A cDNA encoding CNDP-like protein was also isolated from tilapia brain.. On the other hand, ‘serum’ carnosinase with a narrow Results and Discussion Purification and characterization of
Trang 1Shoji Yamada, Yoshito Tanaka and Seiichi Ando
Faculty of Fisheries, Kagoshima University, Japan
Na-Acetylhistidine is found in high concentration
exclusively in the brain, retina, lens, and occasionally
the heart of poikilothermic vertebrates (bony fishes,
amphibians and reptiles) excluding jawless and
cartila-ginous fishes, but is absent from these tissues in
homothermic vertebrates (birds and mammals) [1–3]
However, little is known about its biological roles in
poikilothermic vertebrates It is synthesized from l-His
and acetyl-CoA by histidine acetyltransferase (EC 2.3.1.33) in the brain and lens [4], and hydrolyzed to histidine by anserinase (Xaa-methyl-His dipeptidase,
EC 3.4.13.5) in the brain and eye [5,6] Baslow & Lenney [5] isolated the enzyme that deacetylates Na-acetylhistidine from the brain of skipjack tuna Katsuwonus pelamis, and thus this enzyme was tempor-arily classified as ‘Na-acetylhistidine deacetylase’ (EC
Keywords
acetylhistidine; anserinase; carnosinase;
cytosolic nonspecific dipeptidase; MEROPS
M20A metallopeptidase
Correspondence
S Yamada, Faculty of Fisheries, Kagoshima
University, 4-50-20 Shimoarata, Kagoshima
890-0056, Japan
Fax: +81 99 2864015
Tel: +81 99 2864172
E-mail: yamada@fish.kagoshima-u.ac.jp
Enzyme
EC 3.4.13.5; recommended name:
Xaa-methyl-His dipeptidase; other names:
anserinase, aminoacyl-methylhistidine
dipeptidase, acetylhistidine deacetylase,
N-acetylhistidine deacetylase,
a-N-acetyl-L -histidine aminohydrolase, X-methyl-His
dipeptidase
Note
The nucleotide sequences reported in this
paper have been submitted to
DDBJ ⁄ EMBL ⁄ GenBank databank with
accession numbers AB179777 for anserinase
and AB219566 for CNDP-like protein.
(Received 7 August 2005, revised 2
September 2005, accepted 23 September
2005)
doi:10.1111/j.1742-4658.2005.04991.x
Anserinase (Xaa-methyl-His dipeptidase, EC 3.4.13.5) is a dipeptidase that mainly catalyzes the hydrolysis of Na-acetylhistidine in the brain, retina and vitreous body of all poikilothermic vertebrates The gene encoding anserinase has not been previously identified We report the molecular identification of anserinase, purified from brain of Nile tilapia Oreochromis niloticus The determination of the N-terminal sequence of the purified anserinase allowed the design of primers permitting the corresponding cDNA to be cloned by PCR The anserinase cDNA has an ORF of 1485 nucleotides and encodes a signal peptide of 18 amino acids and a mature protein of 476 amino acids with a predicted molecular mass of 53.3 kDa Sequence analysis showed that anserinase is a member of the M20A metal-lopeptidase subfamily in MEROPS peptidase database, to which ‘serum’ carnosinase (EC 3.4.13.20) and cytosolic nonspecific dipeptidase (EC 3.4.13.18, CNDP) belong A cDNA encoding CNDP-like protein was also isolated from tilapia brain Whereas anserinase mRNA was detected only
in brain, retina, kidney and skeletal muscle, CNDP-like protein mRNA was detected in all tissues examined
Abbreviations
CNDP, cytosolic nonspecific dipeptidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Trang 2ions and has broad specificity, with ability to
hydrolyze many kinds of substrates such as
Na-ace-tylhistidine, N-acetylmethionine, anserine, carnosine,
homocarnosine (c-aminobutyrylhistidine),
alanylhisti-dine, glycyl-leucine and leucylglycine [6,8,9]
