Vol. 69, No. 4, 1976

BIOCHEMICAL

Lipotropin:

precursor A.F.

Mill Received

D.G.

February

active

Smyth and C.R.

Institute

Hill,

RESEARCH COMMUNICATIONS

to two biologically

Bradbury,

National

AND BIOPHYSICAL

for

London,

Medical

peptides.

Snell.

Research,

NW7 IAA.

23,1976

SUMMARY Lipotropin

appears

a peptide

P-M=,

with

a peptide

with

formed

determined

is

central

might

of

basis be the 2

Gilardeau conversion

lipotropin

, on the

If

remain

of

secretory

on the biosynthetic

isolated

peptides

Copyright All rights

using

precursor

of the

origin

pulse P-MSH,

950

of

and the

LPH

and

labelling formation

of

the fragments To obtain

gland.

of S-MSH, we have

in a search

prohormone.

that

to demonstrate

by release

prohormone

from pituitary

0 I976 by Academic Press, Inc. of reproduction in any form reserved.

of

the polypeptide.

Lis

were unable

particle

evidence

of the

a 91 residue

Bertagna,

be accompanied of the

within

suggested

@-MSH.

hand,

activating

18 residue

contained

(LPH),

LPH is the

fragments

in the

C-fragments

is

colleagues'

other

the hormone should

of the

P-MSH, the

of LFW to @-MSH in vitro

techniques.

contiguous

of

hormone,

prohormone

The product

specificity

sequence

to

and C-Fragment,

activity.

by the

Li and his

common precursor

activity,

opiate

stimulating

region

I'> Gn this ._ "

lipolytic

potent

enzymes. ;c I.-d * The amino acid melanocyte

to be the

for

the N- and

Vol. 69, No. 4, 1976

Table

BIOCHEMICAL

1.

AND BIOPHYSICAL

Polypeptides

isolated

gland

LPH Residue

pig.

1-91

LPH

1.1

l-58

y-LPH

1.7

l-38

N-fragment

0.7

41-58

P-MSH

1.6

61-87

C -fragment

0.7

61-91

C-fragment

0.9

glands

were obtained

and precipitation

chromatography

and gel

constituent active

Each of the the

action

peptides

(Fig.1) of

appears

chain

at

none of the residues.

fragments

with

enzymes that

generate

supports

view

the

that

a series

of

present

were the

comprising

the carboxyl

side

amino acids B enzyme.

of the the insulin the

the biologically

N- and C-fragments

(Table

of

1).

It

is

pituitary

specificity

of

951

basic

notable at single

that basic

enzymes involved the

from proinsulin. LPH fragments

paired

removed by the

was formed by cleavage

The specificity identical

yielded

exchange

to have been formed by cleavage

and the basic

a carboxypeptidase

and after ion

lipotropin,

P-MSH and the

lipotropin

residues

of

1200 pigs to Li'

among those

fragments

peptide

from

according

filtration

homogeneous peptides:

thus

of the

pituitary

Yield (Umole)

homogenization

is

from the

Number

Pituitary

of

RESEARCH COMMUNICATIONS

pancreatic 4

This

are released

in a

Vol. 69, No. 4, 1976

BIOCHEMICAL

1

38

LPH

ILys

N-fragment

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

41

58

61 Lys

LYS

Arg

Lys

1

c -fragment

I I

fragments

of

organised

diverse

lipotropin

process

degrading

lipotropin

their

corresponds

was observed

tyrosine

of the

that

conditions 30 min.,

pituitary

place either pH 7.4,

specificity

of LPH (residues (residues

the

the

peptide

rapidly

enzyme that

bond between

l-58)

trypsin

by

(I),

57-62 arginine

cleavage

both

of the

was digested

under

or with

the

in the

and the

finding

complementary

were present

952

by trypsin granules

same peptide

(enzyme substrate pituitary

in LPH. and

from secretory

trypsin

was implicit

activates

hexapeptide

and specifically,

when lipotropin

61-91)

of

from positions

enzyme isolated

37°C)

to

Gly

sequence

with

glands.

formed by exposure

specificity

the

Specific

pituitary

isolation.

on the synthetic

trypsin-like

pituitary.

bond took

y-LPH

to

was cleaved

and by the

same

of the

actions

from porcine

and were not

Lys Asp Lys Arg Tyr

which It

isolated

was compared with

I.

I

1.

enzymes during

The specificity

studying

I

1

Fig.

highly

Lys-

I

I

C-fragment

Peptide

91

I

P-MSH

y-LPH

87

mild

ratio

enzyme: that

1:4000, and the

the C-fragment

polypeptide

in substantial

quantity

in

Vol. 69, No. 4, 1976

the

gland.

BIOCHEMICAL

On this

pituitary

the

first

bond at positions Tryptic

which

of

Ala

cleavage

of LPH is

of the

to the place

the hexapeptide. the

acid.

terminus,

it

@-MSH from

the

value

the

weight

in the for

cleavage of the

l-38)

@-MSH is

released

requirement

the for

(II),

that

the

P-M%

lysine

pituitary,

consecutive

by enzymic

differences

cleavage sequence

when at

takes

of LPH

does confirm

hydrolysis

of

953

residues

under

that

lipotropin.

enzyme has an absolute basic

place

on the N-terminal

the N-fragment

however,

of

was unaffected

be detected

residues of

to have

action

of these

by trypsin:

The presence

pituitary

peptides

no P-MSH could

paired

releases

compared with

and the

In view

at the

at the NH~-

optimum pH of the

trypsin:

synthetic

and

specificity

100,000,

the

than

within

in vivo

acid

lysine

enzyme which

of

heavily

in the

specifically

enzyme was found

region

in

than hydrolysis

has a different

sites

hormone.

