Purification and sequence identification of anserinase
Shoji 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

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.
FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing 6001
3.5.1.34). Subsequently, Lenney et al. [6] reported that
the substrate specificity of Na-acetylhistidine deacety-
lase purified from brain and eye of skipjack tuna
resembled that of anserinase purified from skeletal
muscle of codling Gadus callarias. In 1981, therefore,
Na-acetylhistidine deacetylase was judged to be identi-
cal with anserinase by NC-IUB, and its EC number
was deleted. Anserinase was discovered by Jones [7,8],
who found that anserine (b-alanyl-1-methylhistidine)
was hydrolyzed by this enzyme in skeletal muscle of
G. callarias. Anserinase is activated by bivalent metal
ions 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].

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

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

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
.
S. Yamada et al. Purification and sequence identification of anserinase
FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing 6003
‘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

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.
Purification and sequence identification of anserinase S. Yamada et al.
6004 FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing
Cloning 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

Na-Acetyl-
L-Cys 0 0
Na-Acetyl-
L-Trp 3 30
Na-Acetyl-
L-Leu 58 110
Na-Chloroacetyl-
L-Leu 755 1235
L-Leu-b-naphthylamide 0 0
Anserine 39 16
Carnosine 74 83
Homocarnosine 155 67
L-Ala-L-His 447 105
Gly-
L-His 327 166
L-His-Gly 58 6
Gly-
L-Leu 443 823
Gly-
D-Leu 0 0
L-Leu-Gly 360 101
L-His-L-Leu 26 0
Gly-Gly 543 385
L-Cys-Gly 0 0
L-Pro-Gly 301 76
L-Ala-L-Pro 46 0
Gly-Gly-
L-Leu 0 0
Gly-
L-Leu-L-Tyr 0 0

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

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.
S. Yamada et al. Purification and sequence identification of anserinase
FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing 6007
CNDP-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

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 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 30 °C for 1 h with
1mm Na-acetylhistidine in 150 mm N-ethylmorpholine ⁄ HCl
buffer, pH 6.5, containing 1 mm CoSO
4
[9]. The reaction
was terminated by the addition of HClO
4
at 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 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-

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 CoSO
4
at 7 °C. 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,

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). Protein
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
() [19] using ‘unnamed protein’
sequence (DDBJ ⁄ EMBL ⁄ GenBank accession number
CAF95589) of spotted river puffer Tetraodon nigroviridis,

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 95 °C for 2 min, followed by
40 cycles of denaturation at 95 °C for 20 s, annealing at
50 °C for 30 s, and extension at 72 °C for 1 min, final
extension step at 72 ° C 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
Purification and sequence identification of anserinase S. Yamada et al.
6010 FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing
tilapia Oreochromis mossambicus, which is classified in the
same genus as Nile tilapia, was extracted from the MER-
OPS database. For PCR amplification of a partial cDNA
for CNDP-like protein of Nile tilapia, the following primers

72 °C for 2 min. This was followed by a final extension step
at 72 °C for 7 min.
DNA sequencing
PCR products were subcloned into pGEM-T easy vector
(Promega, Madison, WI, USA). cDNA clones obtained
were sequenced by the dideoxy-chain termination method
using BigDye Terminator v3.1 Cycle Sequencing kit and an
ABI 3100 DNA sequencer (Applied Biosystems).
Computer assisted analysis
The Fugu (puffer) Takifugu rubripes genome database
( was screened for
potential homologues to Nile tilapia anserinase or CNDP-
like protein by tblastn search. The genscan program
( was then used to
predict Fugu anserinase-like protein and CNDP-like protein
genes in scaffold files extracted from the genome database.
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
( databases are as follows:
human Homo sapiens, ‘serum’ carnosinase (NM_032649)
and CNDP (BC003176); mouse Mus musculus, ‘serum’ car-
nosinase-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’ carno-

