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J. Biol. Chem., Vol. 277, Issue 21, 19080-19086, May 24, 2002
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From the Department of Animal Breeding, Graduate School of
Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1,
Bunkyo-ku, Tokyo 113-8657 and the § Department of Integrated
Biosciences, Graduate School of Frontier Sciences, The University
of Tokyo, Kashiwa City, Chiba 277-8562, Japan
Received for publication, January 29, 2002, and in revised form, March 11, 2002
L-Amino acid oxidase
(LAO) was purified from mouse milk. LAO reacted with
L-amino acids in an apparent order of Phe > Met, Tyr > Cys, Leu > His The mammary gland starts milk synthesis at the end of pregnancy to
supply milk to the newborn neonate. Milk contains vital nutrients such as proteins, carbohydrates, lipids, minerals, and vitamins together with bioactive substances including immunoglobulins, bioactive peptides, peptide and steroid hormones, and growth factors (1-4). Antibacterial factors are present in milk as well (5, 6). The
presence of these substances in milk may confer the biological
effect on both the mother and her offspring's survival. Some of these
substances begin to be synthesized upon the onset of milk synthesis in
the mammary gland. It is well established that prolactin
(PRL)1 is a key hormone to
regulate lactose and casein syntheses. PRL is able to stimulate the
mammary gland at the end of pregnancy since the PRL receptor gene is
expressed half a day before parturition (7). To find the genes
expressed highly in the late pregnant mouse mammary gland, mRNA
screening was carried out using differential display methods. One
cDNA fragment was obtained, and the entire nucleotide sequence from
the 5'- to 3'-end was determined in the present experiments. The
nucleotide sequence of this cDNA has high similarity to those for
snake venom L-amino acid oxidase (LAO) (8), snake venom
Apoxin 1 (9), and Fig1 protein in the mouse B cell (10). LAO
catalyzes the oxidative deamination of particular L-amino
acids (i.e. Cys, Phe, Met, Leu, Ile, Pro, and Tyr) and
converts them into hydrogen peroxide (H2O2),
ammonia, and keto acids (11). It has been demonstrated that
administration of LAO isolated from snake venom results in the
depletion of murine plasma amino acids such as Phe, Leu, Tyr,
Met, Ile, and Val (12, 13). As a marker of both the
H2O2 production and the amino acid conversion,
we intended to isolate LAO from mouse milk.
In 1963, Armstrong and Yates (14) reported on concentrations of free
amino acids in human and cow milk and showed that the concentrations of
most free amino acids (except Glu) in milk are lower compared with
those of the serum amino acids of the mothers who produce the milk
samples. The lactating mammary gland almost equally adsorbs all kinds
of free amino acids from the circulating plasma in the rat (15) and sow
(16). Except Cys, Gly, and Leu, the milk of a human, baboon, rhesus
monkey, horse, cow, and pig contains almost equal amounts of amino
acids presented mostly as the protein constituents (17). Although the
concentrations of individual free amino acids in milk are not equally
dependent upon the stage of lactation in the human (18) and sow (19), the imbalance of free amino acids in milk has similarly been observed in a number of species (20). As compared with the amino acid compositions of mouse caseins (21), it is evident that free amino acids
of LAO-convertible species (12, 13, 22) are few in mouse milk
(20). The presence of LAO in milk remains unknown, but our hypothesis
is that the imbalance of the free amino acid composition of milk is due
to the LAO-catalyzed reaction of particular amino acids in the mammary gland.
Here we demonstrate that mouse milk contains LAO and prove our
hypothesis in the present experiments. The physiologically important
role of LAO in milk is also discussed.
