Because Na+⁄ K+-ATPase can hydrolyze p-nitrophenylphos-phate pNPP, a reaction that is ouabain-sensitive and K+-dependent, the pNPPase activity is presumed to be a partial reaction of Na+
Trang 1Na+⁄ K+-ATPase by NaCl with oligomycin or ATP
Haruo Homareda1and Makoto Ushimaru2
1 Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo, Japan
2 Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo, Japan
Na+⁄ K+-ATPase (Na+⁄ K+-exchanging ATPase;
EC 3.6.3.9) is a membrane-integrated protein that
act-ively transports Na+ from the inside of cells to the
outside and transports K+ in the reverse direction,
coupled with ATP hydrolysis Na+⁄ K+-ATPase has
two conformations, the E1 conformational state and
the E2 conformational state Na+ and ATP bind to
E1 The NaE1ATP formed is phosphorylated to the
high-energy phosphoenzyme with Na+ (NaE1P) and
then transformed to the low-energy phosphoenzyme
(E2P), accompanied by Na+ release E2P is K+
-dependently dephosphorylated to E2+ Pi[1] Because
Na+⁄ K+-ATPase can hydrolyze
p-nitrophenylphos-phate (pNPP), a reaction that is ouabain-sensitive and
K+-dependent, the pNPPase activity is presumed to be
a partial reaction of Na+⁄ K+-ATPase [2–5] About
30 years ago, it was reported that NaCl with oligo-mycin or ATP stimulated K+-dependent pNPPase activity, although NaCl and ATP individually inhibited the activity and oligomycin had little effect [6–11] Oligomycin and ATP are an inhibitor and a substrate for Na+⁄ K+-ATPase, respectively [2–5] Because it remains unclear why both the inhibitor and the sub-strate activate the K+-dependent pNPPase activity in the presence of Na+[2], we investigated this question
to clarify the activation mechanism
Keywords
diprotomer; Na + ⁄ K + -ATPase; oligomycin;
p-nitrophenylphosphate (pNPP);
p-nitrophenylphosphatase (pNPPase)
Correspondence
H Homareda, Department of Biochemistry,
Kyorin University School of Medicine,
Mitaka, Tokyo 181-8611, Japan
Fax & Tel: +81 422 76 7651
E-mail: homareda@kyorin-u.ac.jp
(Received 5 July 2004, revised 24 October
2004, accepted 19 November 2004)
doi:10.1111/j.1742-4658.2004.04496.x
It is known that the addition of NaCl with oligomycin or ATP stimulates ouabain-sensitive and K+-dependent p-nitrophenylphosphatase (pNPPase) activity of Na+⁄ K+-ATPase We investigated the mechanism of the stimu-lation The combination of oligomycin and NaCl increased the affinity of pNPPase activity for K+ When the ratio of Na+ to Rb+ was 10 in the presence of oligomycin, Rb+-binding and pNPPase activity reached a maxi-mal level and Na+was occluded Phosphorylation of Na+⁄ K+-ATPase by p-nitrophenylphosphate (pNPP) was not affected by oligomycin Because oligomycin stabilizes the Na+-occluded E1 state of Na+⁄ K+-ATPase, it seemed that the Na+-occluded E1 state increased the affinity of the phos-phoenzyme formed from pNPP for K+ On the other hand, the combina-tion of ATP and NaCl also increased the affinity of pNPPase for K+and activated ATPase activity Both activities were affected by the ligand condi-tions Oligomycin noncompetitively affected the activation of pNPPase by NaCl and ATP Nonhydrolyzable ATP analogues could not substitute for ATP As NaE1P, which is the high-energy phosphoenzyme formed from ATP with Na+, is also the Na+-occluded E1state, it is suggested that the
Na+-occluded E1 state increases the affinity of the phosphoenzyme from pNPP for K+through the interaction between a subunits Therefore, mem-brane-bound Na+⁄ K+-ATPase would function as at least an (ab)2 -dipro-tomer with interacting a subunits at the phosphorylation step
Abbreviations
AMPPCP, adenylyl-(b,c-methylene)-diphosphonate; E1P, high-energy phosphoenzyme; E2P, low-energy phosphoenzyme; EP,
phosphoenzyme; K 0.5 , concentration giving half-maximal activation; pNPP, p-nitrophenylphosphate; pNPPase, p-nitrophenylphosphatase.