Previ-ously, we reported that this enzyme, purified from the
brain of rainbow trout Oncorhynchus mykiss to
appar-ent homogeneity, is a homodimeric protein with a
subunit of 55 kDa [9] It is commonly believed that
anserinase is universally distributed in poikilothermic
animals containing Na-acetylhistidine in their tissues
[5,6,9–11]
Mammalian tissues contain another peptidase
called carnosinase Carnosinase resembles anserinase in
hydrolytic ability against carnosine, anserine and
homo-carnosine, which are unusual dipeptides containing
non-a-amino acids (i.e b-alanine and c-aminobutyric
acid) No other enzymes except anserinase and
carno-sinase can hydrolyze these three dipeptides Human
tis-sues were aggressively investigated for carnosinase
because its deficiency has been associated with
neuro-logical deficits including intermittent seizures and
men-tal retardation [12,13] Carnosinase exists as two types:
‘tissue’ carnosinase (Xaa-His dipeptidase, EC 3.4.13.3)
and ‘serum’ carnosinase (b-Ala-His dipeptidase, EC
3.4.13.20) [14–16] As ‘tissue’ carnosinase with broad
specificity is present in every human tissue, it has been
suggested that this enzyme is identical with cytosolic
nonspecific dipeptidase (CNDP, EC 3.4.13.18) [16] On
the other hand, ‘serum’ carnosinase with a narrow
Results and Discussion
Purification and characterization of anserinase from Nile tilapia brain
The procedure for the purification of the enzyme from Nile tilapia is summarized in Table 1 The brains were collected from 1000 specimens of Nile tilapia (com-mercial size) Crude extracts from the fish brains were first subjected to ammonium sulfate fractionation Anserinase was recovered from the 50–60% saturated ammonium sulfate fraction The active fraction was then subjected to hydrophobic interaction chromato-graphy on octyl-Sepharose CL-4B When the fractions were screened for hydrolysis against Na-acetylhistidine, the activity was recovered as an unbound fraction (data not shown) The unbound fraction containing anserinase was then subjected to gel filtration using Superdex 200 HR (Fig 1A) The molecular mass of anserinase as determined by gel filtration was
120 kDa The active fractions were pooled, and subjec-ted to anion-exchange chromatography using Resource
Q The bound enzyme was eluted from the column as
a single peak of enzyme activity when the salt concen-tration was 0.30 m (Fig 1B) The active fractions were concentrated, and applied to a preparative native PAGE (Fig 2) As shown in Fig 2A, several protein bands were detected on the native PAGE When the samples extracted from the gel slices were assayed for hydrolysis against Na-acetylhistidine, the activity was
Table 1 Purification of anserinase from brain of Nile tilapia One enzyme unit is defined as that activity of enzyme that catalyzes the hydro-lysis of 1 lmol Na-acetylhistidine in 1 h under the standard conditions.
Step Fraction Total protein (mg) Total activity (U) Specific activity (UÆmg)1) Purification (fold) Yield (%)
Trang 3mainly recovered from gel slice numbers 13 and 14
(Fig 2B) Gel slices 11–15 were subjected to SDS⁄
PAGE under reducing conditions Protein bands were
visualized with silver staining (Fig 2C) The visualized
intensity of the 55-kDa protein band, indicated by an
arrow in Fig 2C, correlated with enzyme activity levels
shown in Fig 2B Moreover, we previously reported
that anserinase purified from brain of rainbow trout
consists of a subunit of 55 kDa [9] Therefore, the
55-kDa protein was assumed to be a single subunit
of Nile tilapia anserinase As the molecular mass of
