Lipotropin: Localization by radioimmunoassay of endorphin precursor in pituitary and brain

Vol. 75, No. 2, 1977

BIOCHEMICAL

LTPO'IROPIN:

AND BIOPHYSICAL

LOCALIZATION

BY RADIOIMMUNOASSAY

OF ENDORPHIN PRECURSOR IN PITUITARY

Frank

LaBella2,

Department University

of Pharmacology and Therapeutics of Manitoba, Faculty of Medicine Winnipeg, Manitoba, Canada

Clinical

February

AND BRAIN1

Gary Queen and Jan Senyshyn

Martin

Received

RESEARCH COMMUNICATIONS

Lis

and Michel

Chretien

Research Institute of Montreal 110 avenue des Pins Ouest Montreal, Quebec, Canada

8,1977 SUMMARY

Beta-Lipotropic Hormone (LPH) was estimated in pituitary and brain by a radioimmunoassay specific for the N-terminal, non-opiate portion of the protein, and endorphin activity by an opiate radioreceptor assay. The intermediate pituitary is most concentrated in LPH and endorphin. Gel filtration indicated the presence in the pituitary of intact LPH and N-terminal fragments but only intact LPH in brain. Endorphin activity in pituitary is associated primarily with a component of 3000 daltons and to a lesser extent with one of about 2000 daltons. Brain endorphin activity is mostly accounted for by a 2000 dalton component, enkephalins representing an apparently minor activity. In pituitary and brain LPH and endorphin are entirely associated with a 12,000 g/10 min fraction. A family from

brain

N-terminal peptide

ization

(l-5).

pentapeptide

sequence

in

of paired

is

found

0 I977

active

in several contained

by the Medical of the MRC

by Academic

of reproduction

in any

Research

Press, Inc.

form

reserved.

within Council

(endorphins)

share

members

in

acid

vary

residue

cells

a common

length.

(6-9).

Thus,

LPH and the and NMUD/Health

The beta-

The localsuggests

points

of this

structure

and the major (10).

isolated

protein,

gland

A protein

the prohormone

contained

has been

in the LPH sequence

fragments.

endocrine

actions

pituitary

residues

peptide

within

peptide

a 91-amino in the

acid

other

peptides

and individual

within

amino

morphine-like

These

identified

basic

one hormonal

1 Supported 'Associate

contained (LPH)

yielding

each case is

sents

Copyright All rights

is

Hormone

of cleavage type

with

and pituitary

sequence

Lipotropic

of peptides

hormone

structure

beta-MSH

repre-

endorphin

sequence,

and Welfare

Canada

350 ISSN

0006-291X

Vol. 75, No. 2, 1977

another. tered

BIOCHEMICAL

In analogy within

with

a secretory

and active

peptide

associated

with

pituitary.

the granules.

chymal

cells

is

work

one would

consisting

of the

our

granules all

expect

of prohormone,

obtained

endorphin

intermediate

of granules

laboratory

of the activity

Pituitary

the distribution

lobe

in brain

in

fraction

pituitary

is

remains

LPH to be sequescleaving

endorphin

enzyme(s)

activity

from homogenates tissues

apparently pituitary

is

derived gland,

pituitary

that

was

of brain

and

contained from

whereas

to be determined.

LPH parallels

the intermediate

from

(11,12)

in these

of the

of immunoreactive

most concentrated

same granule

from

cytoplasmic

within

that

package

Essentially

source

hormones,

fragments.

In previous

cellular

other

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

parenthe

We now show of endorphin;

and associated

with

it the

or brain.

EXPERIMENTAL Antibodies to ovine beta-LPH were developed in rabbits against the hormone conjugated to ovalbumin with carbodiimide. The ratio of LPH to ovalbumin in the conjugate was 1O:l. (We are grateful to Dr. P. Schiller for preparation of the conjugate). Three days before immunization animals were injected subcutaneously with 0.5 ml of crude Bordella pertussis vaccine. The first immunization was carried out with 500 ug LPH in complete Freund's adjuvant intradermally, and a booster given at six weeks. A second injection contained 250 us LPH in incomplete adjuvant. Methionine5,enkephalin (LPI-I 6165) and leucine enkephalin were obtained from Peninsula Laboratories, San Carlos, CA, and alpha-endorphin (LPH 61-76) from Dr. R. Guillemin. Bovine pituitary glands and brains were obtained from freshly killed cattle of unspecified age and sex and transported on ice to the laboratory. About 2 hr elapsed between death of the animal and extraction of tissue. For estimation of LPH, 10% homogenates of tissues in ice cold water were extracted with a final concentration of 2 M acetic acid and serial dilutions of the extracts made in 0.05 M TRIS buffer, pH 7.4. Dilutions of tissue extracts were assayed by solid phase radioimmunoassay, as previously described for other pituitary For gel filtration the 480,000 g-min fraction, containing hormones (13). essentially all the tissue LPH from 0.5 - 1.0 g tissue was extracted in 10 volumes of 2 N acetic acid, freeze-dried, and the residue in 1 ml of 2 N acetic acid was applied to the column. The sample was eluted with 2 N acetic collected. Column acid, with a flow rate of 0.2 ml/min and 0.5 ml fractions dimensions: G-15 and G-50, 1 x 47 cm, and G-25, 1.5 x 66 cm. Endorphin activity was estimated by a radioreceptor assay with 3H-naloxone as previously Centrifugation pellets were suspended in 2 N acetic acid, described (11,12). stirred at room temperature for 30 min, centrifuged, and the supernatant assayed. RESULTS AND DISCUSSION A schematic sequences