Trang 2In the first part of this paper, we describe the effect
of oligomycin with NaCl on pNPPase activity The
lig-and combination induced high-affinity K+-dependent
pNPPase activity When the ratio of Na⁄ Rb in the
presence of oligomycin was 10, the binding of Rb+, a
congener of K+, and pNPPase activity reached a
max-imal level and Na+ remained occluded Today, it is
well known that oligomycin occludes Na+ within the
Na+⁄ K+-ATPase molecule, so that this antibiotic
inhibits Na+ transport and Na+⁄ K+-ATPase activity
but not K+-dependent pNPPase activity [12–19]
Therefore, the present data suggest that the Na+
-occluded E1 state increased the affinity of pNPPase
for K+
In the second part, the effect of ATP with NaCl on
pNPPase activity is described The ligand combination
induced high-affinity K+-dependent pNPPase activity
and ATPase activity at the same time Variation of the
ligand conditions affected both activities ADP and
adenylyl (b,c-methylene)diphosphonate (AMPPCP)
could not substitute for ATP Because NaE1P, which
is formed from ATP with Na+, is also the Na+
-occlu-ded E1 state [20], the present results suggest that the
Na+-occluded E1 state increases the affinity of the
phosphoenzyme (EP) from pNPP for K+ We explain
the present data using a model in which
membrane-bound Na+⁄ K+-ATPase functions as an (ab)2
-dipro-tomer with interacting a subunits
Results
Effect of pH on ouabain-insensitive pNPPase
activity
Although the ouabain-insensitive ATPase activity of
the Na+⁄ K+-ATPase preparation used in this study
was less than 5% in the presence of 0.1 mm ouabain,
16% of the total pNPPase activity was
ouabain-insen-sitive at pH 7.4 To find experimental conditions in
which the ouabain-insensitive pNPPase activity was
minimized, pNPPase activity was measured at pH 6.4,
7.4 and 8.4 The ouabain-insensitive activity was 32%,
16% and 10% at pH 6.4, 7.4 and 8.4, respectively, as
shown by Nagai et al [21] In addition to the change
in pH, the ouabain concentration was increased from
0.1 to 1 mm to completely depress the increase in the
ouabain-insensitive activity due to increasing the KCl
concentration On the other hand, increasing the Na+
concentration had no effect on the
ouabain-insensitiv-ity of pNPPase activouabain-insensitiv-ity The concentration giving
half-maximal activation (K0.5) of pNPPase for K+ was
1 mm, and the Vmax was 2.8 lmolÆmin)1Æmg)1 in the
presence of 10 mm KCl, 5 mm MgCl2 and 2.5 mm
pNPP at pH 8.4 and 37C The K0.5 was similar to that at pH 7.4 (data not shown)
PNPPase activity in the presence of oligomycin and NaCl
The effect of oligomycin with NaCl on pNPPase activ-ity was observed in the presence of 0–3 mm KCl (Fig 1) In the presence of 3 mm KCl without oligo-mycin, NaCl gradually inhibited pNPPase activity (Fig 1A) The addition of 10 lm oligomycin, which appears to be the maximal concentration in an aque-ous solution including 1% (v⁄ v) ethanol [19], streng-thened the inhibitory effect of NaCl on pNPPase activity in the presence of NaCl up to 20 mm In the presence of NaCl at concentrations of more than
20 mm, however, the pNPPase activity with oligomycin was higher than that without the antibiotic In the presence of 1 mm KCl, pNPPase activity with oligo-mycin was higher than that without the antibiotic in the presence of NaCl at concentrations of more than
4 mm (Fig 1B) In the presence of 0.3 mm KCl, the inhibitory effect of Na+was absent in the absence of oligomycin (Fig 1C) The addition of oligomycin stimulated the activity fivefold in the presence of 10–30 mm NaCl These results show that, when the
Fig 1 Effect of oligomycin on pNPPase activity in the presence of NaCl and KCl Ouabain-sensitive pNPPase activity in the absence (s) or presence (n) of 10 l M oligomycin was assayed in a mixture containing 5 lg (A) or 10 lg (B–D) Na + ⁄ K + -ATPase, the standard lig-ands [5 m M MgCl 2 , 50 m M Tris ⁄ Tes (pH 8.4 at 23 C), 2.5 m M
pNPP, 1 m M EDTA, and with or without 1 m M ouabain], 1% etha-nol, 0–300 m M NaCl, and (A) 3 m M KCl, (B) 1 m M KCl, (C) 0.3 m M
KCl, or (D) 0 m M KCl Data represent the means of two independ-ent experimindepend-ents.
Trang 3ratio of Na⁄ K was higher than 4, oligomycin activated
pNPPase In the absence of KCl, oligomycin slightly
enhanced pNPPase activity in the presence of NaCl at
concentrations of more than 10 mm (Fig 1D)
Figure 2 shows the activation of pNPPase by KCl in
the presence of NaCl with or without oligomycin In
the absence of oligomycin, increasing the NaCl
con-centration decreased pNPPase activity (Fig 2A) In
the presence of oligomycin, increasing the NaCl
con-centration obviously increased the affinity of pNPPase
for K+(Fig 2B) In the presence of 10 lm oligomycin
and 30 mm NaCl, the K0.5 for K+ was 0.3 mm The
combination of 10 lm oligomycin and 10 mm NaCl
started to demonstrate an activation of pNPP
hydro-lysis with two phases This activation was clearly
con-firmed by the combination of 30 mm NaCl and 10 lm
oligomycin (Fig 2B) The K0.5 and Vmax were 0.3 mm
and 0.8 lmolÆmin)1Æmg)1 for the high-affinity K+
-dependent pNPPase activity, and were 5 mm and
1.6 lmolÆmin)1Æmg)1 for the low-affinity K+
-depend-ent pNPPase activity, respectively (Fig 3)
The relation between pNPPase activity and ion-binding
pNPPase activity and ion binding were measured in reaction mixtures containing 0.1 mm RbCl, 0.5 mm pNPP and 0–10 mm NaCl (Fig 4) In this experiment, the MgCl2 concentration was reduced to 1 mm, and the reaction temperature lowered to 0C because a high Mg2+ concentration inhibited the binding of
Na+and K+ [14,15] and a low temperature increased the affinities of Na+⁄ K+-ATPase for Na+ and K+ (H Homareda, unpublished data) Specific (oua-bain-sensitive) Na+ binding was very low in the absence of oligomycin, which increased the affinity of
Na+⁄ K+-ATPase for Na+[14] The resultant increase
in Na+binding was regarded as Na+occlusion
In the absence of oligomycin, NaCl gradually inhib-ited pNPPase activity and Rb+binding (Fig 4A,B) In the presence of oligomycin, the binding curve of Rb+ and the activation curve of pNPPase showed a convex shape The peak of both Rb+ binding and pNPPase activity occurred at 1 mm NaCl A plausible explanation
is that the activation of pNPPase by oligomycin with NaCl is due to the increase in K+affinity On the other hand, Na+ occlusion was preserved under the ligand conditions used (Fig 4C) Therefore, the Na+-occluded
E1 state of Na+⁄ K+-ATPase seemed to induce the high-affinity K+-dependent pNPPase activity
Phosphorylation from pNPP
We examined whether the phosphorylation by pNPP was affected by oligomycin or other ligands in the
Fig 2 Activation of pNPPase activity by KCl in the presence of
NaCl with or without oligomycin The pNPPase activity in the
absence (A) or presence (B) of 10 l M oligomycin was assayed in a
mixture containing 10 lg Na+⁄ K +
-ATPase, the standard ligands, 1%
ethanol, 0–3 m M KCl and 0 (s), 1 m M (n), 3 m M (h), 10 m M (b),
30 m M (d), 100 m M (m) or 300 m M NaCl (j) Data represent the
means of two determinations.