GGTACGCG-3¢ (anserinase, reverse); 5¢-TATTCCTCG
CAAGGTCATCGGC-3¢ (CNDP-like protein, forward) and
5¢-GCAGCTTGACTCCCTGAATGTA-3¢ (CNDP-like pro-
tein, reverse); 5¢-GATGCTCCCATGTTCGTCATGGG-3¢
(GAPDH, forward) and 5¢-CAGCATCAAAGATGGA
GGAGTG-3¢ (GAPDH, reverse). The sizes expected were
530 bp, 395 bp, and 517 bp for anserinase, CNDP-like
S. Yamada et al. Purification and sequence identification of anserinase
FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing 6011
protein, and GAPDH, respectively. PCR amplification was
performed in a total volume of 50 lL containing 0.5 lLofa
template, 15 pmol of a forward primer, 15 pmol of a reverse
primer, 1· PCR Gold buffer containing 2.5 mm MgCl
2
,
10 nmol each of dATP, dGTP, dCTP and dTTP, and 1 U
AmpliTaq Gold (Applied Biosystems Japan Ltd). For PCR
the following conditions were used: initial denaturation at
95 °C for 9 min, followed by 45 cycles of denaturation at
94 °C for 20 s, annealing at 56 °C for 30 s, and extension at
72 °C for 1 min, final extension step at 72 °C for 7 min.
Acknowledgements
We thank Mr S. Yamamoto, Ms H. Deguchi and Ms
A. Teshima for technical assistance. We also thank Dr
Steven M. Plakas (FDA, Dauphin Island, AL, USA)
for reviewing the manuscript.
References
1 Baslow MH (1965) Neurosine, its identification with
N-acetyl-l-histidine and distribution in aquatic
vertebrates. Zoologica 50, 63–66.

(1991) Distribution of Na-acetylhistidine-deacetylating
enzyme in tissues of rainbow trout. Nippon Suisan
Gakkaishi 57, 1601.
11 Yamada S, Tanaka Y, Sameshima M & Furuichi M
(1994) Effects of starvation and feeding on tissue
Na-acetylhistidine levels in Nile tilapia Oreochromis nil-
oticus. Comp Biochem Physiol 109A, 277–283.
12 Perry TL, Hansen S, Tischler B, Bunting R & Berry K
(1967) Carnosinaemia: a new metabolic disorder asso-
ciated with neurological disease and mental defect.
N Engl J Med 277, 1219–1227.
13 Murphey WH, Lindmark DG & Mosovich L (1973)
Serum carnosinase deficiency concomitant with mental
retardation. Pediatr Res 7, 601–606.
14 Lenney JF, George RP, Weiss AM, Kucera CM, Chan
PW & Rinzler GS (1982) Human serum carnosinase:
characterization, distinction from cellular carnosinase,
and activation by cadmium. Clin Chim Acta 123,
221–231.
15 Lenney JF, Peppers SC, Kucera-Orallo CM & George
RP (1985) Characterization of human tissue carnosi-
nase. Biochem J 228, 653–660.
16 Lenney JF (1990) Human cytosolic carnosinase: evi-
dence of identity with prolinase, a non-specific dipepti-
dase. Biol Chem Hoppe Seyler 371, 167–171.
17 Jackson MC, Kucera CM & Lenney JF (1991) Purifica-
tion and properties of human serum carnosinase. Clin
Chim Acta 196, 193–205.
18 Teufel M, Saudek V, Ledig JP, Bernhardt A, Boularand
S, Carreau A, Cairns NJ, Carter C, Cowley DJ,

ing assembly of the head of bacteriophage T4. Nature
227, 680–685.
26 Lenney JF, Kan SC, Siu K & Sugiyama GH (1977)
Homocarnosinase: a hog kidney dipeptidase with a
broader specificity than carnosinase. Arch Biochem
Biophys 184, 257–266.
27 Nielsen H, Engelbrecht J, Brunak S & Heijne G (1997)
Identification of prokaryotic and eukaryotic signal pep-
tides and prediction of their cleavage sites. Protein Eng
10, 1–6.
S. Yamada et al. Purification and sequence identification of anserinase
FEBS Journal 272 (2005) 6001–6013 ª 2005 The Authors Journal compilation copyright 2005 FEBS ⁄ Blackwell publishing 6013