A highly sensitive enzymatic assay for D- and total serine detection using D-serine dehydratase from Saccharomyces cerevisiae

Article abstract of DOI:10.1016/j.molcatb.2010.08.002

D-serine acts as a co-agonist of the N-methyl-D-aspartate (NMDA) receptor, an excitatory glutamate receptor in the mammalian brain that is thought to be involved in higher brain functions such as memory and study. A relationship between the concentration of D-serine in cerebrospinal fluid and neurological disorders such as schizophrenia and amyotrophic lateral sclerosis has been hypothesized. A simple and accurate D-serine assay system would be useful in the study and diagnosis of these diseases. Previously, we developed an enzymatic assay of D-serine using D-serine dehydratase from Saccharomyces cerevisiae, lactate dehydrogenase and NADH. Although this method can detect 10 μM D-serine, a 10-fold increase in sensitivity is required for the assay to be useful for the study and diagnosis of neurological disorders. In this study, we increased the sensitivity of this assay to detect submicromolar concentrations of D-serine. In the new assay, D-serine dehydratase converts D-serine to pyruvate, which is in turn oxidized by pyruvate oxidase. Then, in the presence of horseradish peroxidase, hydrogen peroxide formed during the oxidation converts 10-acetyl-3,7-dihydroxy-phenoxazine (Amplex Red) to resorufin, which exhibits a strong fluorescence. We show that this improved assay can be used to determine the concentration of D-serine in calf serum.

Full text of DOI:10.1016/j.molcatb.2010.08.002

Journal of Molecular Catalysis B: Enzymatic 67 (2010) 150–154
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
A highly sensitive enzymatic assay for d- and total serine detection using
d-serine dehydratase from Saccharomyces cerevisiae
∗
Tomoko Naka, Tomokazu Ito, Hisashi Hemmi, Tohru Yoshimura
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Frou-chou, Chikusa-ku, Nagoya, Aichi 464-8601, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 April 2010
Received in revised form 13 July 2010
Accepted 2 August 2010
Available online 10 August 2010
d-Serine acts as a co-agonist of the N-methyl-d-aspartate (NMDA) receptor, an excitatory glutamate
receptor in the mammalian brain that is thought to be involved in higher brain functions such as memory
and study. A relationship between the concentration of d-serine in cerebrospinal fluid and neurological
disorders such as schizophrenia and amyotrophic lateral sclerosis has been hypothesized. A simple and
accurate d-serine assay system would be useful in the study and diagnosis of these diseases. Previously,
we developed an enzymatic assay of d-serine using d-serine dehydratase from Saccharomyces cerevisiae,
lactate dehydrogenase and NADH. Although this method can detect 10 ␮M d-serine, a 10-fold increase
in sensitivity is required for the assay to be useful for the study and diagnosis of neurological disorders.
In this study, we increased the sensitivity of this assay to detect submicromolar concentrations of d-
serine. In the new assay, d-serine dehydratase converts d-serine to pyruvate, which is in turn oxidized by
pyruvate oxidase. Then, in the presence of horseradish peroxidase, hydrogen peroxide formed during the
Keywords:
®
Amplex Red
d-serine
d-serine assay
d-serine dehydratase
®
oxidation converts 10-acetyl-3,7-dihydroxy-phenoxazine (Amplex Red) to resorufin, which exhibits a
strong fluorescence. We show that this improved assay can be used to determine the concentration of
d-serine in calf serum.
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
gresses [6]. This may be the result of glutamate toxicity to neurons
due to the overactivation of the NMDA receptor by excess d-serine.
d-Serine is also found in human urine [7], at a much higher concen-
tration than in mouse or rat urine [8,9]. However, the physiological
function of urinary d-serine is not known.
A rapid and simple method for assaying d-serine would be
useful for studying its physiological role and clinical importance.
d-Amino acids are typically detected by high performance liquid
chromatography (HPLC), after they are derivatized to fluorescent
diastereomers [10,11]. This method is highly sensitive and can
determine the presence of d- and l-amino acids simultaneously,
but it is time consuming and requires proficiency and expensive
equipments.
