Binding of Pyridine Nucleotide Coenzymes to the
-Subunit of
the Voltage-sensitive K+ Channel*
Si-Qi
Liu,
Hongjun
Jin,
Albert
Zacarias,
Sanjay
Srivastava, and
Aruni
Bhatnagar
From the Division of Cardiology, University of Louisville, and
Jewish Hospital Heart and Lung Institute,
Louisville, Kentucky 40202
Received for publication, September 8, 2000, and in revised form, January 8, 2001
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ABSTRACT |
The
-subunit of the voltage-sensitive
K+ (Kv) channels belongs to the aldo-keto
reductase superfamily, and the crystal structure of Kv
2
shows NADP bound in its active site. Here we report that Kv
2 displays a high affinity for NADPH
(Kd = 0.1 µM) and NADP+
(Kd = 0.3 µM), as determined by
fluorometric titrations of the recombinant protein. The
Kv
2 also bound NAD(H) but with 10-fold lower affinity.
The site-directed mutants R264E and N333W did not bind NADPH, whereas,
the KdNADPH of Q214R was 10-fold
greater than the wild-type protein. The KdNADPH was unaffected by the R189M,
W243Y, W243A, or Y255F mutation. The tetrameric structure of the
wild-type protein was retained by the R264E mutant, indicating that
NADPH binding is not a prerequisite for multimer formation. A C248S
mutation caused a 5-fold decrease in
KdNADPH, shifted the
pKa of KdNADPH from
6.9 to 7.4, and decreased the ionic strength dependence of NADPH
binding. These results indicate that Arg-264 and Asn-333 are critical
for coenzyme binding, which is regulated in part by Cys-248. The
binding of both NADP(H) and NAD(H) to the protein suggests that several
types of Kv
2-nucleotide complexes may be formed in
vivo.
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INTRODUCTION |
The voltage-sensitive K+ (Kv) channels
participate in several cellular processes. In excitable tissues, these
channels play an essential role in establishing the resting membrane
potential and in modulating the frequency and the duration of the
action potential (1). In nonexcitable cells, they are involved in cell
volume regulation, hormone secretion, oxygen sensing, and cell
proliferation (2). The functional diversity of these channels is partly
due to variations in their structure. The ion-conducting pore of these
channels is formed by heterotetramers of different, but structurally
related,
subunits (2, 3). Moreover, the cytoplasmic face of the
Kv
proteins associates with auxiliary
-subunits
(Kv
), which do not participate in ion conductance but
can regulate the activity of the channel (4, 5).
Several homologous genes encoding the Kv
proteins have
been described. A comparison of the amino acid sequences of the
-subunit proteins shows that these proteins have a variable N
terminus and a highly conserved C-terminal domain. The
-subunits
have been assigned to three classes: Kv
1 to 3. In
addition, several splice variants of Kv
1, that is,
Kv
1.1, 1.2, and 1.3, have been reported (for review, see
Refs. 4 and 5). Although some of the
-subunits enhance the
inactivation of the Kv
currents (4, 5), the
physiological role of these proteins remains unclear. In heterologous
systems, coexpression of Kv
increases the surface
expression of Kv
, indicating that the
subunits regulate the expression and/or the localization of the
Kv
proteins. Moreover, Kv
2, which is the
most widely distributed of the
-subunits, does not affect
inactivation even though it associates with Kv
, suggesting that the
-subunits may have other undetermined
physiological functions.
Structural analyses support the view that Kv
proteins
may have unique regulatory properties not displayed by accessory
proteins of other ion channels. The primary amino acid sequence of the Kv
proteins is not related to the auxiliary proteins of
other voltage-sensitive channels but, unexpectedly, to the proteins of
the aldo-keto reductase
(AKR)1 superfamily (6, 7).
Within this superfamily, the amino acid sequences of the
Kv
proteins are most closely related to alfatoxin
reductase (AKR7) and morphine dehydrogenase and 2,5-diketogluconate reductase (AKR5). On the basis of this homology, the Kv
proteins have been assigned to the AKR6 family (8). The AKR proteins catalyze the reduction or the oxidation of a broad range of carbonyl substrates, including aldoses, steroids, prostaglandins, and aldehydes derived from lipid peroxidation (8-11). The sequence homology between
the
-subunits and the AKR proteins suggests that the Kv
proteins are catalytically competent oxidoreductases
that couple metabolic changes to membrane excitability.
The crystal structure of Kv
2 shows that the protein
folds into
8/
8 or the triosephosphate isomerase barrel motif
similar to other AKR proteins (12). A single molecule of
NADP+ was found to co-crystallize with each monomer of the
protein (12). The cofactor was bound to the C terminus of
Kv
2 by active site residues, some of which are conserved
within the AKR superfamily. Nonetheless, no functional data are
available on pyridine nucleotide binding to Kv
. In the
present study, we examined the coenzyme specificity and selectivity of
the purified Kv
2 and investigated the role of individual
active site residues involved in binding pyridine nucleotides.
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EXPERIMENTAL PROCEDURES |
Construction of the Expression Vector for
Kv
2--
The cDNA containing the coding sequence
for Kv
2 was a gift from Dr. Min Li. To generate the
Kv
2 cDNA fragment with a NdeI site at the
5' end and a XhoI site at the 3' end, standard polymerase chain reaction procedures were used. The primers for the full-length
-subunit were 5'-CATATGTATCCGGAATCAACC-3' (forward) and
5'-GGATCCTGACTTAGGATCTATAGTCC-3' (reverse) and for the N-terminal
deleted
-subunit were 5'-AGACAGCTCCATATGTACAGGAAC-3' (forward) and
5'-GGATCCTGACTTAGGATCTATAGTCC-3'. The polymerase chain reaction
products were inserted into pCR-TOPO (Invitrogen), and the amplified
vector was further digested by NdeI and XhoI to
isolate the
-subunit fragments, which were ligated to a linearized pET28a vector cleaved by NdeI and XhoI.