Reagents and Chemicals--
o-Dianisidine was
obtained from Tokyo Kasei (Tokyo, Japan); horseradish peroxidase, the
amino acid standard solution (Type H), and oxytocin were from Wako Pure
Chemicals (Osaka, Japan); the silver staining kit and SDS-PAGE
molecular weight markers were purchased from Bio-Rad; Immobilon
was from Millipore Japan (Tokyo, Japan); lysylendopeptidase was from
Roche Diagnostics; 5'- (version 2) and 3'-RACE systems, SuperScript II
reverse transcriptase, oligo(dT) primer, and the RNA size marker were
obtained from Invitrogen; EX Taq DNA polymerase was from
TaKaRa (Kyoto, Japan); the TA cloning kit was from Invitrogen; the auto
cycle sequencing kit and Hybond-N+ membrane were purchased
from Amersham Biosciences; restriction endonucleases and RNA
polymerases were from Toyobo (Osaka, Japan); the DIG RNA labeling kit,
the DIG nucleotide detection kit, and the blocking reagent were from
Roche Molecular Biochemicals; Isogen was from Nippon Gene (Toyama,
Japan); and steroids, amino acids, and the gel-filtration calibration
kit were from Sigma. Primers were obtained from Sawady (Tokyo, Japan).
All other chemicals and reagents were from Wako Pure Chemicals.
Animals--
ICR:JCL mice were purchased from SLC (Shizuoka,
Japan), maintained at 23 ± 1 °C under a lighting schedule of
14 h (lights on 05:00-19:00 h), and given food and water ad
libitum. The day on which a vaginal plug was found was designated
as day 0 of pregnancy. The day of parturition was counted as day 0 of
lactation. On day 13 of pregnancy, adrenalectomy, ovariectomy, and sham
operations were done under pentobarbital anesthesia (23). One mg of
cortisol or progesterone, dissolved in 0.1 ml of sesame oil, was
administered subcutaneously at the completion of the operation (0 h).
Milk Sampling--
Milk was collected from lactating mice on day
8 by mild suction under pentobarbital anesthesia. Oxytocin (0.1 unit)
was injected intraperitoneally before milking. Skim milk was prepared
by centrifugation at 1000 × g for 10 min at 4 °C.
Whey was prepared from skim milk by centrifugation at 100,000 × g for 30 min at 4 °C. All samples were stored at
Determination of the LAO Activity--
The LAO activity was
determined by the production of H2O2.
o-Dianisidine was dissolved in ethanol at 5 mg/ml.
Horseradish peroxidase was dissolved in 0.1 M sodium
phosphate (pH 7.0) at 40 µg/ml. The assay reagent was freshly
prepared and consisted of 1 part o-dianisidine solution and
50 parts peroxidase solution. In the routine assay, L- and
D-Leu (200 µM) were used as a positive and
negative control, respectively. The sample was mixed with a 2.5-fold
volume of the assay reagent. The incubation was carried out at 37 °C
for 120 min unless otherwise indicated in the text. The
absorbance (A) was measured at 420 nm
(A420) in a spectrophotometer. The LAO activity
was expressed on the basis of the o-dianisidine oxidation
(A420).
Purification of LAO--
Proteins were separated on an Amersham
Biosciences FPLC with Superose 12 (HR 16/50) at a flow rate of 0.6 ml/min. The elution buffer consisted of SDS-PAGE and Amino Acid Sequencing--
LAO was denatured in the
presence of 2% SDS with 6% 2-mercaptoethanol at 100 °C for 5 min
and separated by SDS-PAGE on a 7.5% polyacrylamide gel. The gel was
fixed with 7% acetic acid, 20% methanol for silver staining. The
molecular weight of LAO was estimated in comparison with those of
SDS-PAGE molecular weight markers.
The denatured LAO was separated by SDS-PAGE and transferred onto
Immobilon as described above. The protein band stained with Coomassie
Blue was cut out, and amino acid sequence analysis was performed on a
Procise 492 protein sequencing system (Applied Biosystems, Foster City,
CA) under the pulse-liquid phase. For the fragmentation of LAO, the
band was cut out from the gel stained with Coomassie Blue and digested
with lysylendopeptidase for 18 h at 37 °C. The peptide
fragments were separated by HPLC on a Puresil C18 column
(3.9 × 150 mm) (Waters, Milford, MA) with a linear gradient
(0-60%) of 2-propanol:acetonitrile (7:3) in H2O containing 0.1% trifluoroacetic acid in 60 min at the flow rate of 1 ml/min. The amino acid sequence analysis of the peptide fractionated by
HPLC was performed with a Procise 492 protein sequencing system.