anserinase determined by gel filtration is 120 kDa in the present study, Nile tilapia anserinase, like the trout enzyme [9], is apparently a homodimeric protein The sample solutions from gel slices 13 and 14 containing high enzyme activity were pooled and concentrated The separation procedures by preparative native PAGE (step 6) resulted in 794-fold purification and 4% of the enzyme activity However, the enzyme was not homogeneous, as there were several protein bands besides anserinase visualized from gel slices 13 and 14,
as shown in Fig 2A Therefore, the final purification was conducted using preparative SDS⁄ PAGE (step 7) The purified enzyme obtained from step 7 showed a single protein band (55 kDa) on SDS⁄ PAGE (Fig 3) The final recovery of the anserinase protein was
110 lg from 345 g fish brain The sequence of the N-terminal 20 amino acids for the purified Nile tilapia anserinase, determined by automated Edman degrada-tion, was FXYMDLAQYVDSXQDEYVEM In the N-terminal sequence, two amino acids expressed as ‘X’
at the 2nd and 13th residue from the N-terminus failed
to be identified for unknown reasons
In Table 2 the substrate specificities of the Nile til-apia anserinase obtained by step 6 were compared with the previous data obtained from trout anserinase [9], which was purified from the trout brain to apparent homogeneity The Nile tilapia and rainbow trout enzymes had similar broad specificities Both enzymes showed strong hydrolytic activity against Na-chloro-acetyl-l-Leu and Gly-Gly Anserine, carnosine and homocarnosine were also hydrolyzed The Nile tilapia enzyme, however, hydrolyzed l-Ala-l-His, l-Leu-Gly and l-Pro-Gly at a much higher rate than the trout enzyme Moreover, the Nile tilapia enzyme hydrolyzed both l-His-Gly and l-Ala-l-Pro, which were hardly cleaved at all by the trout enzyme From these results,
it is likely that the specificity of Nile tilapia anserinase
is broader than that of rainbow trout anserinase
Molecular cloning of anserinase and CNDP-like protein
Database search Initially we searched for a candidate for anserinase in the GenBank database by blastp using the N-terminal sequence of Nile tilapia anserinase An ‘unnamed pro-tein’ product (DDBJ⁄ EMBL ⁄ GenBank accession num-ber CAF95589) of the spotted river puffer Tetraodon nigroviridis was extracted from the database (Fig 4) The N-terminal amino-acid sequence of Nile tilapia anserinase displayed 13 of 18 residues (excluding two residues of X) identical with the deduced amino-acid sequence 19–38 from the N-terminus of the Tetraodon
Fig 1 Gel filtration and anion-exchange chromatography of Nile
til-apia anserinase (A) Gel filtration on a Superdex 200 HR column
(step 4) The concentrated sample from octyl-Sepharose CL-4B
chromatography was applied to a Superdex 200 HR 10 ⁄ 30 column
equilibrated with 50 m M sodium phosphate buffer, pH 7.0,
contain-ing 150 m M NaCl Fractions of 200 lL each were collected at a
flow rate of 0.4 mLÆmin)1 Standard proteins (molecular mass in
parentheses) were aldolase (158 kDa), BSA (67 kDa), ovalbumin
(43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A
(13.7 kDa) (B) Chromatography on a Resource Q column (step 5).
The pooled active fraction from Superdex 200 HR chromatography
was applied to a Resource Q column (1 mL size) equilibrated with
20 m M Tris ⁄ HCl buffer, pH 7.8 The column was washed with
10 mL of the equilibration buffer A linear NaCl gradient (0–0.5 M ;
20 mL of 20 m M Tris ⁄ HCl buffer, pH 7.8) was then applied
Frac-tions of 1 mL each were collected at a flow rate of 0.4 mLÆmin)1.