Expression and Purification of Kv
2--
The
expression vectors pET28-F
(full-length Kv
2) and
pET28-C
(
NKv
2, encoding amino acid residues
39-367) were transformed into strain BL21 of Escherichia
coli. The transformed bacteria were cultured at 37 °C in LB
medium containing 50 µg/ml kanamycin. When the absorbance of the
culture medium at 600 nm reached ~0.8, the expression of the
Kv
2 protein was induced by the addition of 1 mM isopropyl-
-D-thiogalactoside. Induction
was continued for another 4 h at 25 °C with constant shaking at
280 rpm. The bacteria were lysed by sonication in a buffer consisting
of 20 mM Tris-HCl, pH 7.9, 200 mM NaCl, and 5 mM imidazole. The cell debris was pelleted by
centrifugation, and the supernatant was applied to a nickel
nitrilotriacetic acid Superflow (Qiagen) column that was
pre-equilibrated with the binding buffer. The protein bound to the
column was eluted by a step change in the imidazole concentration from
50 to 300 mM. The Kv
2 protein was identified by its mobility on 12% SDS-polyacrylamide gel electrophoresis. Fractions containing Kv
2 were collected, pooled, and
dialyzed against 0.15 M potassium phosphate, pH 7.4. The
molecular weight of the purified protein was determined by
size-exclusion chromatography using a TSK-GEL G3000SWXL
(TosoHass, Montomeryville, PA) column and a Waters Alliance HPLC. The
column was equilibrated with 0.4 M potassium phosphate, pH
7.4, and calibrated using thyroglobulin (670 kDa),
-globulin (158 kDa), ovalbumin (43 kDa), and myoglobin (17.6 kDa).
Site-directed Mutagenesis--
Site-directed mutants of
NKv
2 were prepared using QuikChange mutagenesis kit
(Stratagene). Mutation sites were introduced by single-mutant primers
in polymerase chain reaction amplification using the Pfu
Turbo DNA polymerase (Stratagene). The following primers were
used: CTGGGGCACATCAATGTGGAGCTCCATGGAG (R189M), GGTGCCATGACCGCGTCCCCTCTGGCGTGC (W243A),
GGTGCCATGACCTACTCCCCTCTGGCGTGC (W243Y),
CTGGTCCCCTCTGGCGTCCGGCATCGTC (C248S),
GTCTCAGGGAAGTTTGACAGCGGGATCCCAC (Y255F),
CCCATCTGCGAGCGAGCGGAATATCAC (Q214R),
CACCCTACTCCGAAGCCTCCCTGAAG (R264E),
CAACTTATGGAGTGGATTGGAGCAATACAG (N333W).
The sequence of the site-directed mutants was confirmed by DNA
sequencing. The mutants were expressed and purified as described above.
Fluorescence Titrations--
Fluorescence spectra were recorded
on a Shimadzu RF-5301 PC fluorescence spectrophotometer. Unless
indicated otherwise, an excitation wavelength of 290 nm and an emission
wavelength of 335 or 345 nm were used for the fluorometric titrations.
Aliquots of the protein were equilibrated with 2.0 ml of 0.15 M potassium phosphate, pH 7.4. The fluorescence of the
protein was measured before and after the addition of 2-20 µl of the
pyridine nucleotides. To minimize nucleotide absorbance, a 5 × 10-mm cuvette was used for titrations with NAD(H). For measuring the pH
dependence of coenzyme binding, a three-buffer system was used that
consisted of MES, MOPS, and Tris. The pK values of the
individual components of the buffer at an ionic strength of 0.2 M and the amount of salt needed to keep the ionic strength
constant throughout the experimental pH range were calculated using a
computer program (13). The protein concentration was measured by the
Bradford dye binding method (14).
Data Analysis--
Fluorescence titration data were fitted to a
binding equation that takes into account the corrections for scatter,
dilution, and cofactor absorbance (15). In this equation, the
fluorescence intensity I is a function of the cofactor
concentration X, the protein concentration P, and
the dissociation constant Kd, as shown below.
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(Eq. 1)
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In this relationship, Ymin and
Ymax are the minimum and maximum fluorescence
intensities, respectively, Ybgnd is the
intensity of the background scatter,
is
Vinitial/( Vinitial + VX) (the dilution factor), and
is the absorbance
coefficient of the cofactor. The fraction of the protein bound to the
cofactor is related to these parameters as follows.
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(Eq. 2)
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Using 0.1-2 µM protein, we first determined the
approximate Kd of the individual nucleotides. Then,
for final measurement of the Kd, the data were
acquired at a protein concentration less than the expected value of
Kd. The concentration of the active protein
[P] was determined by the curve-fitting procedure under
the condition when the total concentration of the protein was more than
Kd. Typically, ~70% of the protein was found to
be active by this method. The absorbance correction used in the
curve-fitting procedure was verified by titrating solutions of
tryptophan (of equal absorbance as the protein) with NADPH.
The pH dependence of coenzyme binding was analyzed using Equation 3, in
which log Y (=1/Kd) decreases at both
high and low pH (16) as follows.
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(Eq. 3)
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where Ka and Kb are the
dissociation constants of the enzyme, and c is the
pH-independent value of Y. Additionally, Equation 4 was used
to analyze data in which the value of Y decreases at low pH
but levels out to a new value.
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(Eq. 4)
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The ionic strength dependence of pyridine nucleotide binding was
analyzed using the Boltzmann relationship as follows.
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(Eq. 5)
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where Y is the dissociation constant, X is
the ionic strength, Ymax is the maximal value of
the parameter, K1/2 is the value of X
at which Y is half-maximal, and C is the slope
factor. In all cases, the best fit to the data was chosen on the basis
of the standard error of the fitted parameter and the lowest value of
, which is the residual sum of squares divided by the degrees of freedom.
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RESULTS |
As shown in Fig. 1, the purified
wild-type (WT) Kv
2, its N terminus deleted form
(
NKv
2), and the indicated site-directed mutants
migrated as single bands on SDS-polyacrylamide electrophoresis gels.
The molecular masses of these proteins were between 38 and 40 kDa. When
examined by size exclusion chromatography, the
NKv
2 eluted with a retention time of 9.8 min, which corresponds to a Strokes
radius of a protein with a molecular mass of 153 kDa, indicating that
under these conditions, the protein exists primarily as a homotetramer.