Fig 3 Activation of pNPPase by KCl in the presence of NaCl and oligomycin The pNPPase activity was assayed in a mixture contain-ing 5 lg Na + ⁄ K + -ATPase, the standard ligands, 1% ethanol, 0–30 m M KCl, and 10 l M oligomycin (m) or 30 m M NaCl plus 10 l M
oligomycin (n) Plots and bars represent the means ± SD from three determinations.
Trang 4presence of ouabain (Table 1) The amounts of EP
were not affected by 10 lm oligomycin, 10 mm NaCl,
0.3 mm KCl or 0.3 mm ATP To determine whether
the phosphorylation by [32P]pNPP was affected by
contaminating32Pi, which is nonenzymatically released
from [32P]pNPP, or was unreactive 32Piin the reaction
mixture for [32P]pNPP synthesis, 0.3 mm nonradioactive
Pi was added to the reaction mixture If the phos-phorylation was due to the contamination by 32Pi, EP must be significantly decreased by the addition of non-radioactive Pi The result showed that EP from 1 mm [32P]pNPP was decreased to 65% by 0.3 mm Pi, whereas EP from 0.3 mm 32Pi was decreased to 36%
by 1 mm pNPP pNPPase activity decreased by only 15% in the presence of 0.3 mm Pi Therefore, the EP formed was not due to 32Pi
The amount of EP from pNPP was 1.9 times greater than that from ATP This value was consistent with the ratio of EP from Pior pNPP to EP from ATP [22–24]
pNPPase activity in the presence of NaCl, KCl and ATP
The effect of ATP with NaCl on pNPPase activity was observed in the presence of 1 mm KCl (Fig 5A) In the absence of NaCl, increasing the ATP concentration from 0 to 1 mm decreased pNPPase activity Increasing the NaCl concentration without ATP inhibited pNPP-ase activity However, simultaneous addition of NaCl and ATP induced convex-shaped activation curves of pNPPase The combination of 10 mm NaCl and 0.1 mm ATP maximally activated pNPPase
In the presence of ATP and KCl, increasing the NaCl concentration induced convex-shaped activation curves of pNPPase (Fig 5B) When 10 mm NaCl and 0.1 mm ATP were present, KCl at 3, 1 and 0.3 mm activated pNPPase by 2.4-fold, 6.4-fold and 20-fold over reactions without NaCl, respectively Conse-quently, the combination of 10 mm NaCl, 0.3 mm KCl
Table 1 Phosphorylation of Na + ⁄ K + -ATPase from pNPP, Pi and ATP For phosphorylation by pNPP or Pi, Na + ⁄ K + -ATPase was incu-bated for 10 min at 37 C in the presence of 5 m M MgCl 2 , 0.5 m M
ouabain, 50 m M Tris ⁄ Tes (pH 8.4 at 23 C), 1 m M [ 32 P]pNPP or 0.3 m M32Piwith or without the ligands shown For phosphorylation
by ATP, Na + ⁄ K + -ATPase was incubated for 30 s at 0 C in the pres-ence of 5 m M MgCl2, 50 m M Tris ⁄ Tes (pH 8.4 at 23 C), 0.1 m M
[ 32 P]ATP with or without 2.5 m M pNPP Data are presented as the means ± SD from three to six determinations.
nmolÆmg)1 %
1 m M [ 32 P]pNPP + 0.5 m M ouabain 1.86 ± 0.05 100 + 10 l M oligomycin 1.85 ± 0.03 99 + 10 l M oligomycin + 10 m M NaCl 1.83 ± 0.04 98 + 10 l M oligomycin + 10 m M NaCl
+ 0.3 m M KCl
1.88 ± 0.02 101 + 0.3 m M ATP 1.63 ± 0.03 88 + 0.3 m M Pi 1.21 ± 0.09 65 0.3 m M32Pi+ 0.5 m M ouabain 1.81 ± 0.05 100
0.1 m M [32P]ATP + 10 m M NaCl 1.05 ± 0.05 100 + 2.5 m M pNPP 1.00 ± 0.05 95
Fig 4 pNPPase activity, Rb + binding and Na + occlusion in the
same ligand condition (A) The pNPPase activity in the absence (s)
or presence (n) of 10 l M oligomycin was assayed in a mixture
con-taining 20 lg Na + ⁄ K + -ATPase, 1 m M MgCl2, 50 m M Tris ⁄ Tes
(pH 8.4 at 23 C), 0.5 m M pNPP, 0–10 m M NaCl, 0.1 m M RbCl, 1%
ethanol, with or without 1 m M ouabain The reaction was started
by the addition of pNPP and followed for 90 min at 0 C (B)
Oua-bain-sensitive 86 Rb + binding in the absence (s) or presence (n) of
10 l M oligomycin was assayed in a mixture containing 30 lg
Na+⁄ K +
-ATPase, 1 m M MgCl 2 , 50 m M Tris ⁄ Tes (pH 8.4 at 23 C),
0.5 m M pNPP, 0–10 m M NaCl, 0.1 m M86RbCl, 1% ethanol, with or
without 0.1 m M ouabain After the addition of pNPP, the mixture
was centrifuged (C) Oligomycin-stimulated 22Na+ binding was
assayed in a mixture containing 30 lg Na + ⁄ K + -ATPase, 1 m M
MgCl2, 50 m M Tris ⁄ Tes (pH 8.4 at 23 C), 0.5 m M pNPP, 0.3, 1 or
3 m M 22NaCl, 0.1 m M RbCl, 1% ethanol, with or without 10 l M
oligomycin After the addition of pNPP, the mixture was
centri-fuged The detailed procedure is described in Experimental
proce-dures Plots and bars in (A), (B) and (C) represent the means ± SD
from three determinations.