Determination of the Amino Acid Content--
Proteins in whey
were precipitated by 5% trichloroacetic acid and removed by
centrifugation at 10,000 × g for 30 min. Free amino
acids and ammonia were analyzed using a Hitachi L-8500A amino acid
analyzer (Tokyo, Japan) according to the manufacturer's instructions.
To determine the conversion of amino acids, the amino acid standard
solution (Type H) was neutralized with 0.1 N NaOH before
the incubation. Amino acids, 125 µM each, were incubated with the test sample at 37 °C. After the incubation, quantities of
free amino acids and ammonia were determined as described above.
Reverse Transcription-PCR and Nucleotide Sequencing--
Total
RNA was extracted from the third thoracic mammary gland using Isogen.
The RNA concentration and protein impurity were determined by measuring
the A260 and
A260/A280, respectively. Total RNA was transcribed to cDNA at 42 °C for 50 min in the
presence of reverse transcriptase and primer using SuperScript II. With cDNA, EX Taq DNA polymerase, and primers, the
PCR was cycled 35 times using a TaKaRa TP2000 thermal cycler. Each
cycle consisted of denaturation at 94 °C for 1 min, annealing at
57 °C for 1 min, and extension at 72 °C for 1 min. The last
reaction was continued for 10 min at 72 °C. The primers used for the
reverse transcription and PCR are given below. Their positions
annealed are also indicated in parentheses.
For differential display, samples were prepared from the mammary glands
on day 16 and 18 of pregnancy. Total RNA was transcribed to cDNA in
the presence of oligo(dT) primer. By use of a set of primers
(5'-CGACTTGA-3' and 5'-ATCGTGCC-3'), cDNA was amplified by the PCR.
One PCR product with about 1.1 kb was detected in the sample collected
on day 18 of pregnancy and sequenced. Based on its partial sequence
information, the nucleotide sequence from the 5'- to 3'-end was
determined as follow. Total RNA was prepared from the mammary gland on
day 18 of pregnancy. For the reverse transcription-PCR, cDNA was
synthesized in the presence of the primer 5'-CGACTTGATGGCGGTGTCTA-3'
(antisense I: 1513-1532 nt). cDNA was amplified by the PCR in the
presence of 5'-GGATGCTGGTCACGAGGTAA-3' (sense I: 263-282 nt) and
5'-CTAGCACTGAGGCCAT-3' (antisense II: 838-853 nt) or in the presence
of 5'-CTGATGAAGGAAGGAACGCT-3' (sense II: 669-688 nt) and
5'-CGACTTGATGGCGGTGTCTA-3' (antisense I). The two PCR products were
used for the DNA sequencing analysis. For the 5'-RACE, cDNA was
prepared in the presence of 5'-CATGACTTCTGAGGCACGAT-3' (antisense III:
487-506 nt). An oligo(dC) was added at the 3'-end using the tailing
system supplied with the kit. The first PCR was carried out in the
presence of abridged anchor primer (supplied with the kit) and
antisense III primer. Nested PCR was performed in the presence of
universal amplification primer (supplied with the kit) and
5'-CCAAGTTCTAAGTACCAGCC-3' (antisense IV: 345-359 nt). DNA obtained by
the nested PCR was used for the sequencing analysis. For the 3'-RACE,
cDNA was prepared in the presence of (dT)15-adaptor
primer (supplied with the kit). The PCR was performed in the presence
of (dT)15-adaptor primer and 5'-TAGACACCGCCATCAAGTCG-3' (sense III: 1513-1532 nt). The PCR product was inserted into the plasmid using a TA cloning kit. The sequence was determined using an
ALF DNA sequencer (Amersham Biosciences) with an auto cycle sequencing
kit. All procedures were carried out according to the instructions
provided with the kit.