With the recent development of analytical methods, several
free d-amino acids have been found to have important physio-
logical functions in eukaryotes, including mammals. For example,
d-serine modulates brain functions by acting as a co-agonist of
the N-methyl-d-aspartate (NMDA) receptor in the mammalian
forebrain [1,2]. Dysfunction of the NMDA receptor may be asso-
ciated with neurodegenerative disorders, such as schizophrenia
and Alzheimer’s disease [3]. Interestingly, the ratio of d-serine to
the total (d- + l-) serine concentration in the cerebrospinal fluid of
schizophrenics [4] and in the serum of Alzheimer’s disease patients
[
5] is significantly lower than in control subjects. This leads the
hypothesis that a lack of d-serine lowers the function of the NMDA
receptor and results in the neuronal disorders. Excess d-serine also
may be related to neuronal disease. For example, in the mouse
model of amyotrophic lateral sclerosis (ALS), the concentration
of d-serine in the cerebrospinal fluid increase as the disease pro-
As a result, we have developed an inexpensive, high-throughput
enzymatic assay method of d-serine [12]. This assay of d-serine uses
d-serine dehydratase from Saccharomyces cereviciae (DsdSC), which
catalyzes the dehydration of d-serine to pyruvate and ammonia
[13]. DsdSC is an eukaryotic d-serine dehydratase that is both
structurally and evolutionally distinct from the bacterial enzyme
discovered by Snell and co-workers [14,15]. Unlike the bacterial
enzymes showing a little activity towards l-serine, DsdSC acts on d-
serine dominantly [13]. Previously we took advantage of the strict
substrate specificity of DsdSC to develop our enzymatic assay of d-
serine. In the assay, DsdSC converts d-serine to pyruvate, which is
detected by a reduction in the concentration of NADH in the cou-
pling reaction with lactate dehydrogenase (LDH) [12]. This assay
Abbreviations: ALS, amyotrophic lateral sclerosis; APF, aminophenyl fluorescein;
DsdSC, d-serine dehydratase of Saccharomyces cerevisiae; HPLC, high performance
liquid chromatography; HRP, horseradish peroxidase; LDH, lactate dehydorogenase;
ꢀ
NMDA, N-methyl-d-asparatate; PLP, pyridoxal 5 -phosphate; XO, xylenol orange.
∗ Corresponding author. Tel.: +81 52 789 4132; fax: +81 52 789 4120.
E-mail address: yosimura@agr.nagoya-u.ac.jp (T. Yoshimura).
1
381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.08.002

T. Naka et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 150–154
151
BY4742 using polymerase chain reaction (PCR) and ligated into the
pET15b vector as described previously in Ref. [13]. The resultant
plasmid encoding the DsdSC with a C-terminal His tag was cloned
6
into Escherichia coli KRX cells (Promega, Madison, WI, USA). DsdSC
was overexpressed and then purified to homogeneity with the Ni-
chelating, anion exchanger DEAE-Toyopearl, and gel permeation
chromatography [13].
Geobacillus stearothermophilus alanine racemase (AR) with an
asparagine mutation at Tyr354 (referred to as “mAR/Y354N”
hereafter) catalyzes the racemization of l-serine at a rate of
8
0 times that of the wild-type enzyme [16]. To construct the
mAR/Y354N gene, the AR gene with N-terminal His6 tag and
thrombin cleavage sequences in the pET15b vector was mutated
with the QuickChange site-directed mutagenesis kit (Agilent Tech-
nologies, Santa Clara CA, USA) using the complementary primer
ꢀ
ꢀ
pairs, 5 -GGAAACGATCAACAACGAAGTGCCTTGC-3 (forward) and
5
ꢀ
ꢀ
-GCAAGGCACTTCGTTGTTGATCGTTTCC-3 (reverse). The mutated
plasmid was cloned into E. coli BL21(DE3) cells to overexpress
mAR/Y354N. Subsequently, mAR/Y354N was purified to homo-
geneity with heat treatment, Butyl-Toyopearl, DEAE-Toyopearl and
gel permeation chromatography [17].
