, 1977
The association and dissociation rate constants for the interaction of [3H]-ouabain with partially purified rat brain (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in vitro were estimated from the time course of the [3H]-ouabain binding observed in the presence of Na+, Mg2+ and ATP by a polynomial approximation-curve-fitting technique. The reduction of the association rate constant by K+ was greater than its reduction of the dissociation rate constant. Thus, the affinity of Na+,K+)-ATPase for ouabain was reduced by K+. The binding-site concentration was unaffected by K+. Consistent with these findings, the addition of KCl to an incubation mixture at the time when [3H]-ouabain binding to (Na+,K+)ATPase is close to equilibrium, caused an immediate decrease in bound ouabain concentration, apparently shifting towards a new, lower equilibrium concentration. Dissociation rate constants which were estimated following the termination of the ouabain-binding reaction were different from ...
Quantitative aspects of the interaction between ouabain and (Na+ + K+)-activated ATPase in vitro
Biochimica Et Biophysica Acta - Biomembranes, 1971
The inhibitory effect of ouabain on (Na + + K+)-activated ATPase (Mg ~+dependent, (Na + + K+)-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was reexamined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabainenzyme mixture in the presence of Na ÷, Mg 2+ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na + + K+)-activated ATPase reaction was subsequently started by the addition of a KC1 solution. When the ouabain-enzyme reaction was brought to near equilibrium prior to the estimation of the ATPase activity, it was kinetically reversible, although overwhelmingly in favor of the ouabain-enzyme complex. This method resulted in a significant shift of the log concentration-response curve to the left. The concentration of ouabain to inhibit 50 % of the (Na + + K+)activated ATPase activity was o.12 #M, whereas it was 0.52 #M with the conventional method for the ATPase assay. Hence, the specific binding of ouabain to the (Na++ K+)-activated ATPase molecule was a slow process, This modified method was not suitable for the study of the effect of p-chloromercuribenzoate on (Na ÷ + K÷)-activated ATPase since the presence of ATP during the preincubation period protected the enzyme from the SH-blocking reagent. With the modified method, the effect of K + to antagonize ouabain inhibition of (Na + + K+)-activated ATPase was markedly reduced, indicating that the welldocumented effect of K ÷ was on the velocity of the ouabain-enzyme complex formation rather than on that of the ouabain-inhibited ATPase reaction. The ouabain-enzyme reaction was competitive with respect to K + at KC1 concentrations below 5 mM, although the competition by K ÷ was not remarkable. Above this concentration, the reaction was non-competitive with respect to K +. Ouabain released from the ouabain-enzyme complex was rebound to the enzyme during an incubation in the presence of Na ÷, Mg 2+ and ATP more easily than ouabain added to the incubation mixture. Abbreviations: PCMB, p-chloromercuribenzoate; S/M ratio, the ratio between the concentration of ouabain in the iooooo × g sediment and that in the medium.
Phosphatase activity of (Na+ + K+)-ATPase. Ligand interactions and related enzyme forms
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1985
The prevailing conformations of partially purified pig kidney (Na++ K +)-ATPase interacting with ligands related to its phosphatase activity were determined following time-dependent trypsin digestion and inactivation as well as the amounts of Rb + or Ca 2+ bound to the enzyme after passage through cation-exchange resin columns. In the presence of 150 mM choline chloride, alone or with 3 mM MgCI 2, 3 mM MnCI 2 or 1 mM CaCi2, the major enzyme conformation was E I. Similar forms were seen with 5 mM p-nitrophenyl phosphate with and without 3 mM MgCI 2. KCI, at 0.5 mM or 150 mM, produced an E 2 enzyme state; the effects of 0.5 mM KCI were completely counteracted by 5 mM p-nitrophenyl phosphate. Under optimal conditions for phosphatase activity (3 mM MgCI2/5 mM p-nitropbenyl phosphate/10 mM KCi) the (Na++ K+)-ATPase was in the E 2 state. At low ionic strength and 20°C and under 85% of maximal RbCI-stimulated phosphatase turnover (1 mM RbCI/3 mM MgCI2/5 mM p-nitrophenyi phosphate) no Rb + occlusion could be detected. Ca 2+, at low ionic strength and in the presence of 3 mM MgCI 2, stimulated an ouabain-sensitive phosphatase activity. The rates of hydrolysis obtained with 1 mM CaCI 2 were similar to those seen with 0.5 mM KCI; under both conditions, similar patterns of trypsin digestion and inactivation of the enzyme were obtained. On the other hand, Ca 2+ could not mimic Rb + in its ability to induce an E2-occluding state. These results suggest that during phosphatase activity of (Na++ K +)-ATPase, the most abundant form is a non-occluding E 2 and that at least one of the mechanisms of potassium stimulation of that activity it to take the enzyme into the E 2 state.