Northern Blot and in Situ Hybridization Analyses--
The 591-bp
DNA, amplified in the presence of sense I and antisense II primers, was
subcloned into the plasmid using a TA cloning kit. The plasmid DNA was
digested by EcoRV or HindIII. The DIG-labeled antisense and sense RNA probes were synthesized using the digested plasmid DNA as a template with SP6 and T7 RNA polymerases,
respectively, using a DIG RNA labeling kit. The DIG-labeled RNA probe
was used as a detection probe.
Five µg of total RNA, dissolved in an electrophoresis buffer (25 mM MOPS (pH 7.0), 6 mM sodium acetate, and 1.2 mM EDTA) containing 60% formamide and 7.7% formaldehyde,
was heated at 65 °C for 10 min. The denatured RNA was separated on a
1.2% agarose gel and transferred onto Hybond-N+ membrane.
The membrane was incubated overnight with the antisense RNA probe.
After incubation with an anti-DIG antibody, signals were detected using
a DIG nucleotide detection kit. Ribosomal RNA stained with ethidium
bromide was used as an internal standard.
In situ hybridization was carried out according to Fujimura
et al. (24). In brief, a fresh mammary gland was fixed with paraformaldehyde, dehydrated in ethanol, and embedded in paraffin to
make a 6-µm section. The deparaffined section was digested with
proteinase K. The sections were hybridized with either antisense or
sense RNA probes. After washing and blocking, each section was
incubated with an alkaline phosphatase-conjugated anti-DIG antibody and
then with nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate. The section was counterstained with methylgreen.
Statistics--
The data are expressed as the mean ± SE.
The correlation coefficient (r) was determined by the linear
correlation analysis. The experiments were repeated three to five times
using different milk samples.
Free Amino Acids in Mouse Milk--
The concentrations of free
amino acids in mouse milk were determined (Table
I, column I). Gly, Ala, Ser, and Glu were
the most abundant species, and their concentrations were higher than 240 µM. Concentrations of Lys, Val, Thr, and Arg ranged
from 67 to 131 µM. All other amino acids (Asp, Leu, Ile,
Cys, His, Phe, Met, Tyr, and Trp) were lower than 25 µM.
In particular, Met, Tyr, and Trp were less than 5 µM.
To examine the conversion of free amino acids in whey, amino acids
added exogenously were incubated (Table I, column II). Gly, Ala, Ser,
Glu, Lys, Thr, and Ile remained unchanged. Leu, Cys, His, Phe, Met, and
Tyr decreased by greater than 60%, and among them, Met, Phe, and Tyr
disappeared completely. The apparent decreasing order was Met, Phe,
Tyr > Cys, Leu > His Characterization of LAO--
As the marker of the LAO activity,
the time-dependent production of
H2O2 was measured (Fig.
1). LAO in the presence of
L-Leu produced H2O2 in a
time-dependent manner, and the H2O2
production reached a maximum at ~120 min. D-Leu showed no
effect on the H2O2 production since the amount
of H2O2 produced was the same as that in the
absence of Leu.
The 113-kDa fraction, shown in Fig. 3, was used below as the source of
LAO since the high background observed in the absence of Leu (Fig. 1)
was no longer observed. LAO produced H2O2 in
the presence of L-His, L-Leu, or
L-Phe, while in the presence of D-His, D-Leu, or D-Phe, the production of
H2O2 was not observed. In the presence of
L-His, L-Leu, or L-Phe at various
concentrations, the incubation was carried out at 37 °C for 120 min.
At low concentrations between 20 and 100 µM,
H2O2 increased linearly as the incubation time
progressed (r > 0.95), and the highest production was
observed in the presence of L-Phe at any concentration. To
obtain the linear relationship between the concentration of amino acid
and the A420, the 113-kDa fraction was diluted
with a 2-fold volume of H2O and incubated with
L-Phe, L-Leu, and L-His (0.4, 0.8, 1.6, and 3.2 mM). The concentration of amino acid was
plotted against the A420 according to the
Lineweaver-Burk procedure (data not shown). Each line was
linear (r > 0.96). The apparent Km
of LAO was 1.0 mM for L-Phe, 8.1 mM
for L-Leu, and 20.2 mM for L-His.
