BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
AMINO ACID SEQUENCE OF CARDIOTOXIN-ANALOGUE
I FROM
THE VENOM OF --NAJA NAJA ATRA* Hayashit*Masayuki
Kyozo
From Department
Takechi, Toyosaku **** C. Y. Lee
of Biological Sciences,
Received
March
Chemistry,
Kyoto
***
Sasaki,
Faculty
University,
and
of Pharmaceutical
Kyoto,
JAPAN
21,197s SUMMARY
Cardiotoxin(CTX)-analogue from
the venom of naja
followed
I was obtained naia
by CM-cellulose
peritoneally toxin
ture
of the skeletal
its muscle
the same venom but differs a number
Recently, sequences
of snake
as a group disulfide tial
consist
cells
venom of Naja filtration
and other atra,
on Sephadex
(l-8).
contrac-
sequence that
of
of CTX from
acid
residues,
G-50 followed
work
tion
and the Tanabe
**
To whom inquiries
***
Himeji Institute Pharmacological
was aided
cytotoxicity
B I9 75 by Academic Press, Inc. of reproduction in any f&-m reserved.
by four by substanto Yoshida
properties active
proteins
(13).
principles
were
isolated
by chromatography
by a grant
in the by gel
on CM-cellulose.
Amino Acid this
from the Ministry
Research article
Taiwan,
Republic 360
of Educa-
Foundation.
should
be directed.
of Technology, Himeji, Japan. Institute, College of Medicine,
Taipei,
acid
cytotoxicity to Yoshida sarcoma cells as neuromuscular block as did CTX (9-
in part about
cross-linked
from neurotoxins
pharmacological basic
on the amino
Cardiotoxins(cytotoxins)
of biologically
possessed as well
This
University,
acid with
have appeared
composition,
several
*
****
amino acid
of study
Some of these proteins and produced contracture
Copyright All rights
of publications
in amino
naja
and produced
The amino
They are distinguished
(9-12)
intra-
body weight.
in 13 positions.
of 60-61
In the course
cells
of homology
venom cardiotoxins
bridges.
differences
sarcoma
CTX.
degree
vg/g
5.3%
G-50,
When injected
(2.45-3.19)
sarcoma
as did
a high
of about
on Sephadex
chromatography.
to Yoshida
I exhibits
filtration
LD50 was 2.8
was cytotoxic
CTX-analogue
by gel
column
in mice,
This
atra
in a yield
of China.
National
Taiwan
BIOCHEMICAL
Vol. 64, No. 1, 1975
12,
14).
In the present
of CTX-analogue some of its
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the venom of Naja
pharmacological
The crude
from
in 1 % acetic
acid
in the same elution
with
of about
to a CM-cellulose
column,
buffer,
pH 5.8.
pH 5.8,
to 0.5 M sodium
top of the column. tate.
Homogeneity
aceate
Acid
buffer
Each fraction
Numbers CB-I
and CB-IV,
pectively;
Co.,
a 0.005
Composition
U. S. A.
on a column
of Sepha-
The protein
fraction and applied
M sodium
acetate
M sodium
acetate
buffer,
, pH 6.5)
was then
applied
at the
by disc
gel
to remove
sodium
ace-
electrophoresis.
I of CTX-Analogue
I and Peptides
by CNBr Cleavage I
represent
the value
and in fragment
CNBr fragments CB-II CB-III
CB-I 2.6(3) 1.9(2) 0.9(l) 0.9(l) 0 1.9(2) -1.0(l) 1.9(2) 0 0 1.9(2) 2.8(3) 0.6(l) trace 0 0 4.3(5) 0 1.0(l) 0
8.7(8) 3.4(3) 3.1(3) 0 4.4(4) -2.0(2) 2.2(2) 4.2(4) 3.0(3) 4.2(4) 6.6(6) 2.1(2) 1.3(l) 0 7.8(8) 7.7(8) 0 0 2.5(2)
in parentheses
sequence with
0.005
was gel-filtered
CTX-Analogue
CM-Cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Half-Cystine Lysine Histidine Homoserine Arginine
medium.
(from
was ascertained
Generated
Sigma Chemical
with
gradient
Table
Amino
acid
6000 - 7500 was freeze-dried
equilibrated
A linear
Amino Acid
amino
is presented
was applied
dex G-50 equilibrated weight
atra
--and Methods
venom was purchased
molecular
complete
naja
properties. Materials
The venom dissolved
the
communication,
I from
nearest
of glycine CB-II,
l.a(l>
0.7(l)
integers.
was taken the value
as 1.0.
861
1.0
CB-IV 3.6(5) 5.3(6) 1.9(2) 1.8(2) 0 1.9(2) -1.0(l) 0 3.4(4) 0 1.8(2) 3.1(3) 0.9(l) 0 0 0 3.6(3) 0 0 1.8(2)
In CTX-analogue as 2.0 and 1.0, res-
of phenylalanine
I,
was taken
BIOCHEMICAL
Vol. 64, No. 1,1975
The first
fraction
having
designated
as CTX-analogue
peritoneal
injection (15).
was used
testing
analyses vage
or tryptic
its
(l&19),
by thin
layer
peptidase Cyanogen acid,
action
toxin
LD50 in mice according
reported
digestion
(24) peptides
were
The fragments
0.2 M acetic acid. column of DEAE-cellulose.
Table Amino Acid Amino Acid
T-l
CM-Cysteine Aspartic acid Threonine Serine Giutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Homoserine Arginine
bers
Composition
were
systems
identified
(20-22).