No monomeric or dimeric forms of the protein were observed (Fig.
1B). The freshly purified
NKv
2 showed a high absorbance at 260 nm and an additional absorbance band centered near 360 nm (Fig. 2A,
inset), indicating that the purified protein remains bound
to NAD(P)H. From the absorbance at 363 nm, a stoichiometry of ~0.9
mol of NADPH bound/mol of the protein was calculated. To confirm that
the purified preparation was indeed a binary complex, the fluorescence
spectrum of the nucleotide-bound protein was recorded. When excited at
290 nm, the freshly isolated protein showed two prominent emission
bands with peaks at 335 and 450 nm (Fig. 2B). Upon extensive
dialysis against 0.15 M potassium phosphate, pH 7.4, the
intensity of the 335-nm band increased with a corresponding decrease in
the emission band at 450 nm. When 1 µM NADPH was added to
the dialyzed protein, the 450-nm band reappeared, whereas the emission
at 335 nm was quenched (data not shown). The emission band at 450 nm
was not restored by the addition of NADP+, although this
did quench the emission at 335 nm. We conclude, based on these
observations, that NADPH remains bound to the freshly purified
NKv
2 and that it is lost from the protein upon
dialysis. These data also show that the formation of a binary complex
between NADPH and
NKv
2 quenches the intrinsic
tryptophan fluorescence of the protein and leads to the appearance of a
new emission band at 450 nm. However, for all subsequent experiments,
we monitored the emission at 335 nm because, in contrast to the
alterations at 450 nm, changes at 335 nm were independent of the redox
state of the nucleotide.

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Fig. 1.
SDS-polyacrylamide gel electrophoresis and
size exclusion chromatography of
Kv 2. A, wild-type
NKv 2 and NKv 2 and its site-directed
mutants were purified from E. coli by a nickel affinity
column and separated on SDS-polyacrylamide electrophoresis gels.
Approximately 2.0 µg of protein was loaded on the gel and visualized
by Coomassie Blue. Lane M, molecular weight markers;
lanes a-g, NKv 2, R189M, W243A, W243Y,
C248S, Y255F, and full-length Kv 2, respectively. B, a
20-µl aliquot of 1 mg/ml NKv 2 was injected into a
TSK-GEL G3000SWXL column equilibrated with 0.4 M potassium phosphate, pH 7.4. The protein eluted with a
retention time of 9.8 min, corresponding to a Strokes radius of a
protein with a molecular mass of ~ 153 kDa. The broken
trace shows the elution pattern of a different run containing the
molecular mass standards; thyroglobulin (670 kDa), -globulin (158 kDa), ovalbumin (43 kDa), and myoglobin (17.6 kDa).
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Fig. 2.
Retention of NADPH in purified
Kv 2. Freshly prepared
NKv 2 (~1 mg/ml) was suspended in 0.15 M
potassium phosphate, pH 7.4, and scanned for absorbance and
fluorescence. A, absorbance scan of NKv 2.
The inset shows the absorbance of the protein between 310 and 400 nm. B, emission scan of NKv 2 in
phosphate buffer before ( ) and after ( ) dialysis against phosphate
buffer for 2 weeks. Identical protein concentrations were used for the
two emission scans.
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The titration of the extensively dialyzed Kv
2 with NADPH
led to a progressive loss of fluorescence at 335 nm (Fig.
3). The change in fluorescence was
saturated at high nucleotide concentration, and the addition of more
than 0.6 µM NADPH caused no further decrease in
fluorescence. Typically, NADPH quenched a maximum of ~30-40% of the
total fluorescence. Because the protein concentration was an
independent variable in the fitting routine, at protein concentration >Kd, we estimate that 60-70% of the total protein
was bound to NADPH. The KdNADPH of the
full-length Kv
2 was 0.08 ± 0.004 µM
and that of
NKv
was 0.10 ± 0.006 µM. These results suggest that Kv
2 has a
high affinity for NADPH that is not affected by the deletion of the N-terminal domain. Thus for all subsequent experiments, the
NKv
2 protein was used.

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Fig. 3.
Binding of pyridine coenzymes to
Kv 2. Dialyzed
NKv 2 was suspended in 0.15 M potassium
phosphate, pH 7.4, and changes in emission were monitored either at 335 nm (for NADPH) or 345 nm (for NADH) using 290 nm as the excitation
wavelength. The protein was titrated with the indicated concentrations
of NADPH (A) or NADH (B). The concentration of
the protein was adjusted to be below the Kd. Data
are shown as discrete points, and the curves are the best fits to the
data estimated as described in the text.
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In addition to NADPH,
NKv
2 also displayed a high
affinity for NADP+, although
KdNADP+ was 3-fold higher than
KdNADPH (Table
I). The nucleotides, NADH, and
NAD+ were also bound to the protein. However, the large
intrinsic absorbance of these nucleotides at the high concentrations
required for the assay precluded the accurate determination of the
KdNAD(H) under conditions identical to
those used for measuring KdNADP(H).
Hence, to optimize emission and to minimize inner filter effects, a
5 × 10-mm cuvette was used for the assay, and instead of 335 nm,
the emission of the protein was measured at 345 nm. Under these
conditions the absorbance of 0.1 mM NAD(H) was less than 0.05 (see "Experimental Procedures"). The
KdNAD(H) values thus determined were in
the low micromolar range (Table I).
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Table I
The binding of pyridine nucleotide coenzymes and analogs to
Kv 2
Recombinant NKv 2 protein was suspended in 2 ml of 0.15 M potassium phosphate, pH 7.4, and changes in emission at
335 or 345 nm were monitored using an excitation wavelength of 290 nm.
The concentration of the protein used was less than the
Kd. Aliquots of the indicated ligands were added to
the cuvette, and steady-state fluorescence was recorded. The
Kd values were determined as described under
"Experimental Procedures." Data are shown as the mean ± S.D.
(n = 3-7). FAD, flavin adenine dinucleotide; NMN,
nicotinamide mononucleotide; N.D., no detectable change in fluorescence
observed after the addition of 100 µM ligand.