Trang 5and 0.1 mm ATP maximally activated pNPPase The
combination of 3 mm NaCl and 0.1 mm ATP slightly
activated pNPPase even in the absence of KCl ADP
and AMPPCP could not substitute for ATP
Figure 6 shows that the combination of 0.1 mm
ATP and 10 mm NaCl increases the affinity of
pNPP-ase for K+, as shown by oligomycin with NaCl
(Figs 2 and 3) The combination decreased the K0.5for
K+from 2 to 0.2 mm, which was one-fifth of the K0.5,
1 mm, under the usual conditions
Competition between oligomycin and ATP
Figure 7 shows the competition between oligomycin
and ATP Oligomycin decreased the Vmax without
affecting the K0.5, suggesting that oligomycin was a
noncompetitive inhibitor of ATP
ATPase activity in the presence of NaCl, KCl
and ATP
The ligand combination of 10 mm NaCl, 0.3 mm KCl,
0.1 mm ATP and 2.5 mm pNPP activated ATPase in
addition to pNPPase (Fig 8A) Omission of KCl signi-ficantly decreased both activities (Fig 8B) Increasing the NaCl concentration inactivated pNPPase more than ATPase irrespective of the absence and presence
of KCl (Fig 8A,B)
In the presence of 10 mm NaCl and 0.1 mm ATP, KCl concentrations up to 1 mm simultaneously activa-ted both activities with a K0.5 of 0.2 mm for K+ (Fig 9A) KCl concentrations greater than 1 mm gradually inactivated ATPase On the other hand, omission of NaCl completely inactivated ATPase (Fig 9B)
Fig 5 Effects of ATP and KCl on pNPPase activity in the presence
of NaCl The pNPPase activity was assayed in a mixture containing
5 lg Na + ⁄ K + -ATPase, the standard ligands, 0–100 m M NaCl, and
(A) 1 m M KCl with 0 (s), 0.01 m M (d), 0.1 m M (n) or 1 m M ATP
(m), or (B) 0.1 m M ATP with 3 m M (s), 1 m M (d), 0.3 m M (j) or
0 m M KCl (h) In (B), b and c represent 0.1 m M AMPPCP and
0.1 m M ADP in the presence of 0.3 m M KCl, respectively Plots and
bars represent the means ± SD from three determinations.
Fig 6 K + -dependent activation curves for pNPPase in the presence
of NaCl with or without ATP pNPPase activity in the absence (s)
or presence (n) of 0.1 m M ATP was assayed in a mixture contain-ing 5 lg Na + ⁄ K + -ATPase, the standard ligands, 10 m M NaCl and 0–30 m M KCl Plots and bars represent the means ± SD from three determinations.
Fig 7 Effect of oligomycin on pNPPase activity in the presence of NaCl and ATP pNPPase activity was assayed in a mixture contain-ing 5 lg Na + ⁄ K + -ATPase, the standard ligands, 10 m M NaCl, 0–30 m M KCl and 0.1 m M ATP (s), 10 l M oligomycin (n) or 0.1 m M
ATP plus 10 l M oligomycin (h) Plots and bars represent the means ± SD from three determinations.
Trang 6In the presence of 10 mm NaCl, 0.3 mm KCl and
0.1 mm ATP, increasing the pNPP concentration
increased pNPPase activity (Fig 10A) The K0.5 for
pNPP was 2 mm This was equivalent to the value under
the usual conditions ATPase activity was decreased by
high pNPP concentrations, although the decrease was
not more than 50% On the other hand, increasing the
ATP concentration had a complex effect on pNPPase
(Fig 10B) ATP concentrations up to 0.3 mm activated
pNPPase, whereas ATP concentrations greater than
0.3 mm completely inhibited it The activation curve of
ATPase was biphasic From a double-reciprocal plot
analysis, the K0.5values for ATP were 0.14 and 2.0 mm
and the Vmaxvalues were 0.7 and 5.0 lmolÆmin)1Æmg)1
Discussion
The affinity of pNPPase for K+is an order of
magni-tude lower than that of Na+⁄ K+-ATPase for K+
[2,5] The combination of NaCl and oligomycin
induced the high-affinity K+-dependent pNPPase
activity (Figs 2 and 3) The K0.5 for K+was 0.3 mm, which was equivalent to that, 0.2 mm, for Na+⁄ K+ -ATPase activity (Fig 9A) The increase in K+affinity caused by oligomycin with Na+was supported by the ion-binding experiment (Fig 4) When the ratio of
Na⁄ Rb in the presence of oligomycin was 10, Rb+ binding and pNPPase activity reached a maximal value, and the occluded Na+ was preserved (Fig 4) Because this antibiotic stabilizes the Na+-occluded E1 state of Na+⁄ K+-ATPase, it seemed that NaE1 –oligo-mycin increased the affinity of pNPPase for K+ The NaE1–oligomycin complex is an arrested form [2–5,12] Therefore, enzyme states other than the complex must hydrolyze pNPP Scheiner-Bobis et al [25–27] and Linnertz et al [28] showed that fluorescein isothio-cyanate, which blocks the high-affinity binding site for ATP, affects Na+⁄ K+-ATPase activity but not pNPP-ase activity and showed that Co(NH3)4ATP, which blocks the low-affinity site for ATP, preserves Na+ -dependent phosphorylation by ATP but inactivates pNPPase They suggested that Na+⁄ K+-ATPase
Fig 8 Na + -dependent activation curves for Na + ⁄ K + -ATPase and
pNPPase in the presence of ATP with or without KCl
Ouabain-sen-sitive activities of ATPase and pNPPase in the presence (A) or
absence (B) of 0.3 m M KCl were assayed in a mixture containing
5 lg Na + ⁄ K + -ATPase, the standard ligands, 0–300 m M NaCl and
0.1 m M ATP (n) or 0.1 m M [ 32 P]ATP (m) n and m represent
pNPP-ase and ATPpNPP-ase activity, respectively Plots and bars represent the
means ± SD from three determinations.
