Isolation and primary structure of endorphin from salmon pituitary glands

Vol. 88, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 27, 1979

Pages 1249-l

254

ISOLATION

AND PRIMARY STRUCTURE OF ENDORPHIN FROM SALMON PITUITARY GLANDS Hiroshi Kawauchi* and Makoto Tubokawa School of Fisheries Sciences, Kitasato University Sanriku, Iwate 022-01, Japan Koji Muramoto Laboratory of Analytical Chemistry, Faculty of Agriculture Tohoku University, Sendai, Miyagi 980, Japan Received

May 14,1979

Summary: Endorphin has been isolated from an acid acetone extract of the-pituitary of the salmon Oncorhynchus keta by ion exchange chromatoqraphy and qel filtration. Sequence analysis revealed it Ac-Tyrto be a nonacosapeptide with following-primary st;ucture: Gly-Gly-Phe-Met-Lys-Pro-Tyr-Thr-Lys-Gln-Ser-His-Lys-Pro-Leu-IleThr-Leu-Leu-Lys-His-Ile-Ti~r-Leu-Lys-Asn-Glu-Gln-OH. It appears that the amino terminal segment which is necessary for analgesic activity is conserved through the evolution of vertebrate except for the blocking of the amino terminal of salmon endorphin. The recent

discovery

activity,

is

Endorphin

has been

porcine amino

an exciting

(2,3), acid

and camel

development

isolated

bovine

sequence endorphin

have been

hormones

in

insight

into

evolutionary

the

structure-function

characterize herein sequence

the

endorphin isolation of endorphin

the

teleost

camel

and the

from

In order

the

it

better

and to study

species. the

of bovine human and

is necessary

of

(1),

complete

to obtain

of endorphin

non-mammalian

and elucidation

from

The structure

each.

relationships,

from

form

and differ

history

agonist

endocrinology.

pituitaries

determined.

two residues

from

purified

(5,6)

identical

porcine

the

human

has opiate

in molecular

in highly

(4),

are

which

of endorphin,

to isolate

and

We report

complete

Oncorhynchus

amino keta

acid

(chum

salmon)

* To whom inquiries

should

be addressed. 0006-291 X/79/1 Copyright

1249

All

rights

@ 1979

21249-06$01.00/O

by Academic

of reproduction

Press,

in any form

Inc. reserved.

BIOCHEMICAL

Vol. 88, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Experiments Salmon glands

endorphin

by the

(7).

cellulose

All

(8).

found

in a fraction

a-MSH

(8).

of

salmon

weight G-75

column.

with

the

of

in

0.1

residues

the

CB-II

was determined

amino

terminal

tyrosine

on polyamide dansylated

tyrosine.

CB-I

Hislv8,

AsP1.Or

of CB-II

Thr2.$,

the

The molecular Sephadex acid

residues

As~l.~,

by the

Thr3.0,

dansyl

(10)

method. cyanogen

consisted

on Sephadex

of five TYroB88

bromide

amino

acid

Gl~~.~p

Pheosgn

* 7. The amino acid sequence of carboxypeptidase method (12). The was identified

layer

hydrolysate gave

tissue.

found

composition:

thin

The yield

(1 mg) was fractionated CB-II

by the

residue

the

were

determination,

+ HomoserinelactoneO

to salmon

1.

HisZsO,

structure

following

was

by exclusion

of 29 amino

(11)

acid.

using

on a calibrated

isothiocyanate

N acetic

with

Homoserine

Ws4.gr

endorphin

gradient

relative

purified

daltons

composition:

salmon

acetate

by radioimmunoassay

wet weight

residues

salmon

by chromatography

retarded

consisted

primary

of

weak immunoreactivity

endorphin

terminal

fluorescein

isolation

G-75 as shown in Figure

to be 3,000

Salmon

pituitary

ammonium

was further

was 110 mg/kg

following

fragments

of

endorphin

on Sephadex

For the

G-50

was slightly

No amino

Phel.O* or the

were tested

which

was estimated

the

using

A very

endorphin

fresh-frozen

was fractionated

antibody(g).