Trang 4‘unnamed protein’ The organization principle of the
MEROPS peptidase database is a hierarchical
classifi-cation in which homologous sets of the proteins of
interest are grouped into families, and the homologous
families are grouped into clans [19] Therefore, the
blastp program of the MEROPS database was used
to search homologous peptidase genes to the
Tetrao-don‘unnamed protein’ Members of CNDP (MEROPS
ID M20.005) and ‘serum’ carnosinase (MEROPS ID
M20.006) in the M20A metallopeptidase family⁄
sub-family of the MH clan were extracted from the
data-base Multiple sequence alignments of the extracted
genes of vertebrates were performed using the clustal
w program to reveal highly conserved amino-acid
sequences for designing degenerate primers for PCR
amplification (data not shown)
Cloning of anserinase cDNA
PCR was performed using a set of primers (A and B)
and the first-strand cDNA for 3¢ rapid amplification
of cDNA ends (RACE), prepared from total RNA of
Nile tilapia brain, as the template The first-round PCR product was then used as a template for nested PCR amplification using a set of nested primers (A and C) As a result, the 281-bp PCR product was spe-cifically amplified A full-length cDNA sequence of Nile tilapia anserinase was finally obtained by 3¢ and 5¢ RACE PCR (Fig 5) The cDNA contained 1840-bp nucleotides including 56 bp of 5¢ UTR, 1482 bp of ORF, and 299 bp of 3¢ UTR The coding region of the sequence was translated into 494 amino acids, which included a typical signal peptide of 18 amino acids and two potential N-glycosylation sites at the 104th and 134th residue (Asn) from the N-terminus The predic-ted N-terminal amino-acid sequence of the ORF exclu-ding signal peptide completely matched the sequence determined by automated Edman degradation cDNA sequence analysis predicted that two unknown amino acids at the 2nd and 13th residue from the N-terminus
of the purified protein were both histidines The cal-culated molecular mass and isoelectric point of the mature protein, excluding the signal peptide, were
53 311 Da and pH 5.3, respectively
C
Fig 2 Preparative native PAGE (step 6) of Nile tilapia anserinase (A) Preparative native PAGE (7.5% running gel and 4.5% stacking gel) was performed as described in Experi-mental procedures Proteins were stained with Coomassie Brilliant Blue R-250 (B) Un-stained gels were cut into 30 gel slices (numbered 1–30), and each fraction was assayed for hydrolytic activity against Na-acetylhistidine (C) The samples of gel slices 11–15 were subjected to SDS ⁄ PAGE (7.5% running gel and 4.5% stacking gel) under reducing conditions Protein bands were visualized with silver staining Standard pro-teins (STD) were phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), and car-bonic anhydrase (30 kDa) The arrow indi-cates the position of the 55-kDa protein that proved to be anserinase.
Trang 5Cloning of the CNDP-like protein cDNA
We also cloned a full-length cDNA encoding Nile
til-apia CNDP-like protein using a partial nucleotide
sequence of Mozambique tilapia (Oreochromis
mossam-bicus) CNDP-like protein for primer design PCR was
performed using a set of primers (D and E) and the
first-strand cDNA for 3¢ RACE as the template As a
result, the 527-bp PCR product was specifically
ampli-fied To obtain the 3¢ and 5¢ terminal segments of the
cDNA, 3¢ and 5¢ RACE were then performed The
ORF of CNDP-like protein coded for a cytoplasmic
protein (no signal peptide) of 474 amino acids with a
calculated molecular mass of 52 807 Da and isoelectric
point of 5.6 (data not shown) The deduced amino-acid
sequence of CNDP-like protein showed 52% identity
with and 66% similarity to that of anserinase from
Nile tilapia (Fig 6)
Tissue distribution of the mRNAs for anserinase and CNDP-like protein in Nile tilapia
The possibility of genomic contamination was elimin-ated by the observation of amplifications spanning the exon–intron boundaries, which were based on the information of the two scaffold files (M001527 for
Fig 3 SDS ⁄ PAGE of purified anserinase (step 7) The purified
enzyme obtained from preparative SDS⁄ PAGE was subjected to
SDS ⁄ PAGE (12.5% running gel and 4.5% stacking gel) under
redu-cing conditions Protein bands were visualized with Coomassie
Bril-liant Blue R-250 Standard proteins (STD) were phosphorylase b
(94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase
(30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin
(14.4 kDa).
Table 2 Substrate specificity of Nile tilapia anserinase.
Substrate
Relative rates of hydrolysis Nile tilapiaa Rainbow troutb
a
The data from this study The semipurified Nile tilapia enzyme obtained from step 6 (Table 1) was incubated at 30 C for 1 h with
1 m M substrate in 150 m M N-ethylmorpholine ⁄ HCl buffer, pH 6.5, containing 1 m M CoSO 4 bThe data from the previous study [9] The anserinase of rainbow trout was purified to apparent homogeneity, and incubated under the same conditions as in the present study.
Fig 4 Alignment of amino-acid sequences of Nile tilapia anserinase N-terminus and ‘unnamed protein’ product (DDBJ ⁄ EMBL ⁄ GenBank accession number CAF95589) of spotted river puffer Tetraodon nigroviridis Vertical lines indicate amino-acid identities, whereas colons indicate conservative substitutions In the N-terminal sequence of Nile tilapia anserinase, two amino acids expressed as
‘X’ at the 2nd and 13th residue from the N-terminus were not able
to be analyzed for unknown reasons.