The Vmax was in the same order with the
A420 of about 3.9. With confirmed amounts and
types of amino acids, LAO was incubated for 240 min (Fig.
2). Leu, Cys, His, Phe, Met, and Tyr
decreased in a time-dependent manner, while other amino
acids remained unchanged. The apparent decreasing order was Phe > Met, Tyr > Leu, Cys > His Purification of LAO--
Skim milk was separated into the whey and
casein fractions by ultracentrifugation.
The whey fraction was used since it had the LAO activity. Gel
filtration showed that the peak of LAO was detected at the position of
apparent Mr 113,000 (Fig. 3). LAO present
in the 113-kDa fraction was further purified by ion-exchange chromatography (Fig. 4). LAO was eluted
at three different fractions. The high LAO activity was found in
fractions I and II. On the protein basis, LAO with the highest purity
was obtained from fraction I. SDS-PAGE followed by silver staining
showed that fraction I contained one protein species of
Mr 60,000 (Fig.
5).
Amino Acid Sequencing of LAO--
LAO in fraction I was used for
the amino acid sequencing analysis. The N-terminal amino acid sequence
of LAO was LYENLVKXFQDPDYEAFLLI. Amino acid sequences of
lysylendopeptidase-digested peptides were LYENLVK, TYVQK, NPGILGY,
YRTDGPTSALHK, and ATRGHTAL. Among them, the peptide fragment ATRGHTAL
ended at the last Leu and was identified as the C terminus of LAO.
Nucleotide Sequence of LAO cDNA--
The nucleotide sequence
of LAO cDNA is shown in Fig.
6a. The cDNA consisted of
1941 nucleotides plus poly(A) at the 3'-end. The N-terminal Leu was
encoded at nucleotides 111-113. The C-terminal residue was Leu. The
CTT codon was present at nucleotides 1599-1601, and coincidentally the
TAG termination codon appeared at nucleotides 1602-1604. LAO was
encoded at nucleotides 111-1601. The first ATG as the translation
start codon was present at nucleotides 33-35. The signal peptide was
encoded at nucleotides 33-110. The AATAAA polyadenylation signal was
at nucleotides 1643-1648, and the poly(A) tail was present at
nucleotide 1942. The amino acid sequence deduced from the cDNA is
shown in Fig. 6b. The amino acid sequences, determined by
the amino acid sequencing analysis, were found at positions 27-32,
27-46, 124-128, 160-166, 300-311, and 516-523 of deduced LAO. LAO
and its signal peptide consisted of 497 and 26 amino acids,
respectively.
Expression of LAO mRNA in the Mammary Gland--
To examine
the tissue- and stage-dependent expression of LAO, the
level of LAO mRNA was determined by Northern blot analysis. Total
RNA extracted from pregnant mice on day 18 was analyzed (Fig.
7). LAO mRNA in the mammary gland was
detected as a clear band with a length of about 2.0 kb, and the faint
band, probably corresponding to hnRNA, was present at the position of
about 4.6 kb (Fig. 7a). Except in the mammary gland, no
expression was found in the brain, heart, liver, lung, muscle, and
placenta (Fig. 7b).
The expression of LAO mRNA was determined in the mammary glands at
various stages of pregnancy and lactation (Fig.
8). The expression of LAO mRNA was
not detected on day 7 of pregnancy, but the faint band was observed on
day 13 of pregnancy. LAO mRNA clearly increased on day 18 of
pregnancy (1 day before parturition) compared with that on day 16 of
pregnancy. LAO mRNA was expressed throughout lactation, but its
expression was weak at the end of lactation.
The expression of LAO mRNA was examined by in situ
hybridization (Fig. 9). On day 13 of
pregnancy, no signal was detected with either antisense (Fig.
9a) or sense probes (Fig. 9b). On day 16 of
pregnancy (Fig. 9e, antisense), some of the mammary epithelial cells showed a strong signal, but most epithelial cells had
a weak or no positive signal. On day 18 of pregnancy (Fig. 9f, antisense), all of the mammary epithelial cells showed a
strong and positive signal.
