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Phosphorylated intermediate of the ouabain-insensitive, Na+-stimulated ATPase in rat kidney cortex and rainbow trout gills
Biochimie 92 (2010) 128e135
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Research paper
Phosphorylated intermediate of the ouabain-insensitive, Naþ-stimulated ATPase in rat kidney cortex and rainbow trout gills V. Ventrella a, J.R. Elvir b, A.R. Borgatti a, 2, G. Trigari a,1, T. Proverbio c, A. Pagliarani a, F. Trombetti a, M. Pirini a, R. Marın c, 2, F. Proverbio c, *, 2 a b c
Department of Biochemistry “G. Moruzzi”, Section of Veterinary Biochemistry, University of Bologna, Via Tolara di Sopra, 50, 40064 Ozzano Emilia, BO, Italy noma de Honduras (UNAH), Tegucigalpa, Honduras Dpto. de Fisiologıa, 2do. Piso Facultad de Ciencias M edicas, Universidad Nacional Auto Laboratorio de Bioenerg etica Celular, Centro de Biofısica y Bioquımica, Instituto Venezolano de Investigaciones Cientıficas (IVIC), A.P. 21827, Caracas 1020A, Venezuela
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 August 2009 Accepted 23 October 2009 Available online 31 October 2009
Several tissues from different animals, including the rat kidney and the freshwater rainbow trout gills, show an ouabain-insensitive, furosemide-sensitive, Naþ-stimulated ATPase activity, which has been associated with the active control of the cell volume. This Na-ATPase is Mg2þ dependent and it is inhibited by vanadate, which can be taken as an indication that this enzyme is a P-type ATPase. The P-type ATPases are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue at the enzyme's substrate site. In the current study, we partially characterized the phosphorylated intermediate of the ouabain-insensitive Na-ATPase of rat kidney cortex homogenates and that of gill microsomes from freshwater rainbow trout. While the kidney cortex homogenates, under our assay conditions, show both Na- and Na,K-ATPase activities, the gill microsomes, when assayed at pH 5.2, only show Na-ATPase activity. Both preparations showed a Mg2þ-dependent, Naþ-stimulated phosphorylated intermediate, which is enhanced by furosemide. Incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH2OH) showed that it is acid-stable and sensitive to hydroxylamine, either when phosphorylated in the presence or absence of furosemide. Addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation. Naþ seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. In a E1eE2 reaction cycle of the Na-ATPase, furosemide seems to be blocking the transition step from Na$E1wP to Na$E2-P. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Ouabain-insensitive Na-ATPase Phosphorylated intermediate Rat kidney cortex Freshwater rainbow trout gills P-type ATPase
1. Introduction Two ways of active ATP dependent Naþ extrusion from the cell have been described in several tissues: Naþ extruded in exchange for Kþ, and Naþ extruded along with Cl and water [1e3]. The former, catalyzed by the ouabain-sensitive Na,K-ATPase, and the latter, catalyzed by the ouabain-insensitive Na-ATPase [1,3e7]. Both enzymes, the Na,K- and the Na- ATPases have been found to be present in the plasma membrane of different cells and, while the Na,K-ATPase has been associated with the maintenance of the Naþ
* Corresponding author. Tel.: þ58 212 504 1395; fax: þ58 212 504 1093. E-mail addresses: [email protected] (V. Ventrella), [email protected] (F. Proverbio). 1 In memoriam. 2 A.R. Borgatti, R. Marın and F. Proverbio are co-senior authors of this article. 0300-9084/$ e see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.10.010
and Kþ gradients across the cell plasma membranes, the Na-ATPase has been associated with the active control of the cell volume [8,9]. Several characteristics of the Na-ATPase have been very well determined. Thus, it has been found that its activity is Mg2þ dependent, Naþ-stimulated, ouabain-insensitive, Kþ-insensitive, furosemide- and triflocin-sensitive. The Na-ATPase is also inhibited by micromolar concentrations of vanadate. All these characteristics have been taken as an indication that the Na-ATPase is a different entity from the Na-K-ATPase [1,10]. The fact that the Na-ATPase is Mg2þ-dependent and inhibited by vanadate, can be taken as an indication that it belongs to the family of the P-type ATPases, since all of them are Mg2þ-dependent and inhibited by vanadate. The P-type ATPases, are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue of the enzyme [11]. Preliminary experiments carried out in our laboratories on rat kidney cortex and freshwater (FW) rainbow trout gills showed in both tissues a phosphorylated intermediate, with biochemical characteristics that allowed us to associate it with
V. Ventrella et al. / Biochimie 92 (2010) 128e135
the Na-ATPase [12,13]. More recently, a phosphorylated intermediate of the ouabain-insensitive Na-ATPase, shown through backdoor phosphorylation of the enzyme with Pi, was reported for small intestinal epithelial cells [14]. Similarly, De Souza et al. [15] showed the formation of a Naþ-stimulated phosphorylated intermediate of the ouabain-insensitive Na-ATPase with ATP in MDCK I cells, using the forward phosphorylation of the enzyme. In both cases, the ATPase activity was inhibited by furosemide, but while the Naþstimulated phosphorylated intermediate of the small intestine epithelial cells (backdoor phosphorylation) was enhanced by furosemide [14], the Naþ-stimulated phosphorylated intermediate of the MDCK I cells (forward phosphorylation) was inhibited by furosemide [15]. Furosemide inhibition of the phosphorylation step during the forward reaction does not preclude the possibility that the enzyme could be labeled through the backdoor phosphorylation procedure used by Thomas et al. [14]. However, in our preliminary experiments with rat kidney cortex and rainbow trout gills [12,13], we found that the forward phosphorylation of the ouabain-insensitive Na-ATPase is enhanced by furosemide. The discrepancy between our results and those in MDCK I cells [15], led us to further characterize the phosphorylation and dephosphorylation steps of the ouabain-insensitive Na-ATPase in rat kidney cortex and FW rainbow trout gills.
2. Materials and methods This study was performed in accordance with the EU (86./609/ EEC) guidelines for the care and use of laboratory animals. The study protocol was approved by the Bioethic Committee of Instituto Venezolano de Investigaciones Cientıficas and Universit a di Bologna.
2.1. Preparation of rat kidney cortex homogenates Healthy male rats of the SpragueeDawley strain (200e300 g, 3 months old) were anesthetized with diethylether and immediately decapitated. The kidneys were removed and collected in a medium containing: 250 mM sucrose; 20 mM TriseHCl (pH 7.2); 0.5 mM dithiothreitol (DTT); 0.2 mM phenylmethylsulfonyl fluoride (sucrose/ Tris/DTT/PMSF solution) at 4 C. Then, slices of the kidney cortex were prepared, weighted and homogenized at 4 C in 3 volumes of the sucrose/Tris/DTT/PMSF solution. The homogenates were filtered through gauze filters and kept at 70 C until use.
2.2. Preparation of rainbow trout gills microsomal fractions The whole procedure was carried out on ice. The rainbow trouts were decapitated, the gills were removed and filaments were immediately trimmed from gill arches, rinsed in ice-cold 0.25 M sucrose, 5 mM EDTA, 16.5 mM Tris, pH 7.4 and carefully homogenized in the same buffer with an Ultraturrax and the microsomal fractions were prepared by following a method described elsewhere [6]. Briefly, the homogenates were centrifuged at 900 g for 10 min. The supernatant was filtered through a double gauze layer and then centrifuged for 10 min at 9,000 g. The supernatant was centrifuged for 90 min at 50,000 g and the obtained pellet recentrifuged at the same speed for 60 min and it was resuspended by gentle stirring using a Potter-Elvehjem homogenizer with a Teflon pestle in the above described buffer. The microsomal preparations (10e15 mg protein/ml) were stored under liquid nitrogen until use.