Trang 6anserinase-like protein and M001163 for CNDP-like
protein) extracted from the Fugu genome database
The RT-PCR products of the expected size for
CNDP-like protein were observed in all tissues examined
(Fig 7) These distributions of CNDP-like protein in
the fish are exactly consistent with those of human
CNDP In mouse, Otani et al [20] recently reported
that Western blotting analysis using the antibody
against the recombinant carnosine-hydrolyzing protein,
which is identical with CNDP-like protein, revealed
the presence of the protein in kidney, liver, brain and
spleen, and weakly in heart muscle and skeletal muscle
Although no enzymological information for CNDP in
fish is at present available, it is likely that fish
CNDP-like protein plays the same role as mammalian CNDP
On the other hand, anserinase mRNA was expressed
strongly in brain, retina, skeletal muscle and kidney,
and slightly in spleen (Fig 7) We could not detect the
RT-PCR products of the expected size for anserinase
mRNA in any other tissues According to our previous
works [11,21], the enzymatic activity of anserinase was
detected strongly in kidney, brain, liver and ocular fluid, and weakly in skeletal muscle and spleen of Nile tilapia The mRNA expression in the brain and kidney obtained in this study are therefore consistent with the distribution of the enzyme activity It seems likely that the enzyme activity in the ocular fluid originates from anserinase secretion from the retina, in which anseri-nase mRNA was strongly expressed Interestingly, anserinase mRNA was not expressed in the liver, which contained the enzyme activity Therefore, the question arises which tissue is the origin of liver anseri-nase Whereas human ‘serum’ carnosinase is expressed only in central nervous system, rat and mouse ortho-logues are found exclusively in the kidney and are not expressed in the central nervous system [18] Margolis
et al [22,23] revealed that tissues with carnosinase activity can be divided into two groups: kidney, uterus and olfactory mucosa represent one group, and central nervous system, muscle, spleen, etc represent the sec-ond Therefore, it can be suggested that mouse kidney carnosinase is translated from a gene orthologous to
Fig 5 Nucleotide sequence and predicted amino-acid sequence of cDNA encoding Nile tilapia anserinase The putative signal sequence is shown in italics, and the N-terminal amino acids as determined by protein sequencing are underlined Potential N-glycosylation sites are surrounded by rectangles The active site residues are surrounded by ovals, and the metal binding sites are also highlighted in black The terminal stop codon is marked with an asterisk.
Trang 7human ‘serum’ carnosinase Interestingly, the mouse
protein predicted from ‘serum’ carnosinase cDNA,
unlike the human ‘serum’ carnosinase, does not have a
typical N-terminal signal peptide The gene expression and protein distribution of either anserinase or ‘serum’ carnosinase are therefore very complicated in verteb-rate animals
Molecular phylogenetic analysis
We aligned the deduced amino-acid sequences of ver-tebrate M20A genes including the gene of Nile tilapia anserinase and constructed the unrooted phylogenetic tree shown in Fig 8 Gene sequences from ascidian and vertebrate animals cluster within three distinct groups, CNDP-like, ‘serum’ carnosinase-like, and anse-rinase-like types Only one gene was extracted from the databases for the ascidian Ciona intestinalis It
is likely that the ascidian gene is grouped as a CNDP-like type The primary structures of all
Fig 6 Amino-acid alignment of Nile tilapia anserinase and CNDP-like genes The putative signal sequence is underlined Identical amino acids are indicated by an asterisk, and chemically similar amino acids are indicated by dots Gaps inserted into the sequences are indicated
by dashed lines The active-site and metal-binding-site residues are highlighted in gray and black, respectively The deduced amino-acid sequence of the ORF-encoded anserinase was aligned with the encoded CNDP-like protein using CLUSTAL W , showing 52% sequence identity and 66% similarity.
Fig 7 Tissue distribution of the mRNAs for anserinase and
CNDP-like protein Total RNA was prepared from Nile tilapia tissues, and
RT-PCR was performed using specific primers The expected sizes
of the amplified bands of anserinase, CNDP-like protein and
GAPDH were 530, 395 and 517 bp, respectively.