Hormonal Control of the LAO Gene Expression--
The alveolus had
a narrow lumen on day 13 of pregnancy (Fig. 9, a and
b). At 16 h after ovariectomy, the lumen expanded
clearly, and numerous milk fat droplets appeared in the mammary gland
(Fig. 9, c and d). LAO mRNA increased clearly
between 8 and 16 h, peaked at around 24 h, and then decreased
considerably (Fig. 10). In
situ hybridization (Fig. 9c, antisense) showed that all
of the mammary cells expressed the LAO mRNA, the expression pattern
being very similar to that seen in the mammary gland on day 18 of
pregnancy (Fig. 9f).
To further determine the progesterone- and/or
glucocorticoid-dependent regulation of the LAO gene
expression, bilateral ovaries and adrenal glands were removed on day 13 of pregnancy. LAO mRNA was not detected in the
ovari-/adrenalectomized mouse mammary gland compared with that in the
ovariectomized control. Administration of cortisol increased LAO
mRNA, while progesterone showed no effect (Fig.
11).
The cDNA encoding mouse milk LAO has a nucleotide sequence
similarity of 53.2, 52.7, and 51.1% with cDNAs for snake venom Apoxin 1 (GenBankTM accession number AF093248), snake venom
LAO (GenBankTM accession number AF071564), and mouse Fig1
protein (GenBankTM accession number U70429), respectively,
as aligned by the Cluster method using the DDBJ program. Recently a
cDNA with 508 bp was cloned from the lactating mouse mammary gland
(GenBankTM accession number BE850855). The same nucleotide
sequence is found in the mouse milk LAO cDNA at nucleotides
1252-1759. FAD is essential in expression of the LAO activity (22,
25). The nucleotide sequence encoding the FAD binding motif (10) was present at nucleotides 207-254.
The mouse mammary gland expressed the LAO gene throughout
lactation. Changes in LAO mRNA expression during lactation mimic those of Mouse milk LAO consisted of two subunits. Three distinct isoenzymes
were detected, the electrically heterogeneous pattern being close to
that seen in snake venom LAO (25, 32). The substrate specificity was
close to that reported for other LAOs (11, 25). Unlike snake venom LAOs
(12, 13, 33, 34), mouse milk LAO hardly oxidized L-Ile.
Mouse milk LAO did not oxidize D-amino acids as seen in the
rat liver and kidney (11). Similar to the other LAO (9, 35), mouse milk
LAO produced H2O2 and ammonia. It is known that
the snake venom LAO becomes inactivated by freezing (35, 36). After the
prolonged storage at It has been shown that LAO in snake venom has potent antibacterial
properties associated with the LAO activity (38).
H2O2-induced DNA damage and cell death have
been attributed to the direct cytotoxicity of
H2O2 and other reactive oxygen species produced
from H2O2 (39, 40). Lactoperoxidase is present
in bovine milk (41). Together with hydrogen peroxide and
SCN We present evidence showing that the lactating mouse mammary gland has
a system capable of producing H2O2 constantly.
As the production of H2O2 is a critical step to
activate the antibacterial system (42), most bacteria do not survive in
the mammary gland. Milk must be stored at low temperatures after
milking. For this reason, it is possible to interpret that LAO is
unable to produce H2O2 since free amino acids
are no longer supplied from outside during storage. Occasionally,
however, bacteria grow in the mammary gland. Mastitis is the most
frequent worldwide disease in the dairy industry and a major cause of
economic loss. It is an inflammatory reaction that most frequently
develops in response to an intramammary bacterial infection (45, 46).
This reaction often results in irreversible damages to the mammary
epithelium even after the successful treatment and in permanent
reduction in milk production (47). In the United States alone, economic
losses are estimated at 2 billion dollars per year (48). In our
preliminary experiments, the LAO activity of bovine milk was lower
compared with that of mouse milk. We speculate that the production of
H2O2 is insufficient to kill bacteria in dairy cows.
*
This work was supported by Grants-in-aid for Scientific
Research 10460122 and 10660266 from the Ministry of Education, Science, Sports and Culture of Japan and by the Tikusan-Gijutu Kyokai at Tokyo.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB034801.