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We next determined the interaction of Kv
2 with different
nucleotide analogs. The Kd of the protein for
3'-acetylpyridine NADP+ was 10-fold greater as compared
with NADP+, indicating that the amide side chain of the
nicotine ring participates in high affinity binding of
NADP+ to the protein. The removal of the 3'-carbonyl from
the nicotine ring also led to a decrease in affinity (compare the
Kd values for 3-aminopyridine NADP+ and
NADP+), suggesting that there are energetically significant
interactions between the 3' side chain of the pyridine ring and the
binding site residues. Other fragments of the pyridine coenzymes such as ADP-ribose, NMN, and nicotinamide displayed poor affinity for
NKv
2. Moreover, the flavin coenzyme, FAD, bound
weakly to the protein, indicating that it is unlikely to be an in
vivo ligand of Kv
2 or to compete with pyridine
coenzymes for binding to the active site of the protein.
The crystal structure of the
NKv
·NADP+
binary complex shows that the coenzyme binds into a deep cleft in the
triosephosphate isomerase scaffolding of the protein (12). When bound,
the cofactor displays an extended conformation and makes several
contacts with the binding site residues. A schematic representation of
these interactions is shown in Fig. 4.
The sequence alignment of the Kv
proteins, using the
program CLUSTLW (17), revealed that most of the residues interacting
with the cofactor in Kv
2 are conserved in other
Kv
proteins (Fig. 5). The
orientation of the nicotinamide ring in Kv
2 is
constrained by H bonding with a basic residue (Arg-189) and
-stacking against an aromatic residue (Trp-243). To examine the
significance of these interactions, site-directed mutants of
NKv
2 were prepared in which Arg-189 was replaced by
methionine, and Trp-243 was replaced by phenylalanine. As shown in
Table II, no significant changes in the
KdNADPH were observed with these
mutations as compared with the WT protein. To confirm that the lack of
change in the KdNADPH was not due to the
retention of hydrophobicity in the tryptophan to phenylalanine
substitution, Trp-243 was replaced with alanine. However, the
KdNADPH of W243A was comparable with
that of W243Y or the WT protein, indicating that ring stacking or the
hydrophobicity of the residue at position 243 does not contribute
to NADPH binding. In contrast, the disruption of the hydrogen bond
between Asn-333 and the adenine ring in the N333W mutant completely
prevented NADPH binding to the protein. Similarly, the replacement of
an arginine replacement of glutamine 214, which interacts with the
hydrogens attached to N7N of the nicotinamide ring,
led to a 20-fold increase in KdNADPH.
This observation indicates that the amide side chain of the pyridine
ring plays a significant role in nucleotide recognition at the
Kv
2 binding site.

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Fig. 4.
Schematic diagram of the interactions between
NADPH and Kv 2. The schematic
is based on the crystal structure of the Kv 2·NADP
binary complex solved by Gulbis et al. (12). The amino acid
residues examined in the present study are shown in boxes.
For clarity, multiple contacts with individual residues are shown
separately. The atoms of NADPH interacting with the indicated residues
are numbered.
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Fig. 5.
The alignment of the amino acids sequences of
the conserved C terminus core of Kv
proteins. The sequences were aligned using the program
CLUSTALW (17). The filled circles indicate the residues
forming contacts with NADP(H). The residues mutated in this study are
boxed. The sequences were obtained from the NCBI protein
data bank: Kv 1 (human, S66503), Kv 2 (rat,
X76724), Kv 3 (rat, S7562), and Kv 4
(mouse, U65593).
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Table II
The binding of NADPH to NKv 2 and its site-directed
mutants
The Kd values of the protein were determined in 0.15 M potassium phosphate, pH 7.4, as described under
"Experimental Procedures." Data are the mean ± S.D.
N.D., no detectable change in fluorescence after the addition of 1 mM NADPH.
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In the
NKv
2·NADP binary complex the oxygen attached
to the ribose phosphate (OP1R) interacts with Tyr-255 via a
water molecule (12), suggesting that this residue may be involved in
coenzyme binding. The replacement of Tyr-255 with phenylalanine,
however, did affect KdNADPH (Table II),
indicating that this residue does not contribute to pyridine nucleotide
binding. In addition to Tyr-255, the water molecule associated with
OP1R forms a hydrogen bond with Cys-248 (12). This cysteine
residue also interacts with the pyrophosphate oxygen
(OP2A1) in a mode reminiscent of the lysine residue
(Lys-262) that is responsible for the tight binding of NADPH to aldose
reductase (AR; Ref. 18). The replacement of Cys-248 by serine, however, increased the affinity of
NKv
2 for NADPH, as evinced
by a decrease in KdNADPH from 100 to 20 nM (Table II).
The coenzyme selectivity of the AKR proteins is in part due to the
presence of basic amino acids in their binding pockets that
accommodates the 2'-phosphate of NADPH (8, 19, 20). The 2'-phosphate
binding pocket of Kv
2 contains only one basic residue,
that is, Arg-264. This residue forms a hydrogen bond with the free
hydroxyl group of the adenine ribose and interacts with
OP4R of the 2'-phosphate (Fig. 4). In our experiments, the replacement of Arg-264 with glutamic acid led to a complete loss of
NADPH binding. The fluorescence of R264E was not quenched even by the
addition of 1 mM of NADPH. These observations suggest that Arg-264 is essential for NADPH binding to Kv
2.
To confirm the results obtained from fluorometric titrations, the
complete fluorescence spectra of the site-directed mutants were
recorded. As expected, the freshly purified N333G and R264E proteins
displayed a much stronger emission band at 335 nm than did equimolar
concentrations of the WT or the C248S protein. Both the WT and C248S
proteins displayed an additional band at 450 nm, which was absent in
the emission spectra of the N333W and the R264E proteins (Fig.
6), indicating that the N333W and R264E proteins do not bind NADPH. When excited at 340 nm (to elicit NADPH
fluorescence), both the WT and the C248S proteins displayed strong
emission near 450 nm, whereas the N333W and the R264E proteins did not;
confirming that the N333W and R264E proteins do not contain NADPH bound
to their active sites. To examine whether the lack of NADPH binding
affects the quaternary structure of the protein, we determined the
Strokes radius of R264E using size exclusion chromatography. The R264E
protein eluted from the HPLC column with a retention time of 9.7 min
(data not shown), which was similar to the retention time of the WT
protein, indicating that binding of NADPH is not essential for the
formation of the
NKv
2 homotetramers.