129
2.3. ATPase assays Na- and Na,K-ATPase assays of rat kidney cortex homogenates were carried out as described elsewhere [10,16]. The Na-ATPase activity was assayed as follows. A 180 ml aliquot of the incubation medium containing (final concentrations) 50 mM TriseHCl (pH 7.0), 5 mM MgCl2, 2 mM Tris-ATP and 5 mM ouabain, with and without 100 mM NaCl, was preincubated at 37 C for 2 min. The reaction was started by addition of 20 ml of the homogenates (0.5 mg prot/ml) previously treated with SDS. After 10 min incubation, the reaction was arrested by adding 300 ml of a solution containing (final concentrations) 2.8% SDS, 0.48% molybdic acid, 2.8% ascorbic acid and 0.48 N hydrochloric acid. The test tubes were placed on ice for 10 min, and then mixed with 500 ml of a solution containing (final concentrations), 2% sodium arsenite; 2% sodium citrate and 2% acetic acid. The tubes were rewarmed at 37 C for 10 min, and the developed color was measured in a Sunrise (Tecan) spectrophotometer at 705 nm. All samples were run in quadruplicate. The Na-ATPase activity was calculated as the difference between the amounts of inorganic phosphate (Pi) liberated in the presence of Mg2þ þ Naþ þ 5 mM ouabain, minus that liberated in the presence of Mg2þ þ 5 mM ouabain. The Na,K-ATPase activity was determined by using a similar protocol but with an incubation medium containing (final concentrations) 50 mM TriseHCl (pH 7.2), 5 mM MgCl2, 100 mM NaCl, 20 mM KCl and 2 mM Tris-ATP, with and without 5 mM ouabain. The Na,K-ATPase activity was calculated as the difference between the amount of Pi liberated in the presence of Mg2þ þ Naþ þ Kþ minus that liberated in the same medium in the presence of 5 mM ouabain. Activity was expressed as nmoles of Pi liberated per mg of protein per min. The microsomal fractions from FW rainbow trout gills were assayed at, 30 C, in the presence of 75 mM Hepes, 0.64 mM Tris (pH 5.2), 5.0 mM Mg-ATP, 1.0 mM ouabain and 60 mM NaCl as previously reported [6]. At this pH value FW trout gill Na,K-ATPase activity is undetectable [6]. Enzyme activity and Pi determination were carried out as above mentioned for the ATPase assays of the kidney cortex homogenates. 2.4. Phosphorylated intermediate assays The phosphorylated intermediate was assayed following a modification of the method described elsewhere [17]. 40 ml aliquots of the rat kidney cortex homogenates (0.1e0.2 mg prot/ml) were added to 60 ml of the incubation medium at 0 C, in 1.5 ml microcentrifuge tubes. The final concentrations in the reaction medium were 12 mM MgCl2, 50 mM TriseHCl (pH 7.2), 10 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), and according to the experimental design, either 100 mM NaCl or 100 mM TriseHCl (pH 7.2). After 15 s at 0 C, the reaction was stopped by adding 400 ml of a trichloroacetic acid (TCA) solution (0 C) containing 5% TCA, 0.1 mM ATP and 1 mM phosphoric acid. The tubes were then centrifuged at 16,000 g for 2 min. The supernatants were carefully discarded by aspiration and the pellets were washed twice with the same TCA acid solution, then dissolved in 500 ml of 0.2% Triton 100, and transferred to scintillation vials containing 4 ml of Instagel. The Naþ-stimulated phosphorylated intermediate was calculated as the difference in phosphorylation in a medium containing Mg2þþNaþ, minus that obtained in a medium containing Mg2þ alone. The results were expressed as pmoles P/mg protein. The phosphorylated intermediate of the FW rainbow trout gill microsomal fractions was assayed at 0 C, 75 mM Hepes, 0.64 mM Tris (pH 5.2), 12 mM MgCl2, 2.0 mM ATP and 60 mM NaCl according to the experimental design. The following steps for the gill microsomes were the same already described for kidney cortex.
130
V. Ventrella et al. / Biochimie 92 (2010) 128e135
2.5. Hydroxylamine treatment In accordance with a procedure of Knauf et al. [17], the P-labeled membranes, washed once with 0.01 N HC1, were suspended (final concentration 0.5 mg protein/ml) in 0.08 N acetate (pH 5.3) containing 0.6 N freshly dissolved hydroxylamine hydrochloride (Matheson, Coleman, and Bell, Norwood, Ohio). For the control, hydroxylamine was replaced by 0.6 N NaCl. The membranes were exposed to hydroxylamine for 10 min at 23 C, after which 5% TCA was added and the suspension was chilled and centrifuged at 10,000 g. The precipitate was washed three times with 5% TCA, containing 1 mM Pi and 0.1 mM ATP and was then solubilized in 2.5% SDS. 32
2.6. Protein determination The protein content of the different samples was estimated with the Coomassie blue dye binding assay from Bio-Rad Laboratories based on Bradford's reaction [18]. 2.7. Chemicals ATP, ouabain (strophanthin-G), PMSF and dithiothreitol were purchased from the Sigma Chemical Company, Coomassie blue dye binding assay was purchased from Bio-Rad Laboratories (Hercules, CA, USA), [32P]-g-ATP was purchased from Amersham (Amersham, England), and Instagel was purchased from New England Nuclear (Boston, Mass., USA). 2.8. Statistical analysis Comparisons between the different conditions were assessed by one-way ANOVA with the post hoc analysis with the StudentNewmaneKeuls test. All results are expressed as means SE and (n) represents the number of experiments performed with different preparations. In all cases, the Na-ATPase activity was calculated from paired data. A P value < 0.05 was accepted as statistically significant. 3. Results Table 1 shows the effect of ouabain and furosemide on the Naand Na,K-ATPase activities of rat kidney cortex homogenates. It is clear that the Naþ-stimulated ATPase activity is ouabain-insensitive and it is furosemide-sensitive. On the other hand, the (NaþþKþ)stimulated ATPase activity is ouabain-sensitive and is practically furosemide-insensitive. A similar assay was carried out on microsomal preparations from FW rainbow trout gills (Table 2). In spite of the significant Na, K-ATPase activity of this preparation when assayed at pH 7.2, which is around 65 nmol Pi/mg prot. min [5,6], at pH 5.2 the activity of this
Table 1 Effect of 5 mM ouabain or 2 mM furosemide on the Na- and Na, K-ATPase activities of rat kidney cortex homogenates. ATPase
ATPase activity: nmoles Pi/mg prot. min Control
þOuabain
þFurosemide
Na Na,K
16 1 86 4
15 1 3 2**
1 1* 73 4
Assays were carried out at pH 7.2 and 37 C in the presence of (mM): TriseHCl, 50 or 150; Mg:ATP, 5; NaCl, 100; KCl, 20. Values are expressed as means S.E., n ¼ 6. *P < 0.001 vs “Control” or “þOuabain”. **P < 0.001 vs “Control” or “þFurosemide”.
Table 2 Effect of 5 mM ouabain or 2 mM furosemide on the Na- and Na, K-ATPase activities of gill microsomes from freshwater rainbow trout. ATPase
Na Na,K
ATPase activity: nmoles Pi/mg prot. min Control
þOuabain
þFurosemide
74 3 (6) 4.29 0.47 (20)
75 4 (6) 3.15 0.33 (20)
4 2* (6) 3.94 0.44 (20)
Assays carried out at 30 C and pH 5,2, in the presence of (mM): Tris Hepes, 75 or 60; Mg-ATP, 5; NaCl, 60; KCl, 4. Values are expressed as means S.E, n is given between parentheses. *P < 0.001 vs “Control” or “þOuabain”.
enzyme, as shown in Table 2, is almost abolished, and the preparation practically only shows Na-ATPase activity. The Na-ATPase activity of FW rainbow trout gill microsomal preparations shows the same characteristics of the rat kidney cortex homogenates: insensitivity to ouabain and complete inhibition by furosemide. Therefore, all the experiments for the trout Na-ATPase were carried out at pH 5.2. Since vanadate, in micromolar quantities, inhibits the P-type ATPases [19], we studied the effect of 300 mM vanadate on the NaATPase activity of both kidney cortex and gills. The results of this experiment are shown in Table 3. The presence of vanadate in the assay medium strongly inhibited the activity of the Na-ATPase in both preparations. The results on the phosphorylated intermediate of the NaATPase of rat kidney cortex homogenates are shown in Fig. 1: Panel A shows the Naþ-stimulated 32P-incorporation as a function of the Naþ concentration in the assay medium, in the presence and absence of 2 mM furosemide. Interestingly, the presence of furosemide in the medium increased the amount of Naþ-stimulated 32 P-incorporation by the membranes. In both cases, with and without furosemide, the phosphorylated intermediate reaches maximal values at a Naþ concentration around 25 mM. Since furosemide inhibits the Na- but not the Na,K-ATPase activity, it can be proposed that the furosemide-induced Naþ-dependent phosphate incorporation is due to the phosphorylation of the ouabaininsensitive Na-ATPase. The phosphorylation seen in the absence of furosemide would indicate mainly the phosphorylation of the Na,KATPase. The difference in phosphorylation in the presence and absence of furosemide was plotted against the Naþ concentrations and the results are shown in Fig. 1, panel B. This fraction of the phosphorylation is Naþ-dependent and reaches maximal values at around 25 mM Naþ. The Ka for Naþ of the furosemide-induced phosphorylation is 8.08 0.60 mM. If the furosemide-induced, Naþ-dependent, phosphate incorporation into the membranes is due to phosphorylation of the ouabain-insensitive Na-ATPase, it should be insensitive to the presence of Kþ in the incubation medium, as when this ion stimulates the dephosphorylation of the Na,K-ATPase, it does not affect the ouabain-insensitive Na-ATPase activity. To test this possibility
Table 3 Effect of 300 mM vanadate on the Na-ATPase activity of rat kidney cortex homogenates and rainbow trout gill microsomes. Preparation
ATPase activity: nmoles Pi/mg prot. min Control
þVanadate
Rat kidney cortex homogenates Rainbow trout gill microsomes
17 1 73 3
3 1* 15 2*
Assays carried out at 37 C and pH value of 7.2 for the rat kidney cortex homogenates and at 30 C and pH value of 5.2 for freshwater rainbow trout gill microsomes. Values are expressed as means S.E, n ¼ 6. *P < 0.001 vs “Control”.