between

of the LPH molecule

paired

basic

amino

acid

351

is

shown in Figure

residues

indicated.

1, with

the

The LPH anti-

Vol. 75, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

. NH1

i

I

39 41

1 1 1

-

0 COOH 91

58 61

RADIOIMMUNO 39

ASSAY 58 91 OPIATE 61-65 61-76 61

ASSAY

91

FIGURE 1. Schematic of ovine LPH showing location of known and the fragments determined by radioimmunoassay and opiate assay.

peptide products radioreceptor

CPM 9500-

6500-

3500.

0.001

0.01

0.1

10

1.0

100

us/ml

FIGURE 2. Activity radioimmunoassay.

serum

appears

polypeptide does

to

the

intact

sequence

leucine active

be

(Figure

cross-reactivity,

only

of LPH (l-91)

2).

LPH molecule.

The opiate-like

the assay.

Beta-MSH antigenic

(gamma-LPI0 (residues

C-terminal

reacts 41-58)

fragment

Methionine (residues

purified

bovine

352

in

the solid-phase

the N-terminal

determinants

and alpha-endorphin Several

fragments

against

1-58

0.1% cross-reactivity.

enkephalin in

primarily

Sequence

indicating

l-40.

about

directed

and peptide

almost shows

of the

as well very

as

low

to reside

within

the

(residues

61-91)

shows

enkephalin 61-76) anterior

portion

(residues are

61-65),

completely

pituitary

in-

hormones

Vol.

75,

No.

1977

2,

BIOCHEMICAL

mg

AND

Tissue

BIOPHYSICAL

Equiv.

(Wet

0.1

RESEARCH

COMMUNICATIONS

Wt.)

1.0

10 ng LPH/mg INT.

PIT.

690

POST. ANT.

PIT. PIT.

150

CAUDATE

10

I

I

30

100

ng

LPH

FIGURE 3. Dose-response curves brain in the LPH radioimmunoassay.

(NIAMD-USPHS) for

cross-react

LPH estimates

on the other for

the

hand,

sequences

is

tissue

(Figure

As shown

is

intact

3).

concentrated

than

be very

in endorphin.

observed

and these

permits

tissue.

It

products

differs

assessment

can be seen between

curves

by the

brain

(RIA) assay,

and sizes the

and pituitary.

353

pituitary

gland

the assay

for

intermediate Brain

LPH lobe

is much less known to

nucleus

in LPH levels

are

elsewhere.

of the types 4) that

caudate

differences

be presented

the

in LPH.

with

brain

and

of LPH, specifically

in

(11,121

in conjunction

(Figure

The opiate

region

concentrated

Regional will

lobes

the radioimmunoassay

of the bovine

endorphin

as represented

findings

Gel filtration, assay,

least

pituitary,

lobes

dose-response for

lobe

Thus,

pituitary

61. three

show parallel previously

0.1%.

the C-terminal

residue of the

of bovine

and the N-fragment.

for

with

most and the anterior

rich

than

protein

selective

of each

and of brain

of extracts

to less

beginning

Extracts

103 3

the RIA and opiate of LPH fragments

profile

of LPH and its

In the

pituitary,

radioreceptor in a given cleavage intact

LPH

Vol.

75,

No.

2,

1977

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

LPH

Endorphin

h/ml)

(nM

I

Cytochrome

0

Vitamin

from

the G-25

daltons) gel.

is

Volume

antiserum

may represent

endorphin

component,

fragment.

A minor

components

approaching

The position In brain,

about endorphin the

intact

)

(ml )

of bovine is shown

by the

components

fragments

of synthetic only

indicated

Smaller

Equiv.

812

FIGURE 4. Gel filtration of extracts and of brain. LPH in column fractions activity in the right panels.