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Fig. 6.
The fluorescence spectra of the WT, C248S,
R264E, and N333G
NKv 2. The
indicated proteins were purified and suspended in 0.15 M
potassium phosphate, pH 7.4. Aliquots of equal concentrations of the
proteins were excited at either 290 (A) or 340 nm
(B).
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To further characterize coenzyme binding to Kv
2, we
examined the effects of ionic strength and pH. As shown in Fig.
7A, an increase in the ionic
strength of the buffer led to a decrease in
KdNADPH. This dependence was best
described by a Boltzmann function (Equation 5), in which the maximal
value of the KdNADPH
(Ymax) was calculated to be 2.9 ± 0.3 µM, with a K1/2 of 0.59 ± 0.04 M and a slope factor C of 0.15 ± 0.03 × 10-6. The effect of ionic strength on the
KdNADPH of R189M was similar to that
observed with the WT protein. However, the ionic strength dependence
was significantly altered by the C248S mutation. Compared with the WT
protein, C248S was less sensitive to changes in ionic strength. The
best fit of Equation 5 to the data provided the following estimates of
the parameters: Ymax = 1.6 ± 0.4 µM, K1/2 = 0.79 ± 0.15 M, and C = 0.22 ± 0.05 × 10-6. These results support the idea that NADPH binding to
Kv
2 is sensitive to changes in ionic strength within the
physiological range and that this sensitivity is in part due to
Cys-248.

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Fig. 7.
pH and ionic strength dependence of NADPH
binding to Kv 2. A,
The KdNADPH of the WT ( ), R189M
( ), and C248S ( ) NKv 2 was determined in 10 mM HEPES, pH 7.0 at the indicated ionic strength adjusted
with KCl. For clarity, the inset shows a semi-logarithmic
plot of the ionic strength dependence of NADPH binding to the WT
protein. B, the pH-dependence of
KdNADPH of WT ( ) and C248S ( )
Kv 2. Buffers of different pH were prepared using MES,
MOPS, and Tris at a constant ionic strength of 0.2 M. The
data are shown as mean ± S.D. (n = 3), and the
curves are drawn from the best fits of the data
(R2 = 0.96-0.99) to Equation 4 for pH
dependence and Equation 5 for ionic strength dependence of nucleotide
binding.
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The binding of NADPH to Kv
2 was also found to be
sensitive to pH. A systematic evaluation of the effects of pH revealed
that the values of KdNADPH were enhanced
at low pH but decreased at high pH. A plot of log (1/Kd) reached a plateau at low pH, giving rise to a wave-like pH dependence (Fig. 7B). Using Equation 4, a
pKa of 6.9 ± 0.4 was calculated. At high
pH, a slight decrease in Kd was observed, but even
at pH 10, log 1/Kd did not decrease to half its
maximal value, thereby precluding accurate estimates of
pKb. An approximate calculation using Equation 3
indicated that the pKb is near 9.6. This ionization
may be due to Arg-264, but the role of this residue could not be tested
further because the R264E mutant did not bind NADPH. However, our data show that the pKa value depends in part on Cys-248
because the C248S mutation shifted the pKa value
from 6.9 to 7.4 ± 0.2 (Fig. 7B).
 |
DISCUSSION |
The results of this study show that the
-subunit of the
Kv channel preferentially binds NADPH, suggesting that
NADPH may be the most probable ligand bound to Kv
in vivo. Our data further show that NADPH binds to the C
terminus or the conserved AKR core of the protein and that this binding
is not affected by the variable N terminus of the protein. The high
affinity with which Kv
2 binds NADPH is comparable with
the affinity of other AKR proteins for this cofactor (21, 22). Upon
binding NADPH, the intrinsic fluorescence of Kv
2 was
quenched, and an additional emission band appeared that centered around
450 nm. These changes are similar to those observed upon NAPDH binding
to other AKR proteins, such as 3
-hydroxysteroid dehydrogenase
(3
-HSD; Ref. 21) and AR (22). The 450-nm emission of the
3
-HSD·NADPH complex has been suggested to be due to the formation
of a charge-transfer complex between the reduced nicotinamide ring and
Trp-87 located within 10 Å of the ring (21). The Trp-87 of 3
-HSD is
located in the
3 sheet of the triosephosphate isomerase barrel and
is conserved in AR (Trp-79), Kv
(Trp-121), and other AKR
proteins (8), indicating that similar interactions are likely in most
AKRs. Hence, the formation of a low energy charge-transfer complex in the Kv
2·NADPH complex suggests that the functional
characteristics of pyridine coenzyme binding as well as the solution
conformation of the AKR active site are conserved in
Kv
2.
The crystal structure the Kv
2·NADP complex shows
that the coenzyme forms multiple contacts with the protein (12). The
two ends of the coenzyme molecule, the nicotinamide and the adenine rings, interact with residues that are similar to those observed in AR,
3
-HSD, and 2,5-DKGR (23-25). The binding of the nicotinamide ring by 3
-HSD is defined by the interactions between Asn-167 and
O7N, Ser-166 and N7N, and Gln-190
and N7N of NADP+ (24). The corresponding
residues in AR are Asn-160, Ser-159, and Gln-183 (23). Although in
Kv
2 the serine residue (at position 188) and the
glutamine residue (at position 214) are conserved, the O7N
of NADP+ interacts with Arg-189 (12). However, the
interaction between O7N and Arg-189 in Kv
2
does not seem to contribute to the stabilization of the nicotinamide
ring at the binding site, because R189M mutation did not affect
KdNADPH. Thus, the recognition of the
nicotinamide ring appears primarily to be due to the interactions
between the Ser-188 and Gln-214 of Kv
2 and the amino
group of the nicotinamide ring. This view is supported by the
observation that the Kd values of Kv
2
for 3'-acetyl pyridine NADP+ and 3'-amino pyridine
NADP+ were 10-30-fold higher than that for
NADP+ (Table I). Nicotinamide by itself did not bind to the
protein, although ADP-ribose and NMN displayed high, but measurable,
Kd values, suggesting that the coupling of the
adenine and the nicotinamide rings to ribose enhances the binding of
the cofactor to the protein.