Salmon

chromatography

for

column

fractions

human endorphin

from

described

The extract

on a carboxymethyl elution

was extracted

procedure

melanotropins

and Results

chromatography

with

following Seros6,

to be N-acetyl-

authentic amino

Glu3-o,

The amino acid sequence of CB-I TYr0.8* up to the 23rd residue by the fluorescein

1250

by comparison N-acetyl,O-dansyl-

acid

composition:

Fro2.0,

Ile2.0J

(0.3

Lys5.0' Leu4.0,

mg) was determined

isothiocyanate

method

BIOCHEMICAL

Vol. 88, No. 4, 1979

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

IO

20

30

I

40

60

50 TUBE

70

80

90

100

110

NUMBER

Fiqure 1. Sephadex G-75 gel filtration of salmon endorphin fraction obtained from the carboxymethyl cellulose chromatography of the acid acetone extract Column size : 2.64 x 47 cm. Pre-equilibrated and of salmon pituitaries. eluted with 0.05 M ammonium acetate, pH 7.0. Fraction size: 1.5 ml/tube.

(11,13).

To confirm

fractionated

were submitted fluorescein results

Four peptide

were separated solvent

to amino

acid

and sequence

in

n-butanol

(C-II, C-I

Table

1.

layer

saturated

ran

procedure digest

water.

1251

and the of

salmon mapping.

were obtained

together

chromatography with

by both

by two dimensional

C-V and C-VI) which

was

and -V)

analysis

A chymotryptic

fractionated

and C-IV

CB-I

(CB-I-T-I,-II,-III,-IV

and dansyl-Edman

C-III,

by thin

digest

chromatography-electrophoresis

spot

was similarly

map.

paper

each

isothiocyanate

peptides

a trypt.c

from

summarized

endorphin

sequence,

by two dimensional

The eluates

(14).

the

on the on silica

The Rf values

from peptide gel

the map

in

of C-I

the and

BIOCHEMICAL

Vol. 88, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Amino

Acid

Composition Tryptic

1.

(Molar

Peptides

Ratio)

of

CB-I

Peptide

and of

NH*-Terminal

Salmon

Residue

Endorphin.

NH*-Terminal

Composition

Residue

Number

T-I

Lys2.0Thr0.8Prol.3TYr0.6

LYS

T-II

Lys1~9His0~6ThrO~9Sero~5Glu,~,Prol~lIle,~3Leu3~2

---

T-ID

Lys3.6HiSO.BThrl.~SerO.5G'u,.lPro2.211el.lLeu3.2Tyrl.l

LYS

T-IV T-V

His

Lysl.OHis0.7Thr0.7~'el.lLeul.l

Asn*

AsP0.8G1u2.0

* Determined

by

the

fluorescein

isothiocyanate

Table Amino

Acid

Composition

(Molar

Chymotryptic

method.

2. Ratio)

Peptides

Peptide

of

and

NH2-Terminal

Salmon

Residue

NH2-Terminal

Composition

Residue

C-I

Ile

Thrl.011eo.9Leu2.1

c-v

C-VI the

were analyzed

LYS

Lys0.9Asp1.oGlu2.0

LYS

and 0.21, for

to obtain data, is

Lys0.9His0.6Thr0.9*1el.lLeul.l

text.

were 0.27

endorphin

Thr

Lys2.0His0.6Thr0.6Ser0.5G'ul.lprol.0Leu0.9

C-IV

From these

Met

LYs0.9Prol.lMet0.6Tyr0.6

c-m

order

N-Ac-Tyr*

Glyl.9Tyrl.lPhel.0

C-II

* See

of

Endorphin

Number

C-IV

of

respectively.

amino acid the overlaps the complete

proposed

composition

These chymotryptic and partially

and the results amino acid

as shown in Figure

1252

summarized

peptides sequenced

in

in Table

2.

sequence of salmon 2.