Fig 9 K + -dependent activation curves for Na + ⁄ K + -ATPase and pNPPase in the presence or absence of NaCl The activities of ATP-ase and pNPPATP-ase in the presence (A) or absence (B) of 10 m M
NaCl were assayed in a mixture containing 5 lg Na + ⁄ K + -ATPase, the standard ligands, 0–30 m M KCl, and 0.1 m M ATP (n) or 0.1 m M
[32P]ATP (m) n and m represent pNPPase and ATPase activity, respectively Plots and bars represent the means ± SD from three determinations.
Trang 7works as a functional (ab)2-diprotomer, in which the
E1 state and the E2 state coexist [29] The effect of
flu-orescein isothiocyanate resembles the effect of
oligo-mycin Furthermore, our data suggest that the
interaction between NaE1 and E2 increased the affinity
of pNPPase for K+ Mimura et al [30] showed that
octaethylene glycol dodecyl ether-solubilized Na+⁄ K+
-ATPase forms a loosely associated diprotomer in the
E1 state but a tightly associated one in the E2 state
Nakamura et al [31] suggested a monomer–dimer
transition model of Ca2+-ATPase, in which the
cyto-plasmic domains clap like a castanet duet, and
Carv-alho-Alves et al [32] proposed dimerization of the
cytoplasmic domain of Ca2+-ATPase Abe et al [33]
showed that H+,K+-ATPase functions as an oligomer
in the membrane, although a monomer of these P-type
ATPases has ATPase activity [34–37] Therefore, we
attempted to explain the present data using the (ab)2
-diprotomer model with interactive a subunits, in which
the E1 and E2 states coexist depending on the ligand conditions (Fig 11) The consistency between the pro-posed model and the crystal structure is discussed in the last paragraph According to our model [model (2)
in Fig 11], Na+-occluded E1–oligomycin and E2– oligomycin coexist in the presence of 10–30 mm NaCl and 10 lm oligomycin, as shown in Figs 2 and 3 E2– oligomycin is phosphorylated to E2P–oligomycin, which has low-affinity for K+, by pNPP, as shown in Table 1 The Na+-occluded E1–oligomycin complex increases the affinity of E2P–oligomycin for K+ through the interaction between Na+-occluded E1 and
E2P This assumption is supported by the finding that
Na+ transforms the K+-insensitive E2P, which is formed from Pi and has low affinity for K+, to the
K+-sensitive E2P, which has high affinity for K+[38] Increasing the KCl concentration in the presence of NaCl and oligomycin induces the activation curve with two phases (Fig 3) As K+ binding competes with
Na+ binding [15], it is likely that Na+-occluded E1 is transformed to KE2by high K+concentration, so that
Fig 10 Effects of pNPP and ATP on pNPPase and ATPase
activit-ies The activities of ATPase and pNPPase were assayed in a
mix-ture containing 5 lg Na + ⁄ K + -ATPase, 5 m M MgCl2, 50 m M Tris ⁄ Tes
(pH 8.4 at 23 C), 1 m M EDTA, 10 m M NaCl, 0.3 m M KCl, with or
without 1 m M ouabain and (A) 0–30 m M pNPP with 0.1 m M ATP
(n) or 0.1 m M [ 32 P]ATP (m), or (B) 2.5 m M pNPP with 0–3 m M ATP
(n) or [ 32 P]ATP (m) n and m represent pNPPase and ATPase
activ-ity, respectively Plots and bars represent the means ± SD from
three determinations.
Fig 11 Proposed models (1) Activation of pNPPase by K+ and pNPP (2) Activation of pNPPase by Na + , K + , pNPP and oligomycin (3) Activations of pNPPase and ATPase by Na + , K + , pNPP and ATP E1 (Na) represents the Na + -occluded E 1 state E2(s)P and E2(i)P represent the K + -sensitive E2P and K + -insensitive E2P state, respectively lK and hK, (Na) and O represent KCl at low and high concentrations, occluded Na + and oligomycin, respectively P(ATP) and P(pNPP) represent the phosphate transferred from ATP and pNPP, respectively M represents membrane The conformations
of E, E1 (Na) and E2 are referred to the crystal structure of Ca2–E1,
Ca 2 –E1–AMPPCP and E2–thapsigargin, respectively [49–51] The conformation of E2(s) is slightly different from the one of E2(i) The domains including M5, M7 and M10 face each other in a diproto-mer The upper and lower side of the enzyme represent the exter-nal and interexter-nal side of cells, respectively Oligomycin is accessible
to Na + ⁄ K + -ATPase at the external side [52] K + is not transported
by pNPPase activity [53].