Trang 8CNDP-like proteins are relatively conserved; however,
the function of these proteins is unknown except in
humans [18] In both African clawed frog Xenopus
laevis and Atlantic salmon Salmo salar, three genes,
which are separately grouped into CNDP-like, ‘serum’
carnosinase-like, and anserinase-like types, were
extrac-ted from the databases Darmin, the function of which
is unknown, is a secreted protein expressed during
endoderm development in African clawed frog [24]
From our phylogenetic analysis, Darmin protein is
grouped as an anserinase-like type However, as the
phylogenetic divergence of Xenopus Darmin protein is
a relatively long way from fish anserinase, as shown
in Fig 8, the enzymatic properties of Darmin protein
need to be investigated and compared with those
of fish anserinase Another hypothetical MGC68563
protein of African clawed frog is closely related to a human ‘serum’ carnosinase A homologous gene to
‘serum’ carnosinase in Atlantic salmon was also obtained by assembling three EST clones (TC33189, CX353277 and TC29012) extracted from the databases
We therefore suggest that before the vertebrate–ascidian divergence, an ancestral CNDP gene was first duplica-ted to form the original CNDP and the copied CNDP genes The copied CNDP gene was further dupli-cated to form ‘serum’ carnosinase and anserinase genes This second gene duplication event occurred before the divergence of ray-finned fish and tetrapod lineages
In conclusion, we report the molecular identification
of anserinase, and demonstrate that the enzyme is a member of the M20A metallopeptidase subfamily, as well as ‘serum’ carnosinase and CNDP The anserinase
Fig 8 Phylogenetic tree of anserinase and M20A genes of vertebrate animals The tree was constructed by neighbor-joining distance analy-sis Bootstrap values of 1000 resampling are indicated for all nodes on the tree The scale bar corresponds to the estimated evolutionary dis-tance units In Fugu Takifugu rubripes, the homologous genes to anserinase-like and CNDP-like proteins were located on scaffolds M001527 and M001163, respectively The accession numbers of the homologues extracted from the DDBJ ⁄ EMBL ⁄ GenBank or the TIGR (http:// www.tigr.org/tdb/tgi/) databases are as follows: human Homo sapiens, ‘serum’ carnosinase (NM_032649) and CNDP (BC003176); mouse Mus musculus, ‘serum’ carnosinase-like (NM_177450) and CNDP-like (NM_023149); chicken Gallus gallus, ‘serum’ carnosinase-like (BX931960) and CNDP-like (TC188297); African clawed frog Xenopus laevis, MGC68563 protein (BC060450), Darmin protein (AY166869) and CNDP-like (BC056069); zebrafish Danio rerio, CNDP-like (AY391414); medaka Oryzias latipes, CNDP-like (TC30957); salmon Salmo salar,
‘serum’ carnosinase-like (assembled using TC33189, CX353277 and TC29012), anserinase-like (assembled using CK873786 and TC31285) and CNDP-like (assembled using CK884742, CX352802 and TC22931); ascidian Ciona intestinalis, CNDP-like (TC64855).