V. Ventrella et al. / Biochimie 92 (2010) 128e135
A
8
+ Na -stimulated phosphorylated intermediate pmoles P/mg prot
+ Na -stimulated phosphorylated intermediate pmoles P/mg prot
A
7 6
Furosemide
5 4
Control
3 2 1 0 0
20
40
60
80
100
131
8 +KCl
7 6 5 4 3 2 1 0 0
+
10
20
[Na ], mM
2.0
1.50
1.5
Ka= 8.08 ± 0.60 mM
1.25 1.00
1.0
0.75 0.50 0.25
0.5
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
1/S
0.0 0
20
40
60
80
100
+
[Na ], mM þ
Fig. 1. Panel A. Na -stimulated P-incorporation from [g-32P]ATP in rat renal cortex homogenates, as a function of the Naþ concentration of the medium, in the presence or absence of 2 mM furosemide. The phosphorylation was carried out at 0 C for 15 s. The final concentrations in the reaction medium were 12 mM MgCl2, 50 mM TriseHCl (pH 7.2), 10 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), and varying concentrations of NaCl. Panel B shows the differences between the two conditions. Values are expressed as means S.E., n ¼ 6. 32
Kþ was added to the medium, in a final concentration of 20 mM, after 15 s of a phosphorylation time, in the presence and absence of furosemide. Samples were then taken after 10, 20 and 30 s, in order to measure the Naþ-stimulated phosphorylated intermediate. The results are shown in Fig. 2. Addition of Kþ to the incubation medium produces a clear dephosphorylation of the membranes, regardless of the presence or absence of furosemide. However, while in the presence of furosemide (Fig. 2, panel A), addition of Kþ produces only a partial dephosphorylation of the membranes, in the absence of the drug (Fig. 2, panel B), the Kþ-induced dephosphorylation is almost complete. We also tested the effect of 300 mM vanadate on the Naþstimulated phosphorylation of the kidney cortex preparations. Aliquots of the kidney cortex homogenates were incubated at 0 C in a medium containing 12 mM MgCl2, 50 mM TriseHCl (pH 7.2), 10 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), 50 mM NaCl, 20 mM KCl, 2 mM furosemide in the presence and absence of 300 mM vanadate. After 15 s, the assay was stopped and we measured the phosphorylation of the proteins. It was found that
Na+-stimulated phosphorylated intermediate pmoles P/mg prot
B
2.5
1 /V
Furosemide-induced phosphorylated intermediate pmoles P/mg prot
B
30
40
50
30
40
50
Time, s
8 7 6 + KCl
5 4 3 2 1 0 0
10
20 Time, s
Fig. 2. Effect of the addition of 20 mM Kþ on the Naþ-stimulated 32P-incorporation from [g-32P]ATP in rat renal cortex homogenates. The experiment was performed in the presence (panel A) and absence (panel B) of 2 mM furosemide. Kþ was added after 15 s of phosphorylation. The phosphorylation was carried out at 0 C. The final concentrations in the reaction medium were 12 mM MgCl2, 50 mM TriseHCl (pH 7.2), 10 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), and 100 mM of NaCl. Values are expressed as means S.E., n ¼ 6.
the Naþ-stimulated phosphorylation was partially blocked by the presence of vanadate, reaching values of 1.94 0.12 pmol P/mg protein in the absence of vanadate and 0.31 0.06 pmol P/mg protein in its presence. Fig. 3, panel A, indicates that the FW rainbow trout gill microsomes show a Naþ-stimulated phosphorylation, with a similar behavior to that shown by the rat kidney cortex homogenates (Fig. 1, panel A). It can also be seen that the phosphorylation in the absence of furosemide reaches a peak at around 25 mM Naþ, and then drops for higher Naþ concentrations. Phosphorylation differences in the presence and absence of furosemide were plotted as a function of the Naþ concentration, and the results are shown in Fig. 3, panel B. The furosemide-induced phosphorylation is Naþdependent, and reaches maximal values at around 50 mM Naþ. The Ka for Naþ of the furosemide-induced phosphorylation is 7.97 0.40 mM.
132
0.8
0.6 Furosemide 0.4
0.2 Control
20
40
60
80
0.4
0.2
+
0.5
0.4
12 10
1/V
0.2
Ka= 7.47 ± 0.40 mM
8 6 4
0.1
2 0.0
0.1
0.2
0.3
0.4
0.5
1/S
0.0 0
20
40
+Mg2+
-Mg2+
+Mg2+
Fig. 4. Mg2þ-dependence of the Naþ-stimulated 32P-incorporation from [g-32P]ATP in gill microsomes from freshwater rainbow trout. The experiment was performed at pH 5.2 in the presence and absence of 2 mM furosemide. The phosphorylation was carried out at 0 C for 15 s. The final concentrations in the reaction medium were 75 mM Hepes, 0.64 mM Tris (pH 5.2), 2.0 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), and 60 mM NaCl, in the presence and absence of 12 mM MgCl2. Values are expressed as means S.E., n ¼ 6.
[Na ], mM
0.3
-Mg2+
100
60 + [Na ], mM
80
100
Fig. 3. Naþ-stimulated 32P-incorporation from [g-32P]ATP in gill microsomes from freshwater rainbow trout, as a function of the Naþ concentration of the medium. The experiment was performed at pH 5.2 in the presence and absence of 2 mM furosemide. The phosphorylation was carried out at 0 C for 15 s. The final concentrations in the reaction medium were 75 mM Hepes, 0.64 mM Tris (pH 5.2), 12 mM MgCl2, 2.0 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), and 60 mM NaCl. Values are expressed as means S.E., n ¼ 6.
Freshwater rainbow trout gill microsomes made it possible to further characterize the Naþ-stimulated phosphorylation of the ouabain-insensitive Na-ATPase. Its Mg2þ-dependence and its chemical nature were tested, in order to identify whether this phosphorylated intermediate is an acyl phosphate. Fig. 4 shows that the Naþ-stimulated phosphorylation is Mg2þ-dependent, either when the phosphorylation was carried out in the absence or in the presence of furosemide. Besides, as shown in Fig. 5, incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH2OH) showed that it is sensitive to NH2OH treatment, either when phosphorylated in the presence or absence of furosemide. The functionality of the enzyme complex was tested by adding non-radioactive ATP or ADP to the phosphorylated enzyme. As shown in Fig. 6, addition of 100 mM non-radioactive Mg-ATP to the reaction medium containing phosphorylated membranes, resulted in a diminution of the Naþ-stimulated phosphorylation when the experiment was carried out in the absence of furosemide. No diminution of the Naþ-stimulated phosphorylated intermediate was seen after addition of non-radioactive ATP, when the experiment was carried out in the presence of 2 mM furosemide. On the
other hand, as seen in Fig. 7, addition of 100 mM MgADP to the assay medium, after 15 s of phosphorylation of the microsomes in the presence of furosemide, resulted in a diminution of the Naþ-stimulated phosphorylated intermediate. If, as shown above, the enzyme complex is functional under the phosphorylation conditions, one should be able to measure an ATPase activity under those conditions. The experiment was performed and the results are shown in Fig. 8. In fact, there is a Naþstimulated ATPase activity, which is not seen when Kþ substitutes Naþ in the incubation medium. Addition of Kþ to the medium with Naþ does not result in any further stimulation of the ATPase activity. Furthermore, the Naþ-stimulated ATPase activity is totally abolished by furosemide.
+ Na -stimulated phosphorylated intermediate pmoles P/mg prot
0
Furosemide-induced phosphorylated intermediate pmoles P/mg prot
0.6
0.0
0.0
B
+Furosemide
Control
0.8
1.0
Na+-stimulated phosphorylated intermediate pmoles P/mg prot
+ Na -stimulated phosphorylated intermediate pmoles P/mg prot
A
V. Ventrella et al. / Biochimie 92 (2010) 128e135
Control
0.8
+Furosemide
0.6
0.4
0.2
0.0 none
NaCl
NH2OH
none
NaCl
NH2OH
Incubations after phosphorylation Fig. 5. Effect of incubation with 0.6 N hydroxylamine (NH2OH) on the Naþ-stimulated phosphorylated intermediate in gill microsomes from freshwater rainbow trout. The gill microsomes were previously phosphorylated as indicated in Fig. 3 and then treated or not with NH2OH as indicated in the Materials and methods section. The phosphorylation was carried out in the presence and absence of 2 mM furosemide. Values are expressed as means S.E., n ¼ 6.
+
0.8 0.7
5 ATPase activity
0.6 Addition of 100 M non-radioactive Mg:ATP
0.5 0.4 0.3 0.2
4 3 2 1
0.1 0
0.0
+
Na
0
5
10
15
20
25
30
Time, s Fig. 6. Effect of the addition of 100 mM non-radioactive Mg:ATP on the Naþ-stimulated 32 P-incorporation from [g-32P]ATP, in gill microsomes from freshwater rainbow trout. The experiment was carried out at pH 5.2, in the presence and absence of 2 mM furosemide. The gill microsomes were phosphorylated as indicated in Fig. 3. Nonradioactive Mg:ATP was added after 15 s of phosphorylation. Values are expressed as means S.E., n ¼ 6.