Biochimica Et Biophysica Acta - Biomembranes, 1978
Effects of commonly used purification procedures on the yield and specific activity of (Na ÷ + K*)-ATPase (Mg2÷-dependent, Na* + K÷-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP<tependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na*+ K*)-ATPase and the ratio between (Na*+ K*)-ATPase and Mg2÷-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na'+ K*)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na*+ K*)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzyme preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain • enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purification procedure. These results indicate that the presently used purification procedures may alter * To whom reprint requests should be sent. the properties of membrane (Na*+ K÷)-ATPase and affect the interaction between cardiac glycosides and the enzyme. The effect of a given treatment depends on the source of the enzyme. For the in vitro studies involving purified (Na ÷ + K÷)-ATPase preparations, the influence of the methods used to obtain the enzyme preparation should be carefully evaluated.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1991
Coafermationai changes between E t and E 2 enzyme forms of a dog kidney Na+/K +-ATPase preparation labeled with .ledeacetamldofluoresceinwerefollowedwithastopped.flowfluorimeter,intermsoftherateconstant,kobs,andthesteady−statemagnitude,.ledeacetamldofluorescein were followed with a stopped.flow fluorimeter, in terms of the rate constant, kob s, and the steady-state magnitude, %AF of fluorescence change. On rapid mixing of enzyme pies Mg 2+ plus Na + with saturating (0.5 raM) ATP in the absence of K +, kob. varied with Na + concentration ir~ tat: range 0-155 mM, with a !£,/2 of 10 raM, while %AF was relatively insensitive to Na +, with a Kl/2 of 0..ledeacetamldofluoresceinwerefollowedwithastopped.flowfluorimeter,intermsoftherateconstant,kobs,andthesteady−statemagnitude, raM. Oiigomycin reduced kob s by 98-99% for Na+> 10 raM, but only by 00% for Na +-1 raM; %AF was reduced at most by 20%. At 155 mM Na +, both k.t, and %AF changed if K + was present with the enzyme, kob s decreased by 00% when K + was increased from 0 to 0A raM, but increased when K + was varied in the range 0.2-$ raM. K + increased %AF by a factor of 3 with a !~/z of 0.3-0.5 mM as measured in both stopped-flow and steady-state experiments. These data are censidered in terms of the derived presteady-state equations for two alternate schemes for the enzyme, with the E,P to EzP confermationai change either preceding (Albers.Post) or following (Nerby-Yoda.Skou) Na + transport and release. The analysis indicates that: (i) Na + must be released before the conformational transition, from an E 1 form; (ii) the step in which the second and/or third Na ÷ is released is rate.limiting, but this release is accelerated by Na +; and (ill) the release is also accelerated by K + acting with low affinity (possibly at extracellular sites). intreduetion How the chemical and L.'onformational changes in the Na+/K+.ATPase drive cation transport is a central problem in relating this enzyme to its function as the Na÷/K+-pump. For twenty years, the standard model for the reaction sequence has been the Albers-Post scheme [1-3]. Albers and co-workers demonstrated ADP-sensitive (EtP) and K+-sensitive (E2P) phosphoenzymes, and subsequently proposed a four-step sequence:
Detection of a highly ouabain sensitive isoform of rat brainstem Na,K-ATPase
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
The present work provides evidence for the existence in rat brainstem of a form of the Na,K-ATPase catalytic subunit that displays a high affinity for ouabain (K,, about 10-9 M). Its kinetic identification was made out from studies on dose response curves of ouabain inhibition of Na,K-ATPase activity, ouabain inhibition of Na+-dependent phosphorylation from ATP and ouabain stabilized phosphoenzyme formation from inorganic phosphate (Pi). In all these studies this isoform comprises around 11 percent of the total Na,K-ATPase enzyme. The PAGE electrophoretic mobility of its phosphoprotein obtained from Pi in the presence of ouabain is lower than that of the alpha-l form but it cannot be distinguished from that of alpha-Z Whether this highly ouabain sensitive form corresponds to the alpha-3 isoenzyme or represents the translational product of one of the additional genes described for the large catalytic subunit remains at the moment an open question.