Trang 8KE2 exhibits the low-affinity K+-dependent pNPPase
activity
The combination of ATP and NaCl also induced the
high-affinity K+-dependent pNPPase activity (Fig 6)
The K0.5 for K+ decreased from 1 to 0.2 mm, which
was equivalent to that for K+ in Na+⁄ K+-ATPase
activity (Fig 9A) ADP and AMPPCP could not
sub-stitute for ATP (Fig 5B and [8]) The combination of
ATP and NaCl activated both ATPase and pNPPase,
showing that the equilibrium between the E1 and E2
states is dependent on the ligand conditions (Figs 8–
10) According to the Post-Albers scheme [1], Na+
and ATP bind to the E1 state The NaE1ATP formed
is phosphorylated to NaE1P and then converted to
E2P + Na+ Because NaE1P is the Na+-occluded E1
state [20] and the phosphoenzymes are in closer
con-tact [39], it would be understood that NaE1P increases
the affinity of E2P from pNPP for K+ Nandi et al
[40,41] proposed a working model of Na+⁄ K+
-ATP-ase and H+,K+-ATPase They postulated that pNPP
is accessible to the pNPP hydrolytic sites at the
inter-nal and exterinter-nal surfaces However, Garrahan et al
[42] used acetyl phosphate, a membrane-impermeable
substrate, to show that the pNPP hydrolytic site
locates on the internal side of the cell membrane
Therefore, we presumed in a proposed model [model
(3) in Fig 11] that the E1 state and the E2state have a
high-affinity binding site and a low-affinity binding site
for ATP, respectively [25–28] and that pNPP is
inter-nally accessible at the low-affinity site for ATP in the
E2 state but less accessible at the high-affinity site for
ATP in the E1 state, because of a much lower affinity
of pNPP for the high-affinity ATP site (Fig 5 and
Table 1) Our model facilitates understanding of the
results shown in Figs 8–10 In the presence of high
Na+, low K+, moderate pNPP and low ATP
concen-trations, both pNPPase and ATPase were activated,
and higher Na+ concentrations inhibited pNPPase
activity more than ATPase activity (Fig 8) In this
case, ATP binds to the E1 state, which is
phosphoryl-ated to NaE1P On the other hand, pNPP accesses the
low-affinity site for ATP in the E2state, which is
phos-phorylated to E2P (Table 1) NaE1P increases the
affin-ity of E2P for K+ Consequently, K+ binds to the
high-affinity K+site on K+-sensitive E2P and
acceler-ates its dephosphorylation (Fig 4A) An excess of
NaCl may competitively inhibit the effect of K+ on
pNPPase from the external side [43] The combination
of NaCl with ATP or oligomycin slightly enhanced
pNPPase activity even in the absence of KCl (Figs 1D,
5B and 8B) Nagamune et al [44] have demonstrated
ouabain-sensitive pNPPase activity in the absence of
KCl, so these combinations may stimulate the activity
As another possibility, it is likely that NaE1P formed
by pNPP with Na+, as proposed by Yamazaki et al [22], and NaE1P formed by ATP with Na+are sponta-neously dephosphorylated Figure 9A shows that increasing the KCl concentration activated pNPPase but gradually inactivated ATPase Omission of NaCl activated pNPPase but not ATPase (Fig 9B) In this case, KCl concentrations up to 1 mm activate both pNPPase and ATPase Increasing the KCl concentra-tion over 1 mm accelerates dephosphorylaconcentra-tion of E2P from pNPP, whereas the high KCl concentration or the absence of Na+disturbs the phosphorylation from ATP Figure 10A shows that increasing the pNPP con-centration activated pNPPase but partly decreased ATPase activity In this case, pNPP incompletely inhibits the ATP binding to the high-affinity ATP site
in the E1 state Figure 10B shows that increasing the ATP concentration activated ATPase, whereas pNPP-ase was activated by low ATP concentrations but inactivated by high ATP concentrations Because ATP concentrations up to 0.3 mm inhibit phosphorylation from pNPP little (Table 1), both activities are pre-served ATP above 0.3 mm occupies the low-affinity ATP site in the E2 state, so that Na+⁄ K+-ATPase is activated but pNPPase activity is completely blocked Oligomycin noncompetitively affected pNPPase activity in the presence of Na+and ATP (Fig 7) This antibiotic binds in the N-terminal domain of the a subunit [18], whereas ATP binds in the domain con-taining Lys501 of the a subunit [27] NaE1–oligomycin
is an arrested form, whereas NaE1ATP is an active form in the ATP hydrolysis reaction Therefore, the differences in the binding sites and in the biochemical properties between oligomycin and ATP should lead to the noncompetitive effect of oligomycin on ATP Toyoshima et al [45,46] and Sørensen et al [47] have solved the crystal structures of the Ca2–E1, Ca2– nucleotide–E1 and thapsigarigin–E2 states in Ca2+ -ATPase at a high resolution The structures are classi-fied into two groups depending on the structure of the cytoplasmic domain, i.e an open form and a compact form The Ca2–E1 state is an open form [45–47] Bind-ing of the nucleotide converts it into a compact form [47] The compact form induced by binding of the ADP–AlF4 complex, one of the ATP analogues, resembles the E1P state and occludes Ca2+ [47] The thapsigargin–E2 state is a compact form, although its conformation is slightly different from that of the Ca2– nucleotide–E1 state [47] The crystal structure of