Fig. 1. d-Serine and total (d- + l-) serine assay with DsdSC. The enzymatic assay for
d-serine utilizes three enzymes; DsdSC, pyruvate oxidase and HRP, and the fluores-
cent reagent Amplex® Red. DsdSC converts d-serine to pyruvate, which is oxidized
by pyruvate oxidase. Hydrogen peroxide that is formed during the oxidation is
Pyruvate oxidase and horseradish peroxidase (HRP) were pur-
chased from TOYOBO (Tokyo, Japan) and Sigma–Aldrich (St. Louis,
MO, USA), respectively.
®
detected with HRP and Amplex Red. d-Serine concentrations can be determined
2.2. Reagents
from the fluorescence intensity of the resorufin product. To measure total (d- + l-)
serine content, mutant alanine racemase (mAR/Y354N) catalyzing serine racemiza-
tion can be coupled to the d-serine assay.
Amino acids were purchased from Wako (Osaka, Japan). The
optical purity of the d- and l-serine used in this study was over
9%. His-Bind resin was bought from Novagen (EMD Bioscience,
San Diego, CA, USA). DEAE-Toyopearl 650 M and Butyl-Toyopearl
50 M were obtained from Tosoh (Tokyo, Japan). Amplex® Red was
purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals
were of analytical grade.
9
can also be used for the determination of total (d- + l-) serine
by coupling it with the mutant alanine racemase (mAlaR/Y354N),
which exhibits increased serine racemase activity [16]. l-Serine
concentration could be obtained by the subtraction of the d-serine
concentration from the total serine concentration [12]. With the
LDH-coupling method, we can detect 10–250 ␮M d-serine or total
serine, which is sufficient to detect d-serine in human urine [12].
However, since the concentration of d-serine in human cere-
brospinal fluid and human serum is around 2–5 ␮M [4–6], a 10-fold
higher sensitivity is required for the application of the assay to
the study and diagnosis of neurological disorders by detecting d-
serine in the cerebrospinal fluid or in serum. In this study, we
increased the sensitivity of the assay by using pyruvate oxidase,
horseradish peroxidase (HRP), and a fluorescence reagent, 10-
6
2.3. Equipment and analytical conditions
Fluorescence measurements were made using a RF5399-PC flu-
orospectrophotometer (Shimadzu, Kyoto, Japan) with a 3 mm cell,
or with a Fluoroskan Ascent microplate fluorometer (Thermo Elec-
tron Corporation, Vantaa, Finland). To prepare d- and l-serine for
the HPLC analysis, the amino acids were derivatized to fluores-
cent diastereomers with N-tertbutyloxycarbonyl-l-cysteine and
o-phthaldialdehyde (Boc-l-cysteine/OPA) as described previously
in Refs. [10,11]. The HPLC analysis was performed on a Shimadzu
SLC-10A system (Shimadzu) equipped with a COSMOSIL C-18 col-
umn (Nacalai Tesque, Kyoto, Japan). The derivatized amino acids
were separated by a linear gradient of 0–60% mobile phase B (47%
acetonitrile in a 0.1 M acetate buffer, pH 6.0) in mobile phase
A (7% acetonitrile in a 0.1 M acetate buffer, pH 6.0) for 120 min
at a flow rate of 0.8 ml/min at room temperature. Elution was
monitored with an RF-10A fluorescence detector (Shimadzu) with
excitation and emission wavelengths of 344 and 443 nm, respec-
tively.
®
acetyl-3,7-dihydroxyphenoxazine (Amplex Red) (Fig. 1). In the
new assay, d-serine is converted to pyruvate with DsdSC, followed
by the oxidization of pyruvate with pyruvate oxidase to produce
hydrogen peroxide. Finally, in the presence of HRP, the hydro-
®
gen peroxide oxidizes Amplex Red to resorufin, which exhibits
a strong fluorescence. Using this assay, we can detect submicro-
molar concentration of d-serine, and determine the d- and total
serine concentrations in calf serum.
2
. Experimental
2.1. Preparation of enzymes
2.4. Enzymatic assay of d- and total serine
In the previous studies, we used the DsdSC with an N-terminal
Upon completion of the enzymatic assay of d-serine with DsdSC,
the amount of hydrogen peroxide formed from the oxidation of
pyruvate with pyruvate oxidase was measured with Amplex®
Red and HRP. For the analysis of d-serine, the reaction mixture
consisted of100 mMsodium phosphatebuffer (pH7.5), 20 ␮M pyri-
doxal phosphate (PLP), d-serine (sample), 0.1 unit or no DsdSC,
0.4 unit pyruvate oxidase, 1 unit of HRP and 40 ␮M Amplex®
Red, in a final volume of 250 ␮l. For the analysis of total ser-
ine, 0.6 unit of mAlaR/Y354N was added to this reaction mixture.