The major difference in NADPH binding by the AKR proteins is their
interaction with the pyrophosphate backbone and the 2'-phosphate of the
nucleotide. In AR, the pyrophosphate oxygens interact with the basic
residues Lys-21 and Lys-262, and the 2'-phosphate forms electrostatic
links with Arg-268 and Lys-262 (23). These links form a safety belt,
constraining the coenzyme with an unusually high affinity
(KdNADPH = 6 nM) (26). The
cofactor binding loop of AR and the residues involved in tight binding
(that is, Lys-21, Lys-262, and Arg-268) are absent in other AKR
proteins. In 3
-HSD, the 2'-phosphate of the nucleotide forms
hydrogen bonds with Arg-276 and Arg-270, and these interactions are
responsible for the preference of the protein for NADP+
over NAD+ (24). Similarly, a pair of basic residues
(Lys-232 and Arg-238) interacts with the 2'-phosphate of NADPH at the
active site of 2,5-DKGR (25). However, in Kv
2, Arg-264
is the only basic residue interacting with the coenzyme pyrophosphates,
the hydroxyl of the adenine ribose, and, via a water molecule, the
2'-phosphate (12). An arginine residue is also located at the same
position in Kv
1, although Kv
3 and -4 show
a conserved lysine substitution at this site. The predominant role of
Arg-264 in pyridine coenzyme binding to Kv
2 is suggested
by the observation that the R264E mutation prevented NADPH binding
(Kd > 1 mM). Thus, in Kv
2, Arg-264 seems to play a more important role in
coenzyme binding than do the analogous residues of other AKR proteins.
Site-directed mutations of the phosphate-interacting basic residues of
AR (Arg-268 and Lys-262) and 3
-HSD (Arg-276) increase KdNADPH by 50- to 150-fold (27, 28) but
do not completely prevent NADPH binding. In fact, the R276M mutant of
3
-HSD has a 14-fold lower KdNADH than
does the WT enzyme, indicating that residues other than Arg-276 are
more important in binding the pyrophosphate backbone (28). In contrast,
the lack of coenzyme binding to the R264E mutant of Kv
2
demonstrates that the same arginine residue binds to both the 2'-ribose
phosphate and the pyrophosphate backbone of NADPH, as is evident from
the crystal structure (12). The lack of basic residues other than
Arg-264 may also be responsible for the relatively low selectivity of
Kv
2 for NADPH. The Kv
2 binds NADP(H) with
a 10-fold higher affinity than NAD(H) (Table I). In comparison,
3
-HSD (28) and AR (27), which have multiple cofactor-interacting
basic residues, display a 800- to 1000-fold higher selectivity for
NADP(H) as compared with NAD(H).
In contrast to Arg-264, the contribution of other residues interacting
with the coenzyme phosphates appears to be minimal. Although the
phosphate-binding site of Kv
2 contains an aromatic residue (Tyr-255) analogous to Phe-272 in 3
-HSD and Tyr-265 in AR,
the role of this residue in coenzyme binding appears to be limited
because the KdNADPH of Y255F was similar
to that of the WT Kv
2. Interestingly, the OP1R of NADPH, which is bound to Lys-262 in AR (23),
interacts with a cysteine residue (Cys-248) in Kv
2. In
the crystal structure of Kv
2·NADP, Cys-248 also
interacts with the 2'-phosphate via a water molecule (12). The K262M
mutation in AR prevents tight binding of NADPH
(KmNADPH increases > 60-fold) and
leads to an increase in the overall catalytic rate of the enzyme (18).
However, the C248S mutation led to a decrease in
KdNADPH, suggesting that the interaction of this
residue with NADPH prevents tight binding of the cofactor. Moreover,
because the C248S mutation also affected the pH and the ionic strength
dependence of KdNADPH, it appears that
the redox state or the ionization of Cys-248 may be an important
determinant of NADPH binding to Kv
2.
The binding of NADPH to Kv
2 was also prevented by the
N333W mutation. The Asn-333 forms H bonds with N7A and
interacts with the hydrogen attached to N6A. Together with
Glu-332, this residue appears to be important in holding and orienting
the adenine ring at the active site (12). This binding motif is
conserved in all AKR proteins. The corresponding residues are Glu-279
and Asn-280 in 3
-HSD, Glu-217 and Asn-272 in AR, and Asn-242 and
Glu-241 in 2,5-DKGR. The Asn-333 of Kv
2 is also conserved in Kv
1.
However, the corresponding residue in Kv
3 and -4 is a histidine
(Fig. 5). Although the role of these residues in NAD(P)(H) binding to other AKR proteins has not been examined, our results show that Asn-333
is critical for NADPH binding to Kv
2. Moreover, the
interaction of Asn-333 with the adenine ring appears to be more
significant than the corresponding interaction of the amino groups of
the nicotinamide ring with Gln-214 because the Q214R mutation did not
completely prevent binding, even though
KdNADPH was increased 10-fold.
Surprisingly, we found that
-stacking against tryptophan does not
contribute to NADPH binding. It has been suggested that the
-stacking of the nicotinamide ring against an aromatic residue
(Tyr-216 in 3
-HSD, Tyr-209 in AR, and Trp-187 in 2,5-DKGR)
stabilizes the binding of the cofactor at the active site of AKR
proteins (23, 24). However, the KdNADPH
of W243A was similar to that of the WT protein, suggesting that ring
stacking does not make a significant contribution to nucleotide binding. Nonetheless, our results do not rule out the possibility that
this stacking may be important for the proper orientation of the
nicotinamide ring and for facilitating hydride transfer from the B-face
of the cofactor.
Our observation that Kv
displays high affinity for NADPH
further strengthens the view that the
-subunit may be an enzyme with
oxidoreductase properties. Although the catalytic properties of
Kv
have not been reported, the higher affinity of this
protein for NADPH as compared with NADP+ indicates that the
-subunit is more likely to be a reductase rather than an oxidase.