4. Discussion In the present work we studied the furosemide-induced, Naþdependent phosphorylated intermediate of two different tissues, rat kidney cortex and FW rainbow trout gills. Under the tested conditions, both tissues show an ouabain-insensitive, Naþ-stimulated ATPase activity. For both preparations, the Naþ-stimulated ATPase activity is sensitive to furosemide (Tables 1 and 2), and almost totally inhibited by the presence of micromolar quantities of vanadate in the assay medium (Table 3), a condition shared by the P-type ATPases. In addition, vanadate inhibits the Naþ-stimulated phosphorylation of the rat kidney cortex homogenates, which is in
+ Na -stimulated phosphorylated intermediate pmoles P/mg prot
133
6
Control +Furosemide
pmoles Pi/mg prot . min
Na -stimulated phosphorylated intermediate pmoles/mg prot
V. Ventrella et al. / Biochimie 92 (2010) 128e135
1.0
No addition 100 M non-radioactive Mg:ADP
0.8
0.6
0.4
0.2
0.0 0
2
4
6
8
10
12
Time after addition, s Fig. 7. Effect of the addition of 100 mM non-radioactive Mg:ADP on the Naþ-stimulated 32 P-incorporation from [g-32P]ATP, in gill microsomes from freshwater rainbow trout. The experiment was carried out at pH 5.2, in the presence of 2 mM furosemide. The gill microsomes were phosphorylated as indicated in Fig. 3. Mg:ADP was added after 15 s of phosphorylation. Values are expressed as means S.E., n ¼ 6.
+
K
+
+
Na +K
+
Na +furosemide
Fig. 8. Na- and Na,K-ATPase activities of gill microsomes from freshwater rainbow trout carried out under the phosphoryation conditions: 0 C; pH 5.2; 2 mM ATP; 12 mM MgCl2. 50 mM NaCl; and when required 2 mM furosemide; and 4 mM or 50 mM KCl depending upon the Naþ concentration in the assay medium. When Naþ was present, 4 mM KCl was used as a final concentration. Values are expressed as means S.E., n ¼ 6.
agreement with previous results from Thomas et al. [14] and De Souza et al. [15]. However, more experiments are required in order to evaluate the role of the vanadate ions in the reaction cycle of the Na-ATPase. The Naþ-stimulated phosphorylated intermediate behavior of the rat kidney cortex homogenates can be explained as follows: in the absence of furosemide, Naþ stimulates the phosphorylation of both, the Na, K- and the Na- ATPases. The Na,K-ATPase needs Kþ in order to dephosphorylate. The Na-ATPase, on the other hand, dephosphorylates under the same conditions where it is phosphorylated, without any additional cation. In the presence of furosemide, an inhibitor of the Na-ATPase activity, the enzyme is not dephosphorylated. Consequently, the difference in phosphorylation between the two conditions, presence or absence of furosemide, i.e., 1.9 0.1 pmol P/mg prot (Fig. 1), can be considered to mirror the phosphorylated intermediate of the Na-ATPase. If the Naþ stimulated phosphorylation obtained in the presence of furosemide is the result of the phoshorylation of both, the Naand the Na,K-ATPases, the addition of Kþ to the incubation medium after phosphorylation in the presence of furosemide, would result in the dephosphorylation of the Na,K-ATPase and, consequently, the total amount of the EeP complex would be reduced to a value representing only the phosphorylation of the Na-ATPase. Effectively, as shown in Fig. 2, panel A, when Kþ was added to the assay medium where the rat kidney cortex homogenates were previously phosphorylated in the presence of 50 mM Naþ and 2 mM furosemide, the total amount of the EeP complex was significantly reduced from 6.7 0.2 pmol P/mg prot. to 1.8 0.1 pmol P/mg prot. On the other hand, in the absence of furosemide, the addition of Kþ to the incubation medium, after phosphorylation of the rat kidney cortex homogenates with Naþ and no furosemide, reduces the total amount of the EeP complex to values near zero (Fig. 2, panel B). Also in FW rainbow trout gill microsomes (Fig. 3), the dephosphorylation step of the Naþ stimulated phosphorylation was inhibited by furosemide. Consequently, considering that this tissue does not show any activity of the Na,K-ATPase at pH 5.2 (Table 2), we found it convenient to further characterize the phosphorylated intermediate of the Na-ATPase in gill microsomes. The phosphorylated intermediate of the Na-ATPase from the FW rainbow trout gill microsomes showed to be specific and functional and not just an unspecific binding of phosphate to the membranes.
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Thus, when non-radioactive ATP was added to the incubation medium after 15 s of phosphorylation, the phosphorylated intermediate of the Na-ATPase was reduced when furosemide was not present (Fig. 6). Since furosemide blocks the dephosphorylation step of the Na-ATPase, the enzyme cannot go forward in its presence and, therefore, cannot rephosphorylate the enzyme with nonradioactive ATP. On the other hand, addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation, as it happened when the assay was done in the presence of furosemide (Fig. 7). Determination of the ATPase activity under the assay conditions used for the phosphorylation experiments, i.e.: 0 C; pH 5.2; 2 mM ATP; 12 mM MgCl2, in the presence and absence of 2 mM furosemide (Fig. 8), showed a Naþ-stimulated ATPase activity, which is insensitive to Kþ and to ouabain and inhibited by furosemide. Naþ seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. This is based on the results shown in Fig. 3, panel A, where the phosphorylation in the absence of furosemide reaches a peak at around 25 mM Naþ, and then drops for higher Naþ concentrations. This behavior was not seen in the presence of furosemide. On this regard, it is interesting to mention that Kaufman et al. [20,21] have found that Kþ-congener Rbþ is able to accelerate dephosphorylation of the Na,K-ATPase without leading to ion occlusion. Perhaps Naþ might be acting in a similar way in the reaction cycle of the Na-ATPase. More experiments are required in order to evaluate the possible role of Naþ as a ligand that stimulates the dephosphorylation step of the Na-ATPase. Since we did not work with pure preparations of the Na-ATPase, there is a possibility that the EP measured under our conditions could be due to the phosphorylation of other proteins such as the Na,K-2Cl cotransporter. However, the furosemide-induced, Naþ-stimulated phosphorylated intermediate is hydroxylamine-sensitive, indicating that it is an acyl phosphate (Fig. 5). Besides, the Na-ATPase activity (Tables 1 and 2) and its phosphorylated intermediate are sensitive to vanadate (Table 3). These are two very well-known characteristics of the P-type ATPases [19]. On the other hand, the Na,K-2Cl cotransporter is known to be phosphorylated on at least five sites which include serine and threonine residues in the cytoplasmic amino and carboxy-termini, and this phosphorylated protein is not vanadatesensitive [22]. Fig. 9 shows a simplified diagram of the proposed catalytic cycle of the Na-ATPase. E1 and E2 refer to the two major conformational states of the enzyme as it moves Naþ from one side of the membrane to the other. These conformational states are common for the P-type ATPases [23]. The formation of Na$E1wP is
magnesium-dependent (Fig. 4). E1wP (but not E2-P) is readily reversible by ADP in the presence of furosemide (Fig. 7). Naþ stimulates the phosphorylation step of the enzyme, giving at the same time the right conditions for the transition and dephosphorylation steps. According to our results, furosemide does not inhibit the Na$E1wP formation (phosphorylation step) and according to Thomas et al. [14], furosemide does not inhibit the dephosphorylation step from Na$E2-P to E2þPiþNaþ (dephosphorylation). Consequently, both data point out that furosemide blocks the transition step from Na$E1wP to Na$E2-P. However, we cannot explain so far the inhibitory effect of furosemide on the phosphorylation step found by De Souza et al. [15]. One possibility could be the presence of significant amounts of ADP in the assay medium of De Souza et al. [15]. Higher levels of Mg and ATP as well as a higher temperature and a longer incubation time means higher ATPase activities, and evidently higher ADP release. This might be relevant since ADP in the assay medium is able to reverse the furosemide-induced, Naþ-stimulated phosphorylation (Fig. 7) and this could explain the inhibition of the phosphorylated intermediate of the Na-ATPase in the study of De Souza et al. [15]. In summary, both, the rat kidney cortex homogenates and the freshwater rainbow trout gill microsomes show a Naþ-stimulated ATPase activity, which is ouabain-insensitive and inhibited by furosemide. This enzyme is a P-type ATPase and its phosphorylated intermediate is an acyl phosphate, as shown by its NH2OH sensitivity. The Naþ-stimulated phosphorylation is Mg2þ-dependent and the E1eE2 transition step, is either slowed or inhibited by furosemide. Naþ stimulates the phosphorylation step, and gives to the enzyme, at the same time, the right conditions for the transition and dephosphorylation steps. Preliminary experiments indicated that the furosemide-induced, Naþ-stimulated phosphorylated intermediate of the FW rainbow trout gill microsomes, is associated with a polypeptide of 100 kDa [12,13], as elsewhere reported [14]. Further experiments are required in order to unveil unequivocally the reaction cycle of the ouabain-insensitive Na-ATPase.
Acknowledgements This study was supported in part by grant from FONACIT e Venezuela (National Fund of Science and Technology) No. F-2005000222 and by RFO funds from University of Bologna (Italy).
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Fig. 9. Proposed catalytic cycle of Na-ATPase. The formation of Na$E1wP is magnesium-dependent. E1wP (but not E2-P) is readily reversible by ADP in the presence of sodium. Furosemide is presumed to either slow or block the transition step from Na$E1wP to Na$E2-P.