¶
To whom correspondence should be addressed. Tel.:
81-3-5841-5380; Fax: 81-3-5841-8180; E-mail:
asenkiti@mail.ecc.u-tokyo.ac.jp.
Published, JBC Papers in Press, March 20, 2002, DOI 10.1074/jbc.M200936200
The abbreviations used are:
PRL, prolactin;
LAO, L-amino acid oxidase;
r, correlation
coefficient;
RACE, rapid amplification of cDNA ends;
DIG, digoxigenin;
HPLC, high pressure liquid chromatography;
nt, nucleotides;
MOPS, 4-morpholinepropanesulfonic
acid.
Characterization and Expression of L-Amino
Acid Oxidase of Mouse Milk*
,,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
other 11 amino acids
tested and produced H2O2 in a dose- and
time-dependent manner. LAO in milk had a molecular mass of about 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. LAO consisted of two subunits. The N- and C-terminal amino
acid sequence determination followed by cDNA cloning showed that
the 60-kDa protein consisted of 497 amino acids. LAO mRNA spanned
about 2.0 kb, and its expression was found only in the mammary
epithelial cells. Glucocorticoid was essential for LAO gene expression.
Thus, the LAO gene is expressed acutely upon the onset of milk
synthesis. LAO mRNA increased 1 day before parturition, peaked
during early to mid-lactation, and decreased at the end of lactation.
This is the first demonstration showing that LAO is present in milk.
Mastitis is caused by an intramammary bacterial infection. As mouse
milk produced H2O2 using endogenous free amino acids, we suggest that LAO, together with free amino acids, is responsible for killing bacteria in the mammary gland.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
50 °C until use.
20 °C. The protein concentration was determined by measuring the A280.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Amino acid contents of mouse milk (mean ± S.E., n = 5)
Arg, Val other amino
acids. Amino acids with low contents in milk, shown in Table I (column
I), were converted more efficiently and faster than other amino acids
(r = 0.585, p = 0.014).
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Fig. 1.
Time- and L-amino
acid-dependent production of
H2O2. Whey was incubated in the presence
of L- ( 
) and D-Leu (
). The broken
line () shows the absence of Leu.
other amino acids. During
the incubation, the concentration of ammonia increased in a
time-dependent manner (data not shown).
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Fig. 2.
Amino acid species-dependent
conversion. LAO in the 113-kDa fraction was incubated with amino
acids (Type H standard). The 100% value was taken at 0 h. ,
Phe; , Tyr; 
, Met; 
, Leu; 
, Cys; 
, His. Other amino
acids (
) ranged from 92 to 100% at the end of the 4-h incubation
period.
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Fig. 3.
Gel-filtration chromatography of LAO on
Superose 12. The protein concentration ( ,
A280) and LAO activity (filled bars,
A420) are shown with the peak positions of
molecular mass markers.
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Fig. 4.
Ion-exchange chromatography of LAO on
RESOURCE Q. The 113-kDa fraction was applied to the column.
LAO was eluted by a linear gradient (0-500 mM) of NaCl
(dotted line). The protein concentration ( ,
A280) and LAO activity (filled bars,
A420) were determined. Fractions I,
II, and III are indicated by
arrows.
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Fig. 5.
SDS-PAGE and silver staining of LAO.
Lane 1, 113-kDa fraction of gel-filtration chromatography;
lane 2, fraction I of ion-exchange chromatography. The
positions of SDS-PAGE molecular mass markers are indicated.
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Fig. 6.
The nucleotide sequence of LAO cDNA
(a) and deduced amino acid sequence of LAO
(b). The nucleotide and amino acid sequences of
the signal peptide are underlined in panels a and
b, respectively.
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Fig. 7.
Tissue-specific expression of LAO
mRNA. Tissues were collected from the same animal on day 18 of
pregnancy. a, Northern blot of mammary gland RNA. The
positions of RNA size markers are indicated. b, Northern
blot (upper panel) and ethidium bromide staining
(lower panel).