His6 Tag [12,13]. However, we found that the DsdSC with a C-
terminal His6 Tag showed similar properties and more efficient
productivity as compared to those of the N-terminal His-tagged
enzyme [Ito et al. manuscript in preparation]. Therefore, we used
the C-terminal His-tagged DsdSC in the present study. The expres-
sion vector of the C-terminal His-tagged enzyme was constructed
as follows. Briefly, the YGL196W gene (DSD1, gene symbol for Sac-
charomyces Genome Database) was amplified from S. cerevisiae

1
52
T. Naka et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 150–154
One unit of DsdSC is defined as the amount of the enzyme con-
◦
verting 1 ␮mol of d-serine to pyruvate per minute at 30 C with
10 mM d-serine. One unit of mAlaR/Y354N was defined as the
amount of the enzyme forming 1 ␮mol of d-serine per minute
◦
at 37 C with 10 mM l-serine. The reaction was started by the
◦
addition of DsdSC and completed by the incubation at 37 C for
3
0 min. Finally, the fluorescence intensity was measured with
excitation and emission wavelengths of 530 and 580 nm, respec-
tively.
2.5. Enzymatic assay of d- and total serine in calf serum
To demonstrate the usefulness of the enzymatic assay, we mea-
sured the d- and total serine concentrations in calf serum. Calf
serum was obtained from Thermo (Melbourne, Australia). It was fil-
tered with an Amicon Ultra-15 (Millipore, Billerica, MA) to remove
proteins, and then treated with borohydride to removal endoge-
nous pyruvate using the following procedure. NaBH4 (1 M) was
Fig. 2. Calibration curves for d- and l-serine standards using the enzymatic assay.
Fluorescence intensity at 580 nm is plotted versus the concentration of l-serine
(
open circle) and d-serine (closed circle). The y-axis shows the difference in the
fluorescence intensity between that obtained with DsdSC and that obtained without
DsdSC. Average values of three measurements were plotted. Other conditions are
described in the text.
diluted 100-fold into filtrated serum, and the solution was incu-
◦
bated for 30 min at 30 C. Then, to remove the unreacted NaBH ,
4
the fluorescence intensities and the concentrations of d- or l-serine
from 0.1 to 5.0 ␮M (Fig. 2). The correlation coefficients for d- and
l-serine were very similar, even when pyruvate or hydrogen per-
oxide was substituted for serine in the assay (data not shown).
These results suggest that both d-serine dehydratase and pyruvate
oxidase catalyzed substrates nearly completely under the assay
conditions.
0
.2 M HCl was added (1% of the volume), and the solution was
◦
incubated at 30 C for another 30 min. The resulting serum solu-
tion was diluted prior to using it in the enzymatic serine assay. The
d- and total serine concentrations in the serum were obtained from
the calibration curve for d- and l-serine in the serum solution (see
Fig. 4). The mean value (y) of the difference in fluorescence inten-
sity between samples with or without DsdSC was plotted against
the amount of d- or l-serine added (x). Then a line, y = ax + b, was
fit to the data. The slope (a) is the fluorescence intensity per unit
concentration of d- or l-serine, and the y-intercept (b) is the flu-
orescence intensity per unit concentration of d- or total serine
naturally occurring in the serum. Therefore, the concentration of
d- or total serine in the assay mixture is calculated from the ratio
b/a.
3.3. Removal of endogenous pyruvate from calf serum
We attempted to apply the enzymatic d-serine assay system to
the measurement of the d-serine concentrations of the calf serum.