Trang 9mRNA was expressed strongly in brain, retina, skeletal
muscle and kidney of Nile tilapia, whereas the CNDP
mRNA was expressed in all tissues It is also expected
that a set of three genes, CNDP-like, anserinase-like
and ‘serum’ carnosinase-like genes, exists in tetrapods
(African clawed frog) and fish (Atlantic salmon)
Fur-ther studies are Fur-therefore required to extensively
investigate the existence of anserinase-like and ‘serum’
carnosinase-like genes in vertebrates
Experimental procedures
Enzyme assay
As Na-acetylhistidine is a major physiological substrate for
anserinase in brain and eye of fish, we used it instead of
anserine as a substrate for the anserinase assay throughout
this study Enzyme activity was assayed as follows: sample
containing enzyme was incubated at 30C for 1 h with
1 mm Na-acetylhistidine in 150 mm N-ethylmorpholine⁄ HCl
buffer, pH 6.5, containing 1 mm CoSO4 [9] The reaction
was terminated by the addition of HClO4at a final
concen-tration of 5% (w⁄ v) The sample was then centrifuged for
15 min at 8000 g to precipitate the protein Released
histi-dine in the supernatant was quantified by HPLC using the
o-phthalaldehyde post-column labeling method [21]
Protein determination
Protein concentration was calculated as the sum of
amino-acid contents after amino-acid hydrolysis (6 m HCl, 24 h) Amino
acid content was determined by HPLC as above
Analytical SDS/PAGE
Electrophoresis in the presence of SDS and
2-mercaptoeth-anol was performed by the method of Laemmli [25], with a
7.5% or a 12.5% polyacrylamide running gel and a 4.5%
polyacrylamide stacking gel Proteins were stained with
0.25% Coomassie Brilliant Blue R-250 in 50% methanol
containing 10% acetic acid or Silver Stain II Kit (Wako
Pure Chemical Industries, Osaka, Japan)
Purification of brain anserinase
All operations were conducted at 0–4C unless otherwise
mentioned Fresh brains (345 g) of Nile tilapia O niloticus
were stored at)20 C
Step 1: extraction
The frozen brains were homogenized with a Polytron
homo-genizer in 10 vol 10 mm sodium phosphate buffer, pH 7.8
The crude homogenate was centrifuged at 20 000 g for 1 h
Step 2: ammonium sulfate precipitation The supernatant was brought to 50% saturation with solid ammonium sulfate and left overnight The precipi-tate was removed by centrifugation (20 000 g, 1 h) and discarded The supernatant was precipitated by increasing ammonium sulfate to 60% saturation and left for 1 h The precipitate was collected by centrifugation (20 000 g,
1 h), dissolved in 67 mL 10 mm N-ethylmorpholine⁄ HCl buffer, pH 7.2, containing 0.1 mm CoSO4 and brought to 30% saturation with solid ammonium sulfate Insoluble material was removed by centrifugation (20 000 g, 1 h) and discarded
Step 3: octyl-Sepharose CL-4B chromatography The supernatant was applied to a column (2.6· 40 cm) of octyl-Sepharose CL-4B (Amersham Pharmacia Biotech) previously equilibrated with 10 mm N-ethylmorpholine⁄ HCl buffer, pH 7.2, containing 30% saturated ammonium sul-fate and 0.1 mm CoSO4 at 7C The column was washed with 500 mL of the equilibration buffer at a flow rate of 1.5 mLÆmin)1, and a linear ammonium sulfate gradient (30–0% saturation; 2 L) was applied The effluent was fractionated into 15-mL portions The active fractions were pooled and concentrated to 1.2 mL by ultrafiltration through a PM-10 membrane (Amicon, Inc.)
Step 4: Superdex 200 HR gel filtration The sample (200 lL) was injected at room temperature into a Superdex 200 HR 10⁄ 30 column (10 · 300 mm; Amersham Pharmacia Biotech) equilibrated with 50 mm sodium phosphate buffer, pH 7.0, containing 150 mm NaCl at a flow rate of 0.4 mLÆmin)1 Fractions of
200 lL each were collected The active fractions were combined and concentrated to 800 lL using a Centricon
10 (Amicon, Inc.) This separation step was separately performed six times (200 lL· 6)
Step 5: Resource Q chromatography The sample was applied at room temperature to a Resource Q column (1 mL; Amersham Pharmacia Biotech) equilibrated with 20 mm Tris⁄ HCl buffer,
pH 7.8, at a flow rate of 1.0 mLÆmin)1 The column was washed with 10 mL of the equilibration buffer for
10 min A linear NaCl gradient (0–0.5 m; 20 mL 20 mm Tris⁄ HCl buffer, pH 7.8) was applied, and 1-mL fractions were collected The active fractions containing the enzyme were concentrated, and the buffer was replaced with