Ca2+-ATPase is suggested to be similar to that of
Na+⁄ K+-ATPase [48–50] Therefore, the conforma-tions of the intermediates that appeared in this study were referred to the crystal structures of Ca2+-ATPase
Trang 9(Fig 11) The E state is drawn as an open form This
state may loosely associate with another E state at the
cytoplasmic domain, as shown by Carvalho-Alves et al
[32] E2(i)P, which corresponds to K+-insensitive E2P,
is drawn as a compact form Because the K+
-activa-tion curve of pNPPase showed a positive co-operativity
[51], pNPPase probably works as a diprotomer [model
(1) in Fig 11] The Na+-occluded E1state is drawn as
a compact form E2(s)P, which corresponds to K+
-sen-sitive E2P, may be slightly different from the E2(i)P
state because the affinity for K+ varies dependently
with the configurations of the 4th, 5th and 6th
trans-membrane segments (M4, M5 and M6) [50] It is likely
that Na+-occluded E1 associates with E2(s)P through
M5, M7 and M10 because (a) these segments are
line-arly arranged in the crystal structure [49], (b) M7 and
M10 are almost unmoved by large movement of the
cytoplasmic domain [45–47], and (c) the association
between the transmembrane domains including these
segments is not disturbed by the cytoplasmic domain
in a compact form [45–47] The conversion of the Ca2–
E1 state into the Ca2+-occluded E1 state accompanies
movement of M1 and M2 [45–47] Oligomycin binds to
the domain including M1 and M2 of Na+⁄ K+-ATPase
a subunit [18] Therefore, it is conceivable that arrest
of the movement by oligomycin stabilizes the Na+
-occluded E1state and inhibits Na+transport
Experimental procedures
Materials
ATP(Na)2 and AMPPCP were purchased from
Roche-Diagnostics (Penzberg, Germany) A portion of the
ATP(Na)2 was converted into the sodium-free ATP form
by passing it through a cation-exchange column
Oligo-mycin B and ouabain were purchased from Sigma Chemical
Company (St Louis, MO, USA) Oligomycin B was stored
as a 1-mm solution in cold ethanol pNPP (ditris salt) was
from ICN Biomedicals Inc (Aurora, OH, USA) 86RbCl
and22NaCl were obtained from Amersham Pharmacia
Bio-tech (Amersham, Bucks, UK) [32P]ATP[cP] and32Pi were
obtained from PerkinElmer Life Sciences Japan (Tokyo,
Japan) Other reagents were purchased from Wako Pure
Chemicals Industries, Ltd (Osaka, Japan)
Preparation of Na+⁄ K+-ATPase
Microsomes were prepared from canine kidney outer
medulla and treated with sodium deoxycholate, as described
by Hayashi et al [54] More than 95% of the total
Na+⁄ K+-ATPase activity (about 9 lmol PiÆmin)1Æmg)1)
was ouabain-sensitive under the usual conditions
Synthesis of [32P]pNPP
[32P]pNPP was synthesized by the method of Guan & Dixon [55] 32Pi (33 MBq in 0.02 m HCl) and 0.2 mmol (20 mg) crystalline phosphoric acid were dissolved in dehy-drated acetonitrile to use as starting materials The amount
of pNPP (cyclohexylamine salt) synthesized was measured from the absorbance at 310 nm The amount and the initial specific radioactivity of [32P]pNPP were 51 lmol and 9000 cpmÆnmol)1, respectively
Assay of pNPPase activity
The standard reaction mixture (0.5 mL) was composed of
5 lg Na+⁄ K+
-ATPase, 5 mm MgCl2, 50 mm Tris⁄ Tes (pH 8.4 at 23C), 1 mm EDTA, 2.5 mm pNPP and NaCl and KCl at the concentrations indicated in the figure legends, with or without 0.1 mm ATP, with or without 10 lm oligo-mycin, and with or without 1 mm ouabain The control experiment for the oligomycin effect was performed in the presence of 1% ethanol without oligomycin The pNPPase reaction was started by addition of the enzyme, and the reac-tion was followed for 5–10 min at 37C and stopped by addition of 2.5 mL 0.1 m NaOH The p-nitrophenol liberated was measured from the absorbance at 420 nm Ouabain-sen-sitive activity was determined from the difference between the activities in the presence and absence of 1 mm ouabain When pNPPase activity was measured at 0C, the amount of Na+⁄ K+-ATPase was changed to 20 lg and the concentrations of MgCl2 and pNPP to 1 and 0.5 mm, respectively The incubation time was extended to 90 min
Assay of Na+⁄ K+-ATPase activity
The same reaction mixture (0.5 mL) as that used for pNPP-ase activity was prepared, except that [32P]ATP[cP] (0.1 MBqÆlmol)1) was used instead of nonradioactive ATP The Na+⁄ K+-ATPase reaction was started by the addition
of the enzyme, followed for 5 or 6 min at 37C and stopped by the addition of 0.1 mL ice-cold 50% (w⁄ v) tri-chloroacetic acid containing 2 mm ATP and 2 mm Pi To isolate the liberated32Pi, 0.2 mL 4 m H2SO4⁄ 40 mm ammo-nium molybdate solution and, next, 0.8 mL of an isobutyl alcohol⁄ benzene solution was added to the mixture, which was mixed for 15 min in a vortex mixer [56] The32Pi isola-ted in the organic layer was measured with a liquid scintil-lation spectrophotometer Ouabain-sensitive activity was determined from the difference between the radioactivities
in the presence and absence of ouabain
Ion-binding assay
86Rb+ binding and 22Na+ binding to Na+⁄ K+-ATPase were measured using the centrifugation method developed
Trang 10by Matsui & Homareda [14–18], with slight modifications.