Addition of the serum in the assay mixture increased the back-
ground and decreased the sensitivity of the assay system. Such
unfavorable effects of the serum were partially eased by the elim-
ination of the serum protein by the filtration with an Amicon
Ultra-15 (data not shown). However, a high background signal was
still observed when the filtered calf serum was added to the assay
system. Specifically, the fluorescence intensity (Fig. 3, bar B) of the
reaction mixture containing the calf serum, pyruvate oxidase, HRP,
and Amplex® Red was about two times greater than that for the
reaction mixture without pyruvate oxidase (bar A). The closed part
of the bar B, which shows the difference in fluorescence intensity
between A and B, is most likely due to endogenous pyruvate in the
calf serum. To eliminate this endogenous pyruvate, the calf serum
was pretreated with NaBH4, which reduces pyruvate to lactate.
The closed part of bar D, which indicates the difference in fluo-
rescence intensity between C and D, is most likely due to residual
pyruvate in the NaBH4-treated calf serum. These results indicate
that pretreatment of calf serum with NaBH4 decreased the con-
centration of endogenous pyruvate by 83%. In Fig. 3, bars E and F
show the fluorescence intensity of the reaction mixtures contain-
ing DsdSC, pyruvate oxidase and HRP, with either the untreated or
NaBH4-treated serum, respectively. The closed parts of bars E and F
show the difference in fluorescence intensity between E and B, and
that between F and D, respectively. These differences correspond
to the amount of endogenous d-serine in the untreated and NaBH4-
treated serum. Since the areas of the closed parts of bars E and F are
nearly equal, it is unlikely that the pretreatment with NaBH4 has
significant effect on the d-serine assay.
3
. Results and discussion
3.1. Comparison of three pyruvate detection methods using
®
Amplex Red, aminophenyl fluorescein and xylenol orange
In this study, we attempted to increase the sensitivity of the
enzymatic assay of d-serine with DsdSC [12] more than 10-fold
to make it useful for clinical studies of neuronal diseases. One
way to increase the sensitivity is to improve the detection of
pyruvate formed from d-serine. We compared the three differ-
®
ent reagents for detecting pyruvate: Amplex Red, aminophenyl
fluorescein (APF), and xylenol orange (XO). All of these reagents
detect the hydrogen peroxide that is formed during the reaction
between pyruvate and pyruvate oxidase. APF is converted by a
hydroxyl radical to fluorescein, which fluoresces at 538 nm. XO
forms a complex with Fe(III) released during the Fenton reaction
with Fe(II) and hydrogen peroxide. The resulting XO–Fe(III) com-
plex absorbs at 580 nm. All three reagents can be used to detect
1
␮M d-serine in a pure solution (data not shown). However, the
presence of calf serum in the assay mixture reduced the sensitivities
of APF and XO, even in the absence of other proteinaceous compo-
nents (data not shown). Among the three reagents, Amplex® Red
method was the least affected by calf serum. Therefore, we used
the Amplex Red reagent in the enzymatic assay of d-serine with
DsdSC.
®
3
.2. Enzymatic assay of d- and l-serine with Amplex® Red
3.4. Enzymatic assay of d- and total serine in the calf serum
d- And l-serine were detected by using the hydrogen peroxide
Despite the pretreatments, i.e., the membrane filtration and the
NaBH4 reduction, the treated calf serum still affected the sensi-
tivity of the d-serine assay system. Slope of each d- and l-serine
formed from the pyruvate oxidase reaction to convert Amplex®
Red to fluorescent resorufin. There is a linear relationship between

T. Naka et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 150–154
153
Table 1
Comparison of the d- and l-serine concentrations of the calf serum obtained by the
enzymatic assay and the conventional HPLC methods. Values obtained with HPLC
method were the average of four measurements. Other conditions are described in
the text. Details of the enzymatic assay were described in the legend of Fig. 4.
Enzymatic assay
HPLC method
l-Serine
d-Serine
d/(d + l) Ratio
119 ␮M
5.84 ␮M
4.68%
108 ± 4.73 ␮M
5.15 ± 0.200 ␮M
4.55%
genase [12]. To perform the accurate serine assay, we obtained the
calibration curve of d- or l-serine for each measurement (Fig. 4).