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Research paper
Phosphorylated intermediate of the ouabain-insensitive, Naþ-stimulated ATPase in rat kidney cortex and rainbow trout gills V. Ventrella a, J.R. Elvir b, A.R. Borgatti a, 2, G. Trigari a,1, T. Proverbio c, A. Pagliarani a, F. Trombetti a, M. Pirini a, R. Marın c, 2, F. Proverbio c, *, 2 a b c
Department of Biochemistry “G. Moruzzi”, Section of Veterinary Biochemistry, University of Bologna, Via Tolara di Sopra, 50, 40064 Ozzano Emilia, BO, Italy noma de Honduras (UNAH), Tegucigalpa, Honduras Dpto. de Fisiologıa, 2do. Piso Facultad de Ciencias M edicas, Universidad Nacional Auto Laboratorio de Bioenerg etica Celular, Centro de Biofısica y Bioquımica, Instituto Venezolano de Investigaciones Cientıficas (IVIC), A.P. 21827, Caracas 1020A, Venezuela
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 August 2009 Accepted 23 October 2009 Available online 31 October 2009
Several tissues from different animals, including the rat kidney and the freshwater rainbow trout gills, show an ouabain-insensitive, furosemide-sensitive, Naþ-stimulated ATPase activity, which has been associated with the active control of the cell volume. This Na-ATPase is Mg2þ dependent and it is inhibited by vanadate, which can be taken as an indication that this enzyme is a P-type ATPase. The P-type ATPases are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue at the enzyme's substrate site. In the current study, we partially characterized the phosphorylated intermediate of the ouabain-insensitive Na-ATPase of rat kidney cortex homogenates and that of gill microsomes from freshwater rainbow trout. While the kidney cortex homogenates, under our assay conditions, show both Na- and Na,K-ATPase activities, the gill microsomes, when assayed at pH 5.2, only show Na-ATPase activity. Both preparations showed a Mg2þ-dependent, Naþ-stimulated phosphorylated intermediate, which is enhanced by furosemide. Incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH2OH) showed that it is acid-stable and sensitive to hydroxylamine, either when phosphorylated in the presence or absence of furosemide. Addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation. Naþ seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. In a E1eE2 reaction cycle of the Na-ATPase, furosemide seems to be blocking the transition step from Na$E1wP to Na$E2-P. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Ouabain-insensitive Na-ATPase Phosphorylated intermediate Rat kidney cortex Freshwater rainbow trout gills P-type ATPase
1. Introduction Two ways of active ATP dependent Naþ extrusion from the cell have been described in several tissues: Naþ extruded in exchange for Kþ, and Naþ extruded along with Cl and water [1e3]. The former, catalyzed by the ouabain-sensitive Na,K-ATPase, and the latter, catalyzed by the ouabain-insensitive Na-ATPase [1,3e7]. Both enzymes, the Na,K- and the Na- ATPases have been found to be present in the plasma membrane of different cells and, while the Na,K-ATPase has been associated with the maintenance of the Naþ
* Corresponding author. Tel.: þ58 212 504 1395; fax: þ58 212 504 1093. E-mail addresses: [email protected] (V. Ventrella), [email protected] (F. Proverbio). 1 In memoriam. 2 A.R. Borgatti, R. Marın and F. Proverbio are co-senior authors of this article. 0300-9084/$ e see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.10.010
and Kþ gradients across the cell plasma membranes, the Na-ATPase has been associated with the active control of the cell volume [8,9]. Several characteristics of the Na-ATPase have been very well determined. Thus, it has been found that its activity is Mg2þ dependent, Naþ-stimulated, ouabain-insensitive, Kþ-insensitive, furosemide- and triflocin-sensitive. The Na-ATPase is also inhibited by micromolar concentrations of vanadate. All these characteristics have been taken as an indication that the Na-ATPase is a different entity from the Na-K-ATPase [1,10]. The fact that the Na-ATPase is Mg2þ-dependent and inhibited by vanadate, can be taken as an indication that it belongs to the family of the P-type ATPases, since all of them are Mg2þ-dependent and inhibited by vanadate. The P-type ATPases, are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue of the enzyme [11]. Preliminary experiments carried out in our laboratories on rat kidney cortex and freshwater (FW) rainbow trout gills showed in both tissues a phosphorylated intermediate, with biochemical characteristics that allowed us to associate it with
V. Ventrella et al. / Biochimie 92 (2010) 128e135
the Na-ATPase [12,13]. More recently, a phosphorylated intermediate of the ouabain-insensitive Na-ATPase, shown through backdoor phosphorylation of the enzyme with Pi, was reported for small intestinal epithelial cells [14]. Similarly, De Souza et al. [15] showed the formation of a Naþ-stimulated phosphorylated intermediate of the ouabain-insensitive Na-ATPase with ATP in MDCK I cells, using the forward phosphorylation of the enzyme. In both cases, the ATPase activity was inhibited by furosemide, but while the Naþstimulated phosphorylated intermediate of the small intestine epithelial cells (backdoor phosphorylation) was enhanced by furosemide [14], the Naþ-stimulated phosphorylated intermediate of the MDCK I cells (forward phosphorylation) was inhibited by furosemide [15]. Furosemide inhibition of the phosphorylation step during the forward reaction does not preclude the possibility that the enzyme could be labeled through the backdoor phosphorylation procedure used by Thomas et al. [14]. However, in our preliminary experiments with rat kidney cortex and rainbow trout gills [12,13], we found that the forward phosphorylation of the ouabain-insensitive Na-ATPase is enhanced by furosemide. The discrepancy between our results and those in MDCK I cells [15], led us to further characterize the phosphorylation and dephosphorylation steps of the ouabain-insensitive Na-ATPase in rat kidney cortex and FW rainbow trout gills.
2. Materials and methods This study was performed in accordance with the EU (86./609/ EEC) guidelines for the care and use of laboratory animals. The study protocol was approved by the Bioethic Committee of Instituto Venezolano de Investigaciones Cientıficas and Universit a di Bologna.
2.1. Preparation of rat kidney cortex homogenates Healthy male rats of the SpragueeDawley strain (200e300 g, 3 months old) were anesthetized with diethylether and immediately decapitated. The kidneys were removed and collected in a medium containing: 250 mM sucrose; 20 mM TriseHCl (pH 7.2); 0.5 mM dithiothreitol (DTT); 0.2 mM phenylmethylsulfonyl fluoride (sucrose/ Tris/DTT/PMSF solution) at 4 C. Then, slices of the kidney cortex were prepared, weighted and homogenized at 4 C in 3 volumes of the sucrose/Tris/DTT/PMSF solution. The homogenates were filtered through gauze filters and kept at 70 C until use.
2.2. Preparation of rainbow trout gills microsomal fractions The whole procedure was carried out on ice. The rainbow trouts were decapitated, the gills were removed and filaments were immediately trimmed from gill arches, rinsed in ice-cold 0.25 M sucrose, 5 mM EDTA, 16.5 mM Tris, pH 7.4 and carefully homogenized in the same buffer with an Ultraturrax and the microsomal fractions were prepared by following a method described elsewhere [6]. Briefly, the homogenates were centrifuged at 900 g for 10 min. The supernatant was filtered through a double gauze layer and then centrifuged for 10 min at 9,000 g. The supernatant was centrifuged for 90 min at 50,000 g and the obtained pellet recentrifuged at the same speed for 60 min and it was resuspended by gentle stirring using a Potter-Elvehjem homogenizer with a Teflon pestle in the above described buffer. The microsomal preparations (10e15 mg protein/ml) were stored under liquid nitrogen until use.