Carboxy-
procedures out
on Sephadex further
(23).
in 70 % formic G-25 in
purified
separated
on a
by high
vol-
II of Tryptic
T-2
Edman pro-
to standard
were
clea-
Peptides
T-3
T-4
T-5
0.7(l) 1.0(l)
0.5(l)
0.9(l) 1.1(l)
of CB-I T-6
CB-I 3 2 1
0.5(l) 0.9(l)
t 0.8(l) 1.2(l) 1.1(l)
0.7(l) 0.9(l)
0.8(l)
1.3(2) 0.7(l)
--1.0(l)
1.0(l)
of the amino
in parenses
peptides
bromide
was carried were
acid
out by standard
acids
fractionated
obtained
Tryptic
carried
solvent
according
Amino
by the modified amino
of RCM-toxin
(16)
(RCM)-CTX-analogue (17).
by cyanogen
were
conducted
in several
was performed cleavage
and the resulting
The values
obtained
were
of Litchfield
muscle.
by Crestfield
of the RCM-toxin
was
preparation
and S-carboxymethylated
and the phenylthiohydantoin
bromide
muscle
cells by intra-
to the method
on the skeletal
degradation
chromatography
sarcoma
(NIH strain)
cervicis
and the peptides
digestion
RESEARCH COMMUNICATIONS
to Yoshida
biventer
of reduced
Sequential
cedure
Its
out by the method
of the
method.
I.
The chick's
The preparation I was carried
cytotoxicity
was determined
and Wilcoxon for
AND BIOPHYSICAL
represent
acids
1.0(l) 0.5(l) -1.0(l)
underlined
the nearest
362
--1.0(l)
were integers.
1.0(l) 1.0 taken
as 1.0.
2 1 2 0 0 2 3 1 trace 0 5 1 0 The
num-
BIOCHEMKAL
Vol. 64, No. 1,1975
tage
paper
solvent
electrophoresis
system
rated
phenol,
AND BIOPHYSICAL
at pH 3.6,
of 1-butanol-acetic
and paper
acid-water
or n-butanol-acetic
RESEARCH COMMUNICATIONS
chromatography = 4:1:5
(v/v),
acid-pyridine-water
water
produced chick
to be 2.8
a marked
biventer
(2.45-3.19)
contracture
cervicis
ration
of 10 ug/ml.
by gel
filtration
yg/g
followed
muscle
to be about
body weight. as did
weight
7000.
Based on this
acid
analysis,
one molecule
of CTX-analogue
residues:
Asp 8.7,
3.4,
2.2,
Half-Cys
Lys 7.8,
7.7,
4.2,
Met 3.0,
Ile
I was estimated
value
and on the about
Glu 0, Pro 4.2,
in the
CTX, at a concent-
I contains
3.1,
I
Leu 6.6,
40 amino
4.4,
Gly 2.0,
Tyr
amino
2.1,
Ala
Phe 1.3,
Arg 2.5.
Twentynine the
Val
Ser
block
of CTX-analogue
acid
Thr
injection CTX-analogue
by neuromuscular
preparation,
The molecular
satu-
= 15:3:10:12.
--RESULTS AND DISCUSSION The LDSU of CTX-analogue I in mice by intraperitoneal was estimated
in a
stepwise
amino-terminal
Edman degradations
sequence
of RCM-CTX-analogue
I revealed
to be : H-Leu.Lys.Cys.Asn.Lys.Leu.Ile*Pro.
Ile.Ala.Ser.Lys.Thr.Cys.Pro.Ala.Gly.Lys.Asn.Leu.Cys,Tyr.Lys.Met.Phe.Met. Met.Ser.Asp*.
The carboxy-terminal
Table Amino Amino
Acid
bers
Composition
T'-1
CM-Cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Homoserine Arginine The value
Acid
sequence
of Tryptic T'-3
1.0(l)
1.2(l) 0.9(l)
1.1(l)
1.0(l)
Peptides
1.2(l)
2.4(2)
by the use of
III
T'-2
1.0(l)
was examined
of CB-IV
T'-4
T'-5
T'-6
CB-IV
1.8(2) 1.5(l)
1.8(2)
0.6(l) 1.0(l)
5 6
2.3(2) 1.1(l)
0.9(l) 1.0(l)
1.0(l)
1.0(l)
1.0(l)
0.8(l)
1 0 1.4(l)
1.2(l) 0.9(l)
-1.0(l)
-1.0(l)
-1,0(l)
1.0
of the
in parentheses
1.00) amino
acid
represent
underlined the nearest
363
2 2 0 2
were
taken
integers.
as 1.0.
4 0 2 3 1 0 3 0 2 The num-
BIOCHEMICAL
Vol. 64, No. 1, 1975
carboxypeptidase
A.
acids
to be Asn:CM.Cys
were
results,
found
3 and 24 hr incubation,
the carboxy-terminal
The sequence from
bromide were
High
weight
chromatography CB-II
in 70% formic
fractionated
molecular
gments
fragments
on a column
procedure
were
paper
chromatography.
of the
further
From the
to be -Cys*Asn-OH.
tryptic
peptide
derived
I was cleaved
by
24 hr at 37* and the resulting
filtration
CB-I
on a column
and CB-IV were
of Sephadex
further
in the column
purified
by paper
acid
compositions
G-25.
purified
Low molecular
appeared
The amino
in Table
was determined
for
of amino
respectively.
of DEAE-cellulose. which
liberation
RCM-CTX-analogue
acid
by gel
and CB-III,
tration given
sequence.
the
and l:l,
by separation
the carboxy-terminal
cyanogen
= 3:l
sequence
was ascertained
peptides
are
After
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
volume
weight
fra-
of gel
fil-
electrophoresis of these
by
and by fragments
I.
1 10 ~-\~~\2--~2-\~H-Leu.Lys.Cys.Asn.Lys.Leu.Ile.Pro.Ile.Ala.Ser.Lys.Thr.Cys.Pro. t-T-l-r-T-5-
-
T-2