(residues

Whether

tyrosine

37-43

and not between

inhibitor.

surprising

sensitive

positions

occurred

trypsin;

was digested

trypsin

arginyl

more slowly

pituitary

enzyme on the

lipotropin

side

the

enzyme was higher

was not

from

pituitary

in vivo

by soya bean trypsin

before

the

Indeed

pituitary

in the

Yl.eptapeptide

P-MSH has aspartic

seems that

of 23,000

isolated

residues

lipotropin

a molecular

sequence

The cleavage

Since

from trypsin.

that

at the

synthetic

300 times

two lysine

aspartic

seems likely

Glu Lys Lys Asp Glu Gly

LPH, took

between

it

it

RESEARCH COMMUNICATIONS

60-61.

corresponds

porcine

evidence

hydrolysis

II.

AND BIOPHYSICAL

in vivo

Vol. 69, No. 4, 1976

conditions between

is not known. Arg

slightly

and Tyr

of the

enzyme,

trypsin

the

in the hexapeptide

(I)

adjacent

cleavage

basic

of the

paired

by the

basic

in vivo

predicting

(residues site the

in LPH. molecule,

at positions enzymic

lies

adjacent

this

the

peptide

the

pituitary occur

that

to

explained of the

empirical

residues

bond between

prohormone rules

of

Tyr.Gly.Gly.Phe

the enzymically located

with

enzyme for

conformation

the

susceptible

on the outside

arginine

understandably

the

the

partially

to

but

peptide

pituitary only

only

E-NH 2

from a knowledge

structure

60 and 61 would

did

for

According

structure

With

of

and tyrogine

be accessible

to

attack.

The C-fragment endogenous terminus termed

to

of

reported

methionine Tyr

correspondence

cleavage

sly

Gly

with

opiate

activity,

Phe Met

of primary

the

a peptide (III)

from upper

enkephalin of

for

enkephalin

was isolated

methionine

LPH (residues 61-91), which is 3 has the same sequence at the NH2-

pituitary,

as that

III,. which

the

a P-bend comprising

61-64)

the

indicate

LPH is

factor.

secondary

5,6,7

that

by the

specificity:

may be an additional

sequence,

of

at the

peptide

than

of cleavage

decreased

With

These results

residues

intrinsic

residue.

less

exhibited

rate

was acetylated

acetylated

residues.

specificity

for

With

lysine

was substantially

paired

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

when the substrate

group

rate

BIOCHEMICAL

is

lipotropin

regions

structure formed

of pig brain. it

in vivo

C-fragment.

954

seems likely

8

From the that

by proteolytic It

has now been

Vol. 69, No. 4, 1976

found

that

BIOCHEMICAL

C-Fragment

receptor,

several

itself

times

pentapeptide.'

AND BIOPHYSICAL

It

has an affinity

greater

is thus

than

clear

activities

reside

lipotropin

molecule.

The enzyme that

lipotropin

@-MSH but the

could

further

study

two activities

specific

will

of

the

enzymic

reported

opiate

for

structure

releases

the

in the

of the C-fragment

in the

be necessary

lipotropin

brain

two potential

be involved

are developed

activation

have ! Idifferent

also

within

for

that

that

biological

from

RESEARCH COMMUNICATIONS

formation

to establish same cell

takes

of

place

whether

or whether

in cells

that

complements.

References

1.

Li,

C.H.,

Barnafi,

Nature, 2.

Bertagna,

208,

X.,

Bradbury,

(1965)

C.,

(1974)

349-358. (1975)

Prohormones

Of @-MSH and ACTH: Structure

and

Activation,

in

Kemmler,

W.,

Steiner, thou,

D.

C.R.

No. 41,

5.

52,

Smyth,

and Cellular

4.

M. and Chung,

M. and Gilardeau,

Biochem.,

A.F.,

Chretien,

1093-1094.

Lis,

Can. J. 3.

L.,

P.Y.

D.G.

and Snell,

"The Peptide

Aspects",

Hormones:

Molecular

CIBA Foundation

Symposium

in press. Peterson, D.F.

J.D.,

(1972)

Rubenstein,

Diabetes,

and Fasman, G.D.

21,

A.H.

and

572-583.

(1974)

Biochemistry,

l3,

(1974)

Biochemistry,

-13

211-222. 6.

thou,

P.Y.

and Fasman, G.D.

222-245. 7.

Lewis,

P.N., Proc.

MOmany, F.A. Natl.

Acad.

and Scheraga,

Sci.

955

U.S.A.,

68,

H.A.

(1971)

2293-2297.

Vol. 69, No. 4, 1976

8.

BIOCHEMICAL

Hughes,

J.,

Smith, Morgan,

L.A., Nature, 9.

Bradbury,

A.F.,

Birdsall,

258,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

T.W., B.A.

Kosterlitz, and Morris,

H.W., H.R.

Fothergill, (1975)

577-579.

Smyth, N.J.M.

D.G.

and Snell,

and Hulme,

Nature.

956

E.C.

C.R. (in

press)