129
2.3. ATPase assays Na- and Na,K-ATPase assays of rat kidney cortex homogenates were carried out as described elsewhere [10,16]. The Na-ATPase activity was assayed as follows. A 180 ml aliquot of the incubation medium containing (final concentrations) 50 mM TriseHCl (pH 7.0), 5 mM MgCl2, 2 mM Tris-ATP and 5 mM ouabain, with and without 100 mM NaCl, was preincubated at 37 C for 2 min. The reaction was started by addition of 20 ml of the homogenates (0.5 mg prot/ml) previously treated with SDS. After 10 min incubation, the reaction was arrested by adding 300 ml of a solution containing (final concentrations) 2.8% SDS, 0.48% molybdic acid, 2.8% ascorbic acid and 0.48 N hydrochloric acid. The test tubes were placed on ice for 10 min, and then mixed with 500 ml of a solution containing (final concentrations), 2% sodium arsenite; 2% sodium citrate and 2% acetic acid. The tubes were rewarmed at 37 C for 10 min, and the developed color was measured in a Sunrise (Tecan) spectrophotometer at 705 nm. All samples were run in quadruplicate. The Na-ATPase activity was calculated as the difference between the amounts of inorganic phosphate (Pi) liberated in the presence of Mg2þ þ Naþ þ 5 mM ouabain, minus that liberated in the presence of Mg2þ þ 5 mM ouabain. The Na,K-ATPase activity was determined by using a similar protocol but with an incubation medium containing (final concentrations) 50 mM TriseHCl (pH 7.2), 5 mM MgCl2, 100 mM NaCl, 20 mM KCl and 2 mM Tris-ATP, with and without 5 mM ouabain. The Na,K-ATPase activity was calculated as the difference between the amount of Pi liberated in the presence of Mg2þ þ Naþ þ Kþ minus that liberated in the same medium in the presence of 5 mM ouabain. Activity was expressed as nmoles of Pi liberated per mg of protein per min. The microsomal fractions from FW rainbow trout gills were assayed at, 30 C, in the presence of 75 mM Hepes, 0.64 mM Tris (pH 5.2), 5.0 mM Mg-ATP, 1.0 mM ouabain and 60 mM NaCl as previously reported [6]. At this pH value FW trout gill Na,K-ATPase activity is undetectable [6]. Enzyme activity and Pi determination were carried out as above mentioned for the ATPase assays of the kidney cortex homogenates. 2.4. Phosphorylated intermediate assays The phosphorylated intermediate was assayed following a modification of the method described elsewhere [17]. 40 ml aliquots of the rat kidney cortex homogenates (0.1e0.2 mg prot/ml) were added to 60 ml of the incubation medium at 0 C, in 1.5 ml microcentrifuge tubes. The final concentrations in the reaction medium were 12 mM MgCl2, 50 mM TriseHCl (pH 7.2), 10 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), and according to the experimental design, either 100 mM NaCl or 100 mM TriseHCl (pH 7.2). After 15 s at 0 C, the reaction was stopped by adding 400 ml of a trichloroacetic acid (TCA) solution (0 C) containing 5% TCA, 0.1 mM ATP and 1 mM phosphoric acid. The tubes were then centrifuged at 16,000 g for 2 min. The supernatants were carefully discarded by aspiration and the pellets were washed twice with the same TCA acid solution, then dissolved in 500 ml of 0.2% Triton 100, and transferred to scintillation vials containing 4 ml of Instagel. The Naþ-stimulated phosphorylated intermediate was calculated as the difference in phosphorylation in a medium containing Mg2þþNaþ, minus that obtained in a medium containing Mg2þ alone. The results were expressed as pmoles P/mg protein. The phosphorylated intermediate of the FW rainbow trout gill microsomal fractions was assayed at 0 C, 75 mM Hepes, 0.64 mM Tris (pH 5.2), 12 mM MgCl2, 2.0 mM ATP and 60 mM NaCl according to the experimental design. The following steps for the gill microsomes were the same already described for kidney cortex.
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2.5. Hydroxylamine treatment In accordance with a procedure of Knauf et al. [17], the P-labeled membranes, washed once with 0.01 N HC1, were suspended (final concentration 0.5 mg protein/ml) in 0.08 N acetate (pH 5.3) containing 0.6 N freshly dissolved hydroxylamine hydrochloride (Matheson, Coleman, and Bell, Norwood, Ohio). For the control, hydroxylamine was replaced by 0.6 N NaCl. The membranes were exposed to hydroxylamine for 10 min at 23 C, after which 5% TCA was added and the suspension was chilled and centrifuged at 10,000 g. The precipitate was washed three times with 5% TCA, containing 1 mM Pi and 0.1 mM ATP and was then solubilized in 2.5% SDS. 32
2.6. Protein determination The protein content of the different samples was estimated with the Coomassie blue dye binding assay from Bio-Rad Laboratories based on Bradford's reaction [18]. 2.7. Chemicals ATP, ouabain (strophanthin-G), PMSF and dithiothreitol were purchased from the Sigma Chemical Company, Coomassie blue dye binding assay was purchased from Bio-Rad Laboratories (Hercules, CA, USA), [32P]-g-ATP was purchased from Amersham (Amersham, England), and Instagel was purchased from New England Nuclear (Boston, Mass., USA). 2.8. Statistical analysis Comparisons between the different conditions were assessed by one-way ANOVA with the post hoc analysis with the StudentNewmaneKeuls test. All results are expressed as means SE and (n) represents the number of experiments performed with different preparations. In all cases, the Na-ATPase activity was calculated from paired data. A P value < 0.05 was accepted as statistically significant. 3. Results Table 1 shows the effect of ouabain and furosemide on the Naand Na,K-ATPase activities of rat kidney cortex homogenates. It is clear that the Naþ-stimulated ATPase activity is ouabain-insensitive and it is furosemide-sensitive. On the other hand, the (NaþþKþ)stimulated ATPase activity is ouabain-sensitive and is practically furosemide-insensitive. A similar assay was carried out on microsomal preparations from FW rainbow trout gills (Table 2). In spite of the significant Na, K-ATPase activity of this preparation when assayed at pH 7.2, which is around 65 nmol Pi/mg prot. min [5,6], at pH 5.2 the activity of this
Table 1 Effect of 5 mM ouabain or 2 mM furosemide on the Na- and Na, K-ATPase activities of rat kidney cortex homogenates. ATPase
ATPase activity: nmoles Pi/mg prot. min Control
þOuabain
þFurosemide
Na Na,K
16 1 86 4
15 1 3 2**
1 1* 73 4
Assays were carried out at pH 7.2 and 37 C in the presence of (mM): TriseHCl, 50 or 150; Mg:ATP, 5; NaCl, 100; KCl, 20. Values are expressed as means S.E., n ¼ 6. *P < 0.001 vs “Control” or “þOuabain”. **P < 0.001 vs “Control” or “þFurosemide”.
Table 2 Effect of 5 mM ouabain or 2 mM furosemide on the Na- and Na, K-ATPase activities of gill microsomes from freshwater rainbow trout. ATPase
Na Na,K
ATPase activity: nmoles Pi/mg prot. min Control
þOuabain
þFurosemide
74 3 (6) 4.29 0.47 (20)
75 4 (6) 3.15 0.33 (20)
4 2* (6) 3.94 0.44 (20)
Assays carried out at 30 C and pH 5,2, in the presence of (mM): Tris Hepes, 75 or 60; Mg-ATP, 5; NaCl, 60; KCl, 4. Values are expressed as means S.E, n is given between parentheses. *P < 0.001 vs “Control” or “þOuabain”.
enzyme, as shown in Table 2, is almost abolished, and the preparation practically only shows Na-ATPase activity. The Na-ATPase activity of FW rainbow trout gill microsomal preparations shows the same characteristics of the rat kidney cortex homogenates: insensitivity to ouabain and complete inhibition by furosemide. Therefore, all the experiments for the trout Na-ATPase were carried out at pH 5.2. Since vanadate, in micromolar quantities, inhibits the P-type ATPases [19], we studied the effect of 300 mM vanadate on the NaATPase activity of both kidney cortex and gills. The results of this experiment are shown in Table 3. The presence of vanadate in the assay medium strongly inhibited the activity of the Na-ATPase in both preparations. The results on the phosphorylated intermediate of the NaATPase of rat kidney cortex homogenates are shown in Fig. 1: Panel A shows the Naþ-stimulated 32P-incorporation as a function of the Naþ concentration in the assay medium, in the presence and absence of 2 mM furosemide. Interestingly, the presence of furosemide in the medium increased the amount of Naþ-stimulated 32 P-incorporation by the membranes. In both cases, with and without furosemide, the phosphorylated intermediate reaches maximal values at a Naþ concentration around 25 mM. Since furosemide inhibits the Na- but not the Na,K-ATPase activity, it can be proposed that the furosemide-induced Naþ-dependent phosphate incorporation is due to the phosphorylation of the ouabaininsensitive Na-ATPase. The phosphorylation seen in the absence of furosemide would indicate mainly the phosphorylation of the Na,KATPase. The difference in phosphorylation in the presence and absence of furosemide was plotted against the Naþ concentrations and the results are shown in Fig. 1, panel B. This fraction of the phosphorylation is Naþ-dependent and reaches maximal values at around 25 mM Naþ. The Ka for Naþ of the furosemide-induced phosphorylation is 8.08 0.60 mM. If the furosemide-induced, Naþ-dependent, phosphate incorporation into the membranes is due to phosphorylation of the ouabain-insensitive Na-ATPase, it should be insensitive to the presence of Kþ in the incubation medium, as when this ion stimulates the dephosphorylation of the Na,K-ATPase, it does not affect the ouabain-insensitive Na-ATPase activity. To test this possibility
Table 3 Effect of 300 mM vanadate on the Na-ATPase activity of rat kidney cortex homogenates and rainbow trout gill microsomes. Preparation
ATPase activity: nmoles Pi/mg prot. min Control
þVanadate
Rat kidney cortex homogenates Rainbow trout gill microsomes
17 1 73 3
3 1* 15 2*
Assays carried out at 37 C and pH value of 7.2 for the rat kidney cortex homogenates and at 30 C and pH value of 5.2 for freshwater rainbow trout gill microsomes. Values are expressed as means S.E, n ¼ 6. *P < 0.001 vs “Control”.