Moreover, because the protein did not bind NADPH as tightly as AR, it
appears likely that the range of the substrates of Kv
may be more restricted than that of AR. The wide substrate specificity
of AR is in part due to its tight binding to NADPH, that provides most
of the energy required to achieve the transition state. The
contribution of the substrate binding step is minimal, thereby enabling
AR to catalyze the reduction of a wide range of aldehydes (29). In
contrast, AKR proteins such as 3
-HSD, which do not bind NADPH very
tightly, recognize a narrower range of structural motifs (30).
Therefore, the Kv
is likely to recognize a limited set
of substrates, making the empirical identification of this set somewhat difficult.
The binding of pyridine nucleotides to Kv
could also
serve noncatalytic functions. Nucleotide binding may be required for the structural stability of the protein or for the formation of Kv
·Kv
or
Kv
·Kv
multimers. Our results
showing that R264E, which does not bind NADPH, is a homotetramer
suggest that nucleotide binding is not a prerequisite for maintaining
structural integrity or for Kv
-Kv
interactions. Additionally, the binding of different nucleotides to
Kv
may be able to differentially regulate Kv
channel activity, thereby allowing the channels to "sense" the
redox state of the cellular pyridine nucleotides pool. The relatively
poor nucleotide discrimination by Kv
2 is consistent with
this idea and indicates that several forms of protein-nucleotide
complexes can exist in vivo.
Although our measurements show that the
KdNADP(H) is 10-fold higher than
KdNAD(H), the nature of the cofactor
bound to Kv
2 in vivo will depend upon the
relative cellular concentration of pyridine coenzymes that will compete
with NADPH for binding. For the NADPH/NADP+ couple this
competition could be described by the following relationship (32).
|
(Eq. 6)
|
where YNADPH is the fraction of the protein
bound to NADPH. At NADPH = 50 µM and
NADP+ = 15 µM, which is near their cellular
concentrations (31), we estimate that more than 90% of the protein
will be bound to NADPH (assuming the Kd values
listed in Table I). However, when NAD+ is the competing
nucleotide, only 75% of Kv
will be bound to NADPH
because the cellular concentration of NAD is 10-fold higher than that
of NADPH (31). Thus, the
-subunit may be capable of sensing the
relative concentrations of pyridine coenzymes such that its
conformation and its ability to bind and modulate Kv
may
depend upon whether it is bound to NADP(H) or NAD(H). Furthermore, because the cytoplasmic concentrations of NADP(H) and NAD(H) vary with
the rate of metabolism and the oxygen concentration (48), differential
nucleotide binding may be relevant to the oxygen-sensing ability of
Kv
that has been demonstrated recently (33).
Despite their low affinity, the binding of nucleotide analogs to
Kv
2 may also be of physiological significance.
Nucleotide analogs such as NMN and ADP-ribose are generated during
cellular metabolism and by DNA degradation (34), for instance,
during apoptosis. Because apoptosis in some cases is mediated by the activation of Kv channels (35) and the bacterial apoptotic
proteins Reaper and Grim increase the inactivation of the Shaker-type
K+ channels (36), the regulation of Kv
by
the metabolites of pyridine coenzymes warrants further investigation.
Additionally, our observation that the high affinity form of
Kv
can change to a low affinity form by changing the
ionic strength of the medium raises the possibility that in
vivo the Kv
protein may exist in two discrete
states. Although demonstrated here in terms of ionic strength, the
transition between these two states may also be regulated by other
conditions, such as coupling with Kv
or the membrane voltage.
In summary, we report here for the first time that
Kv
2 displays high affinity and selectivity for NADPH and
other related nucleotides. The key residues involved in the recognition
and binding of NADPH were identified by site-directed mutagenesis. Specific mutations that can increase (C248S), decrease (Q214E), or
prevent (R264E and N333W) NADPH binding to Kv
2 were
identified. These mutations may be useful for further probing the role
of pyridine nucleotides in regulating the function(s) of
Kv
. Because, with the exception of Asp-333, these
residues are conserved among the Kv
family of proteins,
it appears that the residues identified in Kv
2 play a
similar role in the binding of pyridine nucleotide coenzymes to other
Kv
proteins as well. Further characterization of NADPH
binding to Kv
is necessary to understand the mechanisms by which pyridine nucleotides modulate the activity of the
voltage-sensitive potassium channels and their role in surface
excitability, osmo-regulation, oxygen sensing, and cell survival.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Min Li for providing
the Kv
2 cDNA and Dr. D. K. Wilson for helpful
suggestions and insightful discussions. We also thank Todd Downes for
help in the preparation of the manuscript and the figures.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL55477 and HL59378.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Cardiology,
Dept. of Medicine, Jewish Cardiovascular Research Center, 500 South
Floyd, University of Louisville, Louisville, KY 40202. Tel.:
502-852-4883; Fax: 502-852-2570; E-mail: aruni@louisville.edu.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M008259200
 |
ABBREVIATIONS |
The abbreviations used are:
AKR, aldo-keto
reductase;
HPLC, high pressure liquid chromatography;
MES, 4-morpholineethanesulfonic acid;
MOPS, 4-morpholinepropanesulfonic
acid;
WT, wild type;
AR, aldose reductase;
HSD, hydroxysteroid
dehydrogenase;
2, 5-DKGR, 2-5-diketo-D-gluconic acid
reductase.
 |
REFERENCES |
1.
|
Hille, B.
(1991)
Ionic Channels of Excitable Membranes
, Sinauer Associates, Inc., Sunderland, MA
|
2.
|
Kolb, H. A.
(1990)
Rev. Physiol. Biochem. Pharmacol.
115,
51-91[Medline]
[Order article via Infotrieve]
|
3.
|
Shi, G.,
Nakahira, K.,
Hammond, S.,
Rhodes, K. J.,
Schechter, L. E.,
and Trimmer, J. S.
(1996)
Neuron
16,
843-852[Medline]
[Order article via Infotrieve]
|
4.
|
Xu, J.,
and Li, M.
(1998)
Trends Cardiovasc. Med.