BIOCHEMICAL

Vol. 88, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5 CH3CO-Tyr-Gly-Gly-Phe-Met-Lys-Pro-Tyr-Thr-Lys-Gln-Ser-His-Lyj-Pro-Leu

10

15

, CB-II

CB-I ( CB-I-T-I

.

CB-I-T-II CB-I-T-III

I.

C-I

C-II

'.

20 Ile-Thr-Leu-Leu-Lys-His-Ile-Thr-Leu-Lys-Asn-Glu-Gln-OH

C-III 25

29 )

CB- I --CB-I-T-II

CB-I-T-IV

CB-I-T-V

) CB-I-T-III --C-IV

C-VI

c-v

Figure 2. Proposed amino acid sequence CB: Peptide from cyanogen bromide cleavage. C: Peptide from chymotryptic digestion.

salmon

of salmon endorphin. T: peptide from tryptic

digestion.

:

Figure 3. Comparison that of human, camel, Homology is indicated replacements (x).

of the amino acid sequence of salmon endorphin with bovine, ovine and porcine B-endorphin. by: highly acceptable replacements (-) and unacceptable

Discussion Comparison

of the

mammalian endorphin

salmon endorphin

sequence

have been introduced

into

positions

and 9 highly

terminal

segment which

is

sequence with

shown in Figure

the endorphin.

acceptable

There

replacements.

is necessary

1253

for

analgesic

3.

the known Two gaps

are 13 identical The amino activity

is

BIOCHEMICAL

Vol. 88, No. 4, 1979

conserved.

However,

endorphin

by acetyl

function

of salmon

the

amino

of biological endorphin

terminal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

blocking

of the

group

raises

interesting

endorphin.

In mammalian

of endorphin

activity are currently

amino

terminal questions

assay

and enkephalin

(15).

The biological

under

investigation.

of about

activities

the

blocking

systems

results

salmon

in of

a loss salmon

ACKNOWLEDGEMENTS We thank Professor Choh Hao Li, University San Francisco for radioimmunoassay and Professor University of California, San Francisco for his of the paper.

of California J. Ramachandran, critical reading

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Li, C.H., and Chung, 0. (1976) Proc. Natl. Acad. Sci. U.S.A. 43, 1145-1148. Graf, L., Barat, E., and Patthy, A. (1976) Acta Biochim. Biophys. Acad. Sci. Hung. 11, 121-122. Bradbury,A. F., Smyth, D. G., and Snell, C. R. (1976) Biochem. Biophys. Res. Commun. 69, 950-956. Li, C. H., Tan, L., and Chung, D. (1977) Biochem. Biophys. Res. Commun. 77, 1542-1547. Chrgtien, M., Benjannet, S., Dragon, N., Seidah, N. G., and Lis, M. (1976) Biochem. Biophys. Res. Commun. 72, 472-478. Li, C. H., Chung, D., and Doneen, B. A. (1976) Biochem. Biophys. Res. Commun. 72, 1542-1547. Kawauchi, H., and Muramoto, K. Int. J. Peptide Protein Res. in press. Birk, Y., and Li, C. H. (1964). J. Biol. Chem. 239, 1048-1052. Li, C. H., Rao, J., Doneen, B. A., and Yamashiro, 0. (1977) Biochem. Biophys. Res. Commun. 75, 576-580. Gray, W. R. (1972) Method in Enzymology XXV (ed. by Hirs, C. H. W., and Timasheff, S. N.) pp. 121- 138, Academic Press, New York. Maeda, H., and Kawauchi, H. (1968) Biochem. Biophys. Res. Commun. 31, 188-192. Ambler, R. P. (1972) Method in Enzymology XXV (ed. by Hirs, C. H. W., and Timasheff, S. N.) pp. 143-154, Academic Press, New York. Muramoto, K., Kawauchi, H., and Tuzimura, K. (1978) Agric. Biol. Chem. 42, 1559-1563. Li, C. H., and Chung, D. (1978) Biochem. Biophys. Res. Commun. 81, 900-906. Goldstein, A. (1976) Science 193, 1081-1085.

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