V. Ventrella et al. / Biochimie 92 (2010) 128e135
A
8
+ Na -stimulated phosphorylated intermediate pmoles P/mg prot
+ Na -stimulated phosphorylated intermediate pmoles P/mg prot
A
7 6
Furosemide
5 4
Control
3 2 1 0 0
20
40
60
80
100
131
8 +KCl
7 6 5 4 3 2 1 0 0
+
10
20
[Na ], mM
2.0
1.50
1.5
Ka= 8.08 ± 0.60 mM
1.25 1.00
1.0
0.75 0.50 0.25
0.5
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
1/S
0.0 0
20
40
60
80
100
+
[Na ], mM þ
Fig. 1. Panel A. Na -stimulated P-incorporation from [g-32P]ATP in rat renal cortex homogenates, as a function of the Naþ concentration of the medium, in the presence or absence of 2 mM furosemide. The phosphorylation was carried out at 0 C for 15 s. The final concentrations in the reaction medium were 12 mM MgCl2, 50 mM TriseHCl (pH 7.2), 10 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), and varying concentrations of NaCl. Panel B shows the differences between the two conditions. Values are expressed as means S.E., n ¼ 6. 32
Kþ was added to the medium, in a final concentration of 20 mM, after 15 s of a phosphorylation time, in the presence and absence of furosemide. Samples were then taken after 10, 20 and 30 s, in order to measure the Naþ-stimulated phosphorylated intermediate. The results are shown in Fig. 2. Addition of Kþ to the incubation medium produces a clear dephosphorylation of the membranes, regardless of the presence or absence of furosemide. However, while in the presence of furosemide (Fig. 2, panel A), addition of Kþ produces only a partial dephosphorylation of the membranes, in the absence of the drug (Fig. 2, panel B), the Kþ-induced dephosphorylation is almost complete. We also tested the effect of 300 mM vanadate on the Naþstimulated phosphorylation of the kidney cortex preparations. Aliquots of the kidney cortex homogenates were incubated at 0 C in a medium containing 12 mM MgCl2, 50 mM TriseHCl (pH 7.2), 10 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), 50 mM NaCl, 20 mM KCl, 2 mM furosemide in the presence and absence of 300 mM vanadate. After 15 s, the assay was stopped and we measured the phosphorylation of the proteins. It was found that
Na+-stimulated phosphorylated intermediate pmoles P/mg prot
B
2.5
1 /V
Furosemide-induced phosphorylated intermediate pmoles P/mg prot
B
30
40
50
30
40
50
Time, s
8 7 6 + KCl
5 4 3 2 1 0 0
10
20 Time, s
Fig. 2. Effect of the addition of 20 mM Kþ on the Naþ-stimulated 32P-incorporation from [g-32P]ATP in rat renal cortex homogenates. The experiment was performed in the presence (panel A) and absence (panel B) of 2 mM furosemide. Kþ was added after 15 s of phosphorylation. The phosphorylation was carried out at 0 C. The final concentrations in the reaction medium were 12 mM MgCl2, 50 mM TriseHCl (pH 7.2), 10 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), and 100 mM of NaCl. Values are expressed as means S.E., n ¼ 6.
the Naþ-stimulated phosphorylation was partially blocked by the presence of vanadate, reaching values of 1.94 0.12 pmol P/mg protein in the absence of vanadate and 0.31 0.06 pmol P/mg protein in its presence. Fig. 3, panel A, indicates that the FW rainbow trout gill microsomes show a Naþ-stimulated phosphorylation, with a similar behavior to that shown by the rat kidney cortex homogenates (Fig. 1, panel A). It can also be seen that the phosphorylation in the absence of furosemide reaches a peak at around 25 mM Naþ, and then drops for higher Naþ concentrations. Phosphorylation differences in the presence and absence of furosemide were plotted as a function of the Naþ concentration, and the results are shown in Fig. 3, panel B. The furosemide-induced phosphorylation is Naþdependent, and reaches maximal values at around 50 mM Naþ. The Ka for Naþ of the furosemide-induced phosphorylation is 7.97 0.40 mM.
132
0.8
0.6 Furosemide 0.4
0.2 Control
20
40
60
80
0.4
0.2
+
0.5
0.4
12 10
1/V
0.2
Ka= 7.47 ± 0.40 mM
8 6 4
0.1
2 0.0
0.1
0.2
0.3
0.4
0.5
1/S
0.0 0
20
40
+Mg2+
-Mg2+
+Mg2+
Fig. 4. Mg2þ-dependence of the Naþ-stimulated 32P-incorporation from [g-32P]ATP in gill microsomes from freshwater rainbow trout. The experiment was performed at pH 5.2 in the presence and absence of 2 mM furosemide. The phosphorylation was carried out at 0 C for 15 s. The final concentrations in the reaction medium were 75 mM Hepes, 0.64 mM Tris (pH 5.2), 2.0 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), and 60 mM NaCl, in the presence and absence of 12 mM MgCl2. Values are expressed as means S.E., n ¼ 6.
[Na ], mM
0.3
-Mg2+
100
60 + [Na ], mM
80
100
Fig. 3. Naþ-stimulated 32P-incorporation from [g-32P]ATP in gill microsomes from freshwater rainbow trout, as a function of the Naþ concentration of the medium. The experiment was performed at pH 5.2 in the presence and absence of 2 mM furosemide. The phosphorylation was carried out at 0 C for 15 s. The final concentrations in the reaction medium were 75 mM Hepes, 0.64 mM Tris (pH 5.2), 12 mM MgCl2, 2.0 mM ATP with traces of [32P]-g-ATP (10 mCi/ml specific activity), and 60 mM NaCl. Values are expressed as means S.E., n ¼ 6.
Freshwater rainbow trout gill microsomes made it possible to further characterize the Naþ-stimulated phosphorylation of the ouabain-insensitive Na-ATPase. Its Mg2þ-dependence and its chemical nature were tested, in order to identify whether this phosphorylated intermediate is an acyl phosphate. Fig. 4 shows that the Naþ-stimulated phosphorylation is Mg2þ-dependent, either when the phosphorylation was carried out in the absence or in the presence of furosemide. Besides, as shown in Fig. 5, incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH2OH) showed that it is sensitive to NH2OH treatment, either when phosphorylated in the presence or absence of furosemide. The functionality of the enzyme complex was tested by adding non-radioactive ATP or ADP to the phosphorylated enzyme. As shown in Fig. 6, addition of 100 mM non-radioactive Mg-ATP to the reaction medium containing phosphorylated membranes, resulted in a diminution of the Naþ-stimulated phosphorylation when the experiment was carried out in the absence of furosemide. No diminution of the Naþ-stimulated phosphorylated intermediate was seen after addition of non-radioactive ATP, when the experiment was carried out in the presence of 2 mM furosemide. On the
other hand, as seen in Fig. 7, addition of 100 mM MgADP to the assay medium, after 15 s of phosphorylation of the microsomes in the presence of furosemide, resulted in a diminution of the Naþ-stimulated phosphorylated intermediate. If, as shown above, the enzyme complex is functional under the phosphorylation conditions, one should be able to measure an ATPase activity under those conditions. The experiment was performed and the results are shown in Fig. 8. In fact, there is a Naþstimulated ATPase activity, which is not seen when Kþ substitutes Naþ in the incubation medium. Addition of Kþ to the medium with Naþ does not result in any further stimulation of the ATPase activity. Furthermore, the Naþ-stimulated ATPase activity is totally abolished by furosemide.
+ Na -stimulated phosphorylated intermediate pmoles P/mg prot
0
Furosemide-induced phosphorylated intermediate pmoles P/mg prot
0.6
0.0
0.0
B
+Furosemide
Control
0.8
1.0
Na+-stimulated phosphorylated intermediate pmoles P/mg prot
+ Na -stimulated phosphorylated intermediate pmoles P/mg prot
A
V. Ventrella et al. / Biochimie 92 (2010) 128e135
Control
0.8
+Furosemide
0.6
0.4
0.2
0.0 none
NaCl
NH2OH
none
NaCl
NH2OH
Incubations after phosphorylation Fig. 5. Effect of incubation with 0.6 N hydroxylamine (NH2OH) on the Naþ-stimulated phosphorylated intermediate in gill microsomes from freshwater rainbow trout. The gill microsomes were previously phosphorylated as indicated in Fig. 3 and then treated or not with NH2OH as indicated in the Materials and methods section. The phosphorylation was carried out in the presence and absence of 2 mM furosemide. Values are expressed as means S.E., n ¼ 6.
+
0.8 0.7
5 ATPase activity
0.6 Addition of 100 M non-radioactive Mg:ATP
0.5 0.4 0.3 0.2
4 3 2 1
0.1 0
0.0
+
Na
0
5
10
15
20
25
30
Time, s Fig. 6. Effect of the addition of 100 mM non-radioactive Mg:ATP on the Naþ-stimulated 32 P-incorporation from [g-32P]ATP, in gill microsomes from freshwater rainbow trout. The experiment was carried out at pH 5.2, in the presence and absence of 2 mM furosemide. The gill microsomes were phosphorylated as indicated in Fig. 3. Nonradioactive Mg:ATP was added after 15 s of phosphorylation. Values are expressed as means S.E., n ¼ 6.
