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

Amino acid sequence of cardiotoxin-analogue I from the venom of Naja naja atra.

BIOCHEMICAL Vol. 64, No. 1,1975 AND BIOPHYSICAL RESEARCH COMMUNICATIONS AMINO ACID SEQUENCE OF CARDIOTOXIN-ANALOGUE I FROM THE VENOM OF --NAJA N...
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