For the assay of the total serine in the serum, the calibration curve
for l-serine was obtained (Fig. 4B), because about 95% of serine in
the calf serum is l-enantiomer (see Table 1). As shown in Fig. 4,
there is a linear relationship between the fluorescence intensities
and the concentration of d-serine (Fig. 4A) or l-serine (Fig. 4B) in the
presence of the pretreated calf serum. The linear equations for the
2
d-serine and total serine assays are y = 17.456x + 10.19 (r = 0.999)
2
and y = 30.508x + 7.5954 (r = 0.992), respectively. Difference in the
slopes reflected the difference in the amount of the added calf
serum. The serine concentrations in the assay mixture were cal-
culated by dividing the intercept by the slope. Taking account
of the dilution ratio, the d- and total serine concentrations were
determined to be 5.84 and 124 ␮M, respectively. The l-serine con-
centration determined by subtracting the d-serine concentration
from total serine concentration was 119 ␮M. These values are in
close agreement with those obtained by the conventional HPLC
method, specifically 5.15 and 108 ␮M for d- and l-serine, respec-
tively (Table 1).
Fig. 3. Elimination of endogenous pyruvate in calf serum with NaBH4. The reaction
mixture (250 ␮l) contained 0.1 M potassium phosphate buffer (pH 7.5), 20 ␮M PLP,
4
0 ␮M Amplex® Red, 1 unit of HRP and 25 ␮l of the untreated (A, B, E) or NaBH4-
treated (C, D, F) calf serum. In addition to these ingredients, the reaction mixtures
for B and D included 0.4 unit of pyruvate oxidase, and E and F included 0.4 unit of
◦
pyruvate oxidase and 0.1 unit of DsdSC. After the mixtures were incubated at 37
C
for 30 min, fluorescence intensities were measured with excitation and emission
wavelengths of 530 and 580 nm, respectively.
As mentioned above, d-amino acids are usually detected by
HPLC, after they are derivatized to their fluorescent diastereomers
[10,11]. This method is very sensitive and has the advantage of
being able to determine the concentrations of multiple d- and
l-amino acids simultaneously. However, the HPLC method has
a significant disadvantage in the analysis of biological samples.
Due to the relative abundance of l-amino acids compared to d-
amino acids in organisms, the signals from l-amino acids can mask
those from d-amino acids. In order to accurately measure the
concentrations of d-serine with HPLC, the sample should be co-
chromatographed with a d-serine standard or pretreated with a
d-serine deaminase [18]. Recently, it has been shown that two-
dimensional HPLC (2D-HPLC) can circumvent this inconvenience
by first separating the mixture of d- and l-serine and then sepa-
rating each enantiomer [19]. However, 2D-HPLC is expensive and
impractical for the analyses of many samples. In contrast, our enzy-
matic d-serine assay using DsdSC is relatively inexpensive and can
be used for high-throughput analyses. We anticipate that it will be
useful for studying the relationship between d-serine and various
neurological disorders.
calibration curve changed depending on the amount of the added
serum (see Fig. 4). The reason is not clear, but the serum proba-
bly affects the steps after the DsdSC reaction of the present assay
system (Fig. 1), because the serum did not inhibit our former assay
system consisting of the mAlaR/Y354N, DsdSC and lactate dehydro-
4. Conclusions
We developed a highly sensitive enzymatic d-serine assay
that can detect submicromolar concentrations of d-serine. The
key components of the assay are d-serine dehydratase, pyru-
vate oxidase, horseradish peroxidase, and the fluorescence reagent
Amplex® Red. In the assay, d-serine dehydratase converts d-serine
to pyruvate, which is then oxidized by pyruvate oxidase. Hydro-
gen peroxide that is formed from the pyruvate oxidase reaction
converts Amplex® Red to resorufin, which exhibits a strong fluores-
cence. The assay also can be extended to the measurement of total
Fig. 4. Determination of d- and total serine concentrations of calf serum with the
enzymatic assay. The reaction mixtures for the d-serine assay (A) contained 10%
NaBH4-treated calf serum and 0.1–1.0 ␮M d-serine in a final volume of 250 ␮l. The
reaction mixture for the l-serine assay (B) contained 0.2% NaBH4-treated serum and
(d- + l-) serine assay by including of the mutant alanine racemase
mAR/Y354N. Finally, we showed that this assay could accurately
0
.25–1.5 ␮M l-serine. Average values of three measurements were plotted. Other
conditions are described in the legend of Fig. 2 and the text.
measure d-serine and total serine in calf serum.

1
54
T. Naka et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 150–154
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