4. Discussion In the present work we studied the furosemide-induced, Naþdependent phosphorylated intermediate of two different tissues, rat kidney cortex and FW rainbow trout gills. Under the tested conditions, both tissues show an ouabain-insensitive, Naþ-stimulated ATPase activity. For both preparations, the Naþ-stimulated ATPase activity is sensitive to furosemide (Tables 1 and 2), and almost totally inhibited by the presence of micromolar quantities of vanadate in the assay medium (Table 3), a condition shared by the P-type ATPases. In addition, vanadate inhibits the Naþ-stimulated phosphorylation of the rat kidney cortex homogenates, which is in
+ Na -stimulated phosphorylated intermediate pmoles P/mg prot
133
6
Control +Furosemide
pmoles Pi/mg prot . min
Na -stimulated phosphorylated intermediate pmoles/mg prot
V. Ventrella et al. / Biochimie 92 (2010) 128e135
1.0
No addition 100 M non-radioactive Mg:ADP
0.8
0.6
0.4
0.2
0.0 0
2
4
6
8
10
12
Time after addition, s Fig. 7. Effect of the addition of 100 mM non-radioactive Mg:ADP on the Naþ-stimulated 32 P-incorporation from [g-32P]ATP, in gill microsomes from freshwater rainbow trout. The experiment was carried out at pH 5.2, in the presence of 2 mM furosemide. The gill microsomes were phosphorylated as indicated in Fig. 3. Mg:ADP was added after 15 s of phosphorylation. Values are expressed as means S.E., n ¼ 6.
+
K
+
+
Na +K
+
Na +furosemide
Fig. 8. Na- and Na,K-ATPase activities of gill microsomes from freshwater rainbow trout carried out under the phosphoryation conditions: 0 C; pH 5.2; 2 mM ATP; 12 mM MgCl2. 50 mM NaCl; and when required 2 mM furosemide; and 4 mM or 50 mM KCl depending upon the Naþ concentration in the assay medium. When Naþ was present, 4 mM KCl was used as a final concentration. Values are expressed as means S.E., n ¼ 6.
agreement with previous results from Thomas et al. [14] and De Souza et al. [15]. However, more experiments are required in order to evaluate the role of the vanadate ions in the reaction cycle of the Na-ATPase. The Naþ-stimulated phosphorylated intermediate behavior of the rat kidney cortex homogenates can be explained as follows: in the absence of furosemide, Naþ stimulates the phosphorylation of both, the Na, K- and the Na- ATPases. The Na,K-ATPase needs Kþ in order to dephosphorylate. The Na-ATPase, on the other hand, dephosphorylates under the same conditions where it is phosphorylated, without any additional cation. In the presence of furosemide, an inhibitor of the Na-ATPase activity, the enzyme is not dephosphorylated. Consequently, the difference in phosphorylation between the two conditions, presence or absence of furosemide, i.e., 1.9 0.1 pmol P/mg prot (Fig. 1), can be considered to mirror the phosphorylated intermediate of the Na-ATPase. If the Naþ stimulated phosphorylation obtained in the presence of furosemide is the result of the phoshorylation of both, the Naand the Na,K-ATPases, the addition of Kþ to the incubation medium after phosphorylation in the presence of furosemide, would result in the dephosphorylation of the Na,K-ATPase and, consequently, the total amount of the EeP complex would be reduced to a value representing only the phosphorylation of the Na-ATPase. Effectively, as shown in Fig. 2, panel A, when Kþ was added to the assay medium where the rat kidney cortex homogenates were previously phosphorylated in the presence of 50 mM Naþ and 2 mM furosemide, the total amount of the EeP complex was significantly reduced from 6.7 0.2 pmol P/mg prot. to 1.8 0.1 pmol P/mg prot. On the other hand, in the absence of furosemide, the addition of Kþ to the incubation medium, after phosphorylation of the rat kidney cortex homogenates with Naþ and no furosemide, reduces the total amount of the EeP complex to values near zero (Fig. 2, panel B). Also in FW rainbow trout gill microsomes (Fig. 3), the dephosphorylation step of the Naþ stimulated phosphorylation was inhibited by furosemide. Consequently, considering that this tissue does not show any activity of the Na,K-ATPase at pH 5.2 (Table 2), we found it convenient to further characterize the phosphorylated intermediate of the Na-ATPase in gill microsomes. The phosphorylated intermediate of the Na-ATPase from the FW rainbow trout gill microsomes showed to be specific and functional and not just an unspecific binding of phosphate to the membranes.
134
V. Ventrella et al. / Biochimie 92 (2010) 128e135
Thus, when non-radioactive ATP was added to the incubation medium after 15 s of phosphorylation, the phosphorylated intermediate of the Na-ATPase was reduced when furosemide was not present (Fig. 6). Since furosemide blocks the dephosphorylation step of the Na-ATPase, the enzyme cannot go forward in its presence and, therefore, cannot rephosphorylate the enzyme with nonradioactive ATP. On the other hand, addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation, as it happened when the assay was done in the presence of furosemide (Fig. 7). Determination of the ATPase activity under the assay conditions used for the phosphorylation experiments, i.e.: 0 C; pH 5.2; 2 mM ATP; 12 mM MgCl2, in the presence and absence of 2 mM furosemide (Fig. 8), showed a Naþ-stimulated ATPase activity, which is insensitive to Kþ and to ouabain and inhibited by furosemide. Naþ seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. This is based on the results shown in Fig. 3, panel A, where the phosphorylation in the absence of furosemide reaches a peak at around 25 mM Naþ, and then drops for higher Naþ concentrations. This behavior was not seen in the presence of furosemide. On this regard, it is interesting to mention that Kaufman et al. [20,21] have found that Kþ-congener Rbþ is able to accelerate dephosphorylation of the Na,K-ATPase without leading to ion occlusion. Perhaps Naþ might be acting in a similar way in the reaction cycle of the Na-ATPase. More experiments are required in order to evaluate the possible role of Naþ as a ligand that stimulates the dephosphorylation step of the Na-ATPase. Since we did not work with pure preparations of the Na-ATPase, there is a possibility that the EP measured under our conditions could be due to the phosphorylation of other proteins such as the Na,K-2Cl cotransporter. However, the furosemide-induced, Naþ-stimulated phosphorylated intermediate is hydroxylamine-sensitive, indicating that it is an acyl phosphate (Fig. 5). Besides, the Na-ATPase activity (Tables 1 and 2) and its phosphorylated intermediate are sensitive to vanadate (Table 3). These are two very well-known characteristics of the P-type ATPases [19]. On the other hand, the Na,K-2Cl cotransporter is known to be phosphorylated on at least five sites which include serine and threonine residues in the cytoplasmic amino and carboxy-termini, and this phosphorylated protein is not vanadatesensitive [22]. Fig. 9 shows a simplified diagram of the proposed catalytic cycle of the Na-ATPase. E1 and E2 refer to the two major conformational states of the enzyme as it moves Naþ from one side of the membrane to the other. These conformational states are common for the P-type ATPases [23]. The formation of Na$E1wP is
magnesium-dependent (Fig. 4). E1wP (but not E2-P) is readily reversible by ADP in the presence of furosemide (Fig. 7). Naþ stimulates the phosphorylation step of the enzyme, giving at the same time the right conditions for the transition and dephosphorylation steps. According to our results, furosemide does not inhibit the Na$E1wP formation (phosphorylation step) and according to Thomas et al. [14], furosemide does not inhibit the dephosphorylation step from Na$E2-P to E2þPiþNaþ (dephosphorylation). Consequently, both data point out that furosemide blocks the transition step from Na$E1wP to Na$E2-P. However, we cannot explain so far the inhibitory effect of furosemide on the phosphorylation step found by De Souza et al. [15]. One possibility could be the presence of significant amounts of ADP in the assay medium of De Souza et al. [15]. Higher levels of Mg and ATP as well as a higher temperature and a longer incubation time means higher ATPase activities, and evidently higher ADP release. This might be relevant since ADP in the assay medium is able to reverse the furosemide-induced, Naþ-stimulated phosphorylation (Fig. 7) and this could explain the inhibition of the phosphorylated intermediate of the Na-ATPase in the study of De Souza et al. [15]. In summary, both, the rat kidney cortex homogenates and the freshwater rainbow trout gill microsomes show a Naþ-stimulated ATPase activity, which is ouabain-insensitive and inhibited by furosemide. This enzyme is a P-type ATPase and its phosphorylated intermediate is an acyl phosphate, as shown by its NH2OH sensitivity. The Naþ-stimulated phosphorylation is Mg2þ-dependent and the E1eE2 transition step, is either slowed or inhibited by furosemide. Naþ stimulates the phosphorylation step, and gives to the enzyme, at the same time, the right conditions for the transition and dephosphorylation steps. Preliminary experiments indicated that the furosemide-induced, Naþ-stimulated phosphorylated intermediate of the FW rainbow trout gill microsomes, is associated with a polypeptide of 100 kDa [12,13], as elsewhere reported [14]. Further experiments are required in order to unveil unequivocally the reaction cycle of the ouabain-insensitive Na-ATPase.
Acknowledgements This study was supported in part by grant from FONACIT e Venezuela (National Fund of Science and Technology) No. F-2005000222 and by RFO funds from University of Bologna (Italy).
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[6]
[7]
Fig. 9. Proposed catalytic cycle of Na-ATPase. The formation of Na$E1wP is magnesium-dependent. E1wP (but not E2-P) is readily reversible by ADP in the presence of sodium. Furosemide is presumed to either slow or block the transition step from Na$E1wP to Na$E2-P.
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