10 mm N-ethylmorpholine⁄ HCl buffer, pH 7.2, containing 30% glycerol, using a centrifugal concentrator (Centricon-10)
Trang 10line⁄ HCl buffer, pH 7.2, using a Centricon-10 An aliquot
of the sample obtained from each gel slice was assayed for
enzyme activity The active fractions of gel slices were
con-centrated to 400 lL using a Centricon-10
Step 7: preparative SDS⁄ PAGE
The concentrate was applied over the width of a gel
slab (11· 14 · 0.1 cm, three slabs) and subjected to
SDS⁄ PAGE (7.5% running gel and 4.5% stacking gel) as
described by Laemmli [25] After electrophoresis for 2.5 h
at 30 mA, 1-cm vertical strips were cut from the right
and left sides of the slab using a cheese knife with a
zig-zag shaped blade; these were immediately stained with
Quick-CBB (Wako Pure Chemical Industries) The stained
gel strips were replaced to each original position on the
slab joining along the zigzag edge The horizontal strip
containing the anserinase band was excised from the
unstained gel slab Elution of the protein from the gel
strips was performed electrophoretically using
Electro-Eluter model 422 (Bio-Rad Laboratories), according to
the manufacturer’s instructions The sample solution was
concentrated, and the buffer was completely replaced with
distilled water, using a Centricon-10 for N-terminal
sequence analysis
N-Terminal sequence analysis and BLAST search
Edman degradation was performed on an automated
pro-tein sequencer (model 491; Applied Biosystems) Propro-tein
Sequence Databases were searched for homologies with
N-terminal sequence of anserinase using the world wide
web-based blastp search engine of GenBank (http://
www.ncbi.nlm.nih.gov/BLAST/) A further blastp search
was conducted by an engine of the MEROPS database
(http://merops.sanger.ac.uk) [19] using ‘unnamed protein’
sequence (DDBJ⁄ EMBL ⁄ GenBank accession number
CAF95589) of spotted river puffer Tetraodon nigroviridis,
which was extracted from blastp for N-terminal sequence
of anserinase Multiple sequence alignments were performed
using the clustal w program (http://align.genome.jp/) to
find highly conserved amino-acid sequences
(dT)-adaptor (10 pmol) 5¢-GGCCACGCGTCGACTAG TACTTTTTTTTTTTTTTT-3¢, and reverse transcriptase buffer (20 lL) of the first-strand cDNA synthesis kit (Invi-trogen Corp.) The synthesis reaction was performed at
42C for 50 min The forward primer (A) was designed for PCR; 21-mer degenerate oligonucleotide 5¢-CAR GAYGARTAYGTNGARATG-3¢ corresponding to N-ter-minal amino-acid sequences 14–20 (QDEYVEM) of Nile tilapia anserinase Multiple sequence alignment of the genes belonging to CNDP (MEROPS ID M20.005) and ‘serum’ carnosinase (MEROPS ID M20.006), which were extracted from the MEROPS blastp search using the sequence of Tetraodon ‘unnamed protein’, revealed two highly con-served regions suitable for designing degenerate oligonucleo-tides for amplification of anserinase gene fragments (data not shown) Therefore, two reverse primers were designed for PCR; the 23-mer oligonucleotide 5¢-GAG CCNGWYTCYTCCATBCCYTC-3¢ corresponding to one consensus sequence (EGMEES⁄ TGS) as the outer pri-mer (B), and the 23-mer oligonucleotide 5¢-TCCAG GYTDGCNGGCTGVACRTC-3¢ corresponding to another consensus sequence (DVQPAN⁄ SLD ⁄ E) as the inner pri-mer (C) The first-round PCR was performed using a set of the primers (A and B), and the second-round nested PCR was primed with first-round PCR product and as a tem-plate a set of the primers (A and C) PCR amplification was carried out in a total volume of 50 lL containing 0.75 lL of a template, 150 pmol of a forward primer (A),
150 pmol of a reverse primer (B or C), 1· G-Taq buffer,
10 nmol each of dATP, dGTP, dCTP and dTTP, and 0.5 U G-Taq DNA Polymerase (Cosmo Genetech Co., Seoul, Korea) For PCR the following conditions were used: initial denaturation at 95C for 2 min, followed by
40 cycles of denaturation at 95C for 20 s, annealing at
50C for 30 s, and extension at 72 C for 1 min, final extension step at 72C for 7 min
Isolation of a partial cDNA encoding CNDP-like protein
A partial sequence of CNDP-like protein (DDBJ⁄ EMBL ⁄ GenBank accession number AY260749) of Mozambique