View larger version (59K):
[in a new window]
Fig. 8.
LAO mRNA in the mammary gland during
pregnancy and lactation. A Northern blot (upper panel)
and ethidium bromide staining (lower panel) are shown.
d, day.
View larger version (125K):
[in a new window]
Fig. 9.
In situ hybridization of LAO
mRNA in the mammary gland during pregnancy. Mice (c
and d) were ovariectomized on day 13 of pregnancy.
a, day 13 (antisense); b, day 13 (sense);
c, 16 h after ovariectomy (antisense); d,
16 h after ovariectomy (sense); e, day 16 (antisense);
f, day 18 (antisense). The positive signal was
brown-colored. The sections were counterstained with
methylgreen. The original pictures were taken at the same magnification
(×200).
View larger version (49K):
[in a new window]
Fig. 10.
Time-dependent expression of LAO
mRNA in the mammary gland after ovariectomy. A Northern blot
(upper panel) and ethidium bromide staining (lower
panel) are shown.
View larger version (45K):
[in a new window]
Fig. 11.
Cortisol-dependent expression of
LAO mRNA. Ovariectomy (lanes 1 and 2)
and ovari-/adrenalectomy (lanes 3-6) were performed. Sesame
oil (lanes 1-3), progesterone (lane 4), cortisol
(lane 5), and cortisol plus progesterone (lane 6)
were injected at 0 h. The mammary gland was collected at 0 h
(lane 1) and 16 h (lanes 2-6) after the
injection. A Northern blot (upper panel) and ethidium
bromide staining (lower panel) are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-casein mRNA expression (26) and milk production in mice (27). In ovariectomized mid-pregnant mice, the mammary gland
expresses the PRL receptor and casein genes in a
time-dependent manner after the operation (28). We show
here that the LAO gene is also expressed upon the onset of milk
synthesis. In the circulating plasma, progesterone decreases and
corticosterone increases at the end of pregnancy as well as after
ovariectomy in mice (7). Glucocorticoid (cortisol or corticosterone) is
essential to activate the PRL receptor gene (7) and to maintain the
number of PRL receptors (23). Our data clearly show that the LAO gene
expression is dependent upon cortisol and independent of progesterone.
The similar hormone dependence is shown in the expression of the
mitochondrial Tim23 gene in the mouse mammary gland
(29). In the case of Neurospora crassa, LAO is an inducible
enzyme, and its gene expression is under strong control (30, 31).
Interleukin-4 induces the synthesis of Fig1 in the mouse B cell (10).
It is thus concluded that glucocorticoid is essential for the LAO gene
expression in the mammary gland.
20 or 50 °C, no decrease in the LAO
activity was observed in mouse milk LAO. Similar results are shown in
King cobra (Ophiophagus Hannah) venom LAO (37). The
imbalance of free amino acids in milk is commonly observed among 15 different species examined (20), and we confirmed it in the mouse.
Mouse milk LAO reacted with particular amino acids. We show here that
amino acids convertible by LAO are few in mouse milk. Amino
acids converted by mouse milk LAO could be classified further into
fast-, intermediate- and slow-reactive species. It is expected that the
concentration of H2O2 in milk is kept high and constant.
, lactoperoxidase shows the antibacterial effect (42).
H2O2 with peroxidase also shows the
antibacterial effect (43). As shown here, LAO in the presence of free
amino acids acts as an actual supplier of H2O2.
Because some amino acids are consumed by LAO in the mammary gland,
there is no doubt that their quantities observed at milking are not
parallel with those actually secreted into milk. LAO in whey still
produced H2O2 by utilizing endogenous free
amino acids, the quantity of H2O2 being almost
comparable to that produced in the presence of L-Leu at 200 nmol/ml. LAO produces one molecule of H2O2
through the oxidation of one amino acid (11). We speculate that the
total amount of H2O2 produced in the milk of
the gland is unexpectedly large, probably close to 1 µmol/ml. A high
concentration of H2O2 is also reported in eye
humors of the rabbit (44).
FOOTNOTES
Both authors contributed equally to this work.
ABBREVIATIONS
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