8,
229-234[CrossRef]
|
5.
|
Pongs, O.,
Leicher, T.,
Berger, M.,
Roeper, J.,
Bahring, R.,
Wray, D.,
Giese, K. P.,
Silva, A. J.,
and Storm, J. F.
(1999)
Ann. N. Y. Acad. Sci.
868,
344-355[Abstract/Free Full Text]
|
6.
|
McCormack, T.,
and McCormack, K.
(1994)
Cell
79,
1133-1135[Medline]
[Order article via Infotrieve]
|
7.
|
Chouinard, S. W.,
Wilson, G. F.,
Schlimgen, A. K.,
and Ganetzky, B.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
6763-6767[Abstract]
|
8.
|
Jez, J. M.,
Bennett, M. J.,
Schlegel, B. P.,
Lewis, M.,
and Penning, T. M.
(1997)
Biochem. J.
326,
625-636[Medline]
[Order article via Infotrieve]
|
9.
|
Srivastava, S.,
Harter, T. M.,
Chandra, A.,
Bhatnagar, A.,
Srivastava, S. K.,
and Petrash, J. M.
(1998)
Biochemistry
37,
12909-12917[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Srivastava, S.,
Watowich, S. J.,
Petrash, J. M.,
Srivastava, S. K.,
and Bhatnagar, A.
(1999)
Biochemistry
38,
42-54[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Jez, J. M.,
Flynn, T. G.,
and Penning, T. M.
(1997)
Biochem. Pharmacol.
54,
639-647[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Gulbis, J. M.,
Mann, S.,
and MacKinnon, R.
(1999)
Cell
97,
943-952[Medline]
[Order article via Infotrieve]
|
13.
|
Liu, S. Q.,
Bhatnagar, A.,
and Srivastava, S. K.
(1993)
J. Biol. Chem.
268,
25494-25499[Abstract/Free Full Text]
|
14.
|
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Ward, L. D.
(1985)
Methods Enzymol.
117,
400-414[Medline]
[Order article via Infotrieve]
|
16.
|
Cleland, W. W.
(1979)
Methods Enzymol.
63,
103-138[Medline]
[Order article via Infotrieve]
|
17.
|
Thompson, J. D.,
Higgins, D. G.,
and Gibson, T. J.
(1994)
Nucleic Acids Res.
22,
4673-4680[Abstract]
|
18.
|
Bohren, K. M.,
Page, J. L.,
Shankar, R.,
Henry, S. P.,
and Gabbay, K. H.
(1991)
J. Biol. Chem.
266,
24031-24037[Abstract/Free Full Text]
|
19.
|
Kubiseski, T. J.,
Green, N. C.,
Borhani, D. W.,
and Flynn, T. G.
(1994)
J. Biol. Chem.
269,
2183-2188[Abstract/Free Full Text]
|
20.
|
Matsuura, K.,
Tamada, Y.,
Sato, K.,
Iwasa, H.,
Miwa, G.,
Deyashiki, Y.,
and Hara, A.
(1997)
Biochem. J.
322,
89-93[Medline]
[Order article via Infotrieve]
|
21.
|
Jez, J. M.,
Schlegel, B. P.,
and Penning, T. M.
(1996)
J. Biol. Chem.
271,
30190-30198[Abstract/Free Full Text]
|
22.
|
Kubiseski, T. J.,
Hyndman, D. J.,
Morjana, N. A.,
and Flynn, T. G.
(1992)
J. Biol. Chem.
267,
6510-6517[Abstract/Free Full Text]
|
23.
|
Wilson, D. K.,
Bohren, K. M.,
Gabbay, K. H.,
and Quiocho, F. A.
(1992)
Science
257,
81-84[Medline]
[Order article via Infotrieve]
|
24.
|
Hoog, S. S.,
Pawlowski, J. E.,
Alzari, P. M.,
Penning, T. M.,
and Lewis, M.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
2517-2521[Abstract]
|
25.
|
Khurana, S.,
Powers, D. B.,
Anderson, S.,
and Blaber, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6768-6773[Abstract/Free Full Text]
|
26.
|
Ehrig, T.,
Bohren, K. M.,
Prendergast, F. G.,
and Gabbay, K. H.
(1994)
Biochemistry
33,
7157-7165[Medline]
[Order article via Infotrieve]
|
27.
|
Kubiseski, T. J.,
and Flynn, T. G.
(1995)
J. Biol. Chem.
270,
16911-16917[Abstract/Free Full Text]
|
28.
|
Ratnam, K.,
Ma, H.,
and Penning, T. M.
(1999)
Biochemistry
38,
7856-7864[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Grimshaw, C. E.
(1992)
Biochemistry
31,
10139-10145[Medline]
[Order article via Infotrieve]
|
30.
|
Penning, T. M.
(1999)
J. Steroid Biochem. Mol. Biol.
69,
211-225[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Hoek, J. B.,
and Rydstrom, J.
(1988)
Biochem. J.
254,
1-10[Medline]
[Order article via Infotrieve]
|
32.
|
Segel, I. H.
(1993)
Enzyme Kinetics
, p. 223, John Wiley & Sons, Inc., New York
|
33.
|
Perez-Garcia, M. T.,
Lopez-Lopez, J. R.,
and Gonzalez, C.
(1999)
J. Gen. Physiol.
113,
897-907[Abstract/Free Full Text]
|
34.
|
Ueda, K.
(1987)
in
Pyridine Nucleotide Coenzymes
(Dolphin, D.
, Avramovic, O.
, and Poulson, R., eds)
, pp. 549-598, John Wiley & Sons, Inc., New York
|
35.
|
Yu, S. P.,
Yeh, C. H.,
Sensi, S. L.,
Gwag, B. J.,
Canzoniero, L. M.,
Farhangrazi, Z. S.,
Ying, H. S.,
Tian, M.,
Dugan, L. L.,
and Choi, D. W.
(1997)
Science
278,
114-117[Abstract/Free Full Text]
|
36.
|
Avdonin, V.,
Kasuya, J.,
Ciorba, M. A.,
Kaplan, B.,
Hoshi, T.,
and Iverson, L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11703-11708[Abstract/Free Full Text]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.