Treatment of purifïed sodium and potassium ion-activated adenosine triphosphatase from sheep kiiiney with iV-ethylmaleimide in the présence of KCl and ATP reduced hydrolytie activity by 83% while steady state phosphorylation was inhibited only 24%. At 30°C, in the présence of magnésium plus inorganie phosphate, the ouabain-binding capacity of the TV-ethylmaleimidetreated enzyme was only 15% less than the capacity of the native enzyme. The phosphorylated intermediate formed from the 7V-ethylmaleimide-treated enzyme was less sensitive to décomposition by KCl and more sensitive to ADP when measured at either 0° or 22°C, compared to a native enzyme. The ouabain-binding capacity of native enzyme at 0° and 30°C in the présence of either magnésium plus inorganie phosphate or magnésium plus ATP plus sodium was the same. The ouabain-binding capacity of the iV-ethylmaleimidetreated enzyme in the présence of magnésium plus inorganie phosphate was the same at 0°C as it was at 30°C. However, in the présence of magnésium plus ATP plus sodium, the treated enzyme bound only half as much ouabain at 0°C as it did at 30°C, even when the reaction was extended to several hours. The rates of ouabain binding were approximately equal for the native and iV-ethylmaleimide-treated enzyme when measured in the présence of magnésium alone or magnésium plus ATP. iV-Ethylmaleimide treatment, however, reduced the rates of ouabain binding in the présence of magnésium plus inorganie phosphate, magnésium plus ATP plus sodium, or magnésium plus ATP plus sodium plus potassium. The results indicate that ouabain does interaet with iV-ethylmaleimide-treated enzyme and that multiple conformers of (Na*,K*)-ATPase, as de-
Archives of Biochemistry and Biophysics, 1999
In 40% dimethyl sulfoxide (Me2SO) high-affinity ouabain (O) binding to Na,K-ATPase (E) is promoted by Mg2+ in the absence of inorganic phosphate (Pi) (Fontes et al., Biochim. Biophys. Acta 1104, 215–225, 1995). Furthermore, in Me2SO the EO complex reacts very slowly with Pi and this ouabain binding can therefore be measured by the degree of inhibition of rapid phosphoenzyme formation. Here we found that, unexpectedly, the ouabain binding decreased with the enzyme concentration in the Me2SO assay medium. We extracted the enzyme preparation with Me2SO or chloroform/methanol and demonstrated that the extracted (depleted) enzyme bound ouabain poorly. Addition of such extracts to assays with low enzyme concentration or depleted enzyme fully restored the high-affinity ouabain binding. Dialysis experiments indicated that the active principle had a molecular mass between 3.5 and 12 kDa. It was highly resistant to proteolysis. It was suggested that the active principle could either be a low-molecular-weight, proteolysis-resistant-peptide (e.g., a proteolipid) or a factor with a nonproteinaceous nature. A polyclonal antibody raised against the C-terminal 10 amino acids of the rat kidney γ-subunit was able to recognize this low-molecular-weight peptide present in the extracts. The previously depleted enzyme displayed lower amounts of the γ-proteolipid in comparison to the native untreated enzyme, as demonstrated by immunoreaction with the antibody.
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.