For the86Rb+binding, 30 lg Na+⁄ K+
-ATPase was prein-cubated with or without 0.01 lmol ouabain at room
tem-perature for 30 min in a 80-lL reaction mixture composed
of 0.1 lmol MgCl2, 5 lmol Tris⁄ Tes (pH 8.4 at 23 C),
0–1 lmol NaCl, and 1 lL ethanol with or without 1 nmol
oligomycin The reaction mixture was kept in ice water
After 10 lL 1 mm86RbCl (1 MBqÆlmol)1) and then 10 lL
5 mm pNPP had been added, the reaction mixture (100 lL)
was immediately centrifuged at 368 000 g for 5 min at 2C
in an ultracentrifuge (Hitachi himac CS120FX; Tokyo,
Japan) The supernatant was aspirated, and the inside wall
of each tube was wiped carefully to remove any that
remained Each pellet was dissolved in 0.1 mL 1 m NaOH
by warming at 60C for 20 min; then the entire solution
was transferred to a counting vial, neutralized with
0.15 mL 1 m HCl, which was used to wash the inside of the
tube, and then mixed with a scintillator The radioactivity
was measured using a liquid scintillation
spectrophoto-meter Ouabain-sensitive binding was calculated from the
difference between the radioactivities of the pellets in the
presence and absence of ouabain
For the22Na+binding, 30 lg Na+⁄ K+-ATPase was
sus-pended in a 80-lL reaction mixture composed of 5 lmol
Tris⁄ Tes (pH 8.4 at 23 C), 0.03, 0.1 or 0.3 lmol 22
NaCl (0.2 MBqÆlmol)1), 1 lL ethanol with or without 1 nmol
oligomycin The incubation mixture was kept in ice-cold
pNPP⁄ 10 mm MgCl2had been added, the reaction mixture
(100 lL) was immediately centrifuged
Oligomycin-stimula-ted Na+ binding, which is regarded as the occluded Na+
[13], was calculated from the difference between the
radio-activities of the pellets in the presence and absence of
oligo-mycin
Assay of phosphorylated intermediate
To measure the phosphorylation by pNPP, 50 lg
Na+⁄ K+
-ATPase was preincubated with 0.05 lmol
oua-bain at room temperature for 15–30 min in a 40-lL
reac-tion mixture composed of 0.5 lmol MgCl2 and 5 lmol
Tris⁄ Tes (pH 8.4 at 23 C) Then, the ligands indicated in
Table 1 and 1 lL ethanol with or without 1 nmol
oligomy-cin were added The reaction was started by the addition of
0.1 lmol [32P]pNPP with or without 0.03 lmol Pi(a
reac-tion mixture of 100 lL), followed for 10 min at 37C by
the method of Inturrisi & Titus [57] and stopped by
addi-tion of 1 mL ice-cold 5% (w⁄ v) trichloroacetic acid The
mixture was centrifuged at 14 000 g for 5 min The
precipi-tate was washed three times with 1 mL ice-cold 5%
trichlo-roacetic acid containing 5 mm pNPP and 5 mm Pi and
dissolved in 0.3 mL 1 m NaOH by incubation at 60C for
10 min After neutralization with HCl, the radioactivity of
the precipitate was measured with a liquid scintillation
spectrophotometer The amount of EP was calculated from
the difference between radioactivities of reaction mixtures with native and acid-denatured enzymes
When the phosphorylation by Piwas examined, 0.1 lmol [32P]pNPP and 0.03 lmol Pi in the reaction mixture were replaced by 0.1 lmol pNPP and 0.03 lmol 32Pi, respect-ively The phosphorylation reaction was started by the addition of32Piwith or without pNPP
The reaction mixture (0.5 mL) for phosphorylation by ATP was composed of 50 lg Na+⁄ K+
-ATPase, 5 mm MgCl2, 50 mm Tris⁄ Tes (pH 8.4 at 23 C), 10 mm NaCl, 0.1 mm [32P]ATP[cP] (1 MBqÆlmol)1), with or without 2.5 mm pNPP The phosphorylation reaction was started by addition of the enzyme, followed for 30 s at 0C and stopped by the addition of 0.1 mL ice-cold 5% (v⁄ v) per-chloric acid containing 2 mm ATP and 2 mm Pi The mix-ture was filtered through a membrane filter with a pore size
of 0.45 lm The filter was washed three times with 3 mL ice-cold 5% perchloric acid containing 2 mm ATP and 2 mm Pi, and the radioactivity on the filter was measured with a liquid scintillation spectrophotometer The amount of EP was calculated from the difference between radioactivities of reaction mixtures with native and acid-denatured enzymes
Determination of protein and oligomycin concentration
Protein and oligomycin concentrations were determined as described elsewhere [18,19]
Acknowledgements
We thank Drs R L Post, Y Fukushima and Y Tahara for critical reading and helpful suggestions, Mr S Mik-kaichi for synthesis of [32P]pNPP, and Ms E Hagiwara for technical support
References
1 Post RL (1979) A perspective on sodium and potassium ion transport adenosine triphosphatase In Cation Flux across Biomembranes(Mukohata Y & Packer L, eds),
pp 3–19 Academic Press, New York
2 Glynn IM & Karlish SJD (1975) The sodium pump Annu Rev Physiol 37, 13–55
3 Schwartz A, Lindenmayer GE & Allen JC (1975) The sodium-potassium adenosine triphosphatase: pharmaco-logical, physiological and biochemical aspects Pharma-col Rev 27, 3–134
4 Cavieres JD (1977) The sodium pump in human red cells In Membrane Transport in Red Cells (Ellory JC & Lew VL, eds), pp 1–37 Academic Press, New York
5 Robinson JD & Flashner MS (1979) The (Na++ K+ )-activated ATPase Enzymatic and transport properties Biochim Biophys Acta 549, 145–176