10,000

Noloxone

C

Elution

(about

COMMUNICATIONS

3,000

size

of enkephalin

enkephalin

LPH (l-91)

daltons

354

excluded directed

The major

corresponds

to the 61-91

can be identified,

are detectable

was determined is detectable

N-terminal

respectively.

probably

2,000

of a component

by the

and l-39,

daltons,

of about

presence

detected

1-58

neuro-intermediate pituitary in the left and endorphin

on each

by RIA,

in

but

pituitary.

of the columns.

indicating

that

N-

no

BIOCHEMICAL

Vol. 75, No. 2, 1977

LPH (% of total

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

-kpiT. kE i:/r:;lT.p’T. /~ANT.p 3

12 48

3

s g-min

FIGURE 5. Distribution pituitary lobes.

terminal

fragments

C-terminal 2,000

daltons.

small

proportion

of LPH among subcellular

are degraded

opiate

fragments

or removed

Enkephalin

activity

(Figure

derived

peptide

products

sedimentation (11,121.

are

forces

of 12,000

elaborating lipolytic

peptide activity

pituitary

and

of about

in a relatively

show LPH to be most concentrated We showed

and brain stored

the

within 5) is

g or less.

and endorphin

product

packaging

hormones (17,181

is

(10). this

amino

Although

biological

355

putative

endorphin,

acid

to that

is

the

The

of endorphin compo-

sedimented

at

in LPH and the protein

in several

is

and its

granule.

LPH has been shown property

lobe

in particulate

residues within

pre-

intermediate

to that found

in

prohormone

granules

structures

analogous

in the

identical

of LPI-I-containing The paired

that

the same secretory

of the LPH activity

proportion

of beta-i%H secretion

brain

generation.

component

apparently

most concentrated

of LPH (Figure all

and the great

present

from

their

by a major

(14-16).

also

apparently

pattern

nents,

suggest

pituitary

in

Essentially

recognition

studies

from LPH, is

Furthermore,

(11,12).

is

fractions

4).

of the intermediate

sumably

s

soon after

are represented

Immunohistochemical cells

12 48

x 10 4

sequence other

glands

to possess

not impressive

and,

Vol. 75, No. 2, 1977

until

recently,

enigmatic. is

physiological and his

synthesized

thus

were

done before

In the

present

to be the 61-91

artifacts

of extensively we find

of about

the

2,000

(residues

61-76).

accounted

for

ponent

(about

by Hughes

(1)

LPH-endorphin

components

processed

tissue

component

to predominate,

consistently

61-911,

l-39), are

In contrast,

and two endorphin

of fresh

61-91)

beef

found

upon demand,

(201,

is

last of

only

within

smaller

products

is

another

identified a minor

component.

peptide

The endocrine

products,

i.e.

and beta-endorphin

apparently

yield

the major

granule

in

intact

those

the

LPH only,

opiate

component

may be mobilized from

com-

in brain

a systemic

storage

distinct

activity

daltons;

41-58),

LPH in brain

component

alpha-endorphin

the

than

pituitary

a minor

endorphin

2,000

gland

representing

bovine

although

represents

(residues

in pituitary. to yield

In fresh

the pentapeptide

of brain

both

these

pituitary

apparently

brain

of about

present

extracts

l-58

and the release

in the

as each of the major

apparently

that

to fragment

However,

presumably

presumably

beta-MSH

components,

(4,5).

present,

and Snyder

(residues

synapses

smaller

and Simantov

N-fragment

(residues

fragment,

LPH, as well

rise

of endorphin

endorphin

in pituitary

to propose

(unpublished).

presumably

Intact

pituitary.

fragment.

500 daltons),

secretion.

(residues

the C-terminus

the major

is

has been

the first

and gives

by the component

complex

were

protein

pituitaries

recently

In extracts

chiefly

(9)

studies

61-91

daltons

of the

the discovery

until

appears

glands

colleagues

releasing

was not verified

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

significance

in bovine

presumably

experiments 61-91

the Chretien

beta-LPH (19),

BIOCHEMICAL

at

in pituitary.

REFERENCES 1. 2. 3. 4. 5.

Hughes, .I., Smith, T.W., Kosterlitz, H.W., Fothergill, B.A. and Morris, H.R. (1975). Nature, 258, 577-579. Guillemin, R., Ling, N. and Burgus, R. (1976). C.R. ser. D., 282, 783-785. Bradbury, A.F., Smith, D.G. and Snell, C.R. (1976). Res. Commun., 69, 950-956. Cox, B.M., Goldstein, A. and Li, C.H. (1976). Proc. 73, 1821-1823. Goldstein, A. (1976). Science, 193, 1081-1086.

356

L.A., Acad.

Morgan, Sci.

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(Paris) Biophys.

Acad.

Sci.,

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6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Graf, L. and Li, C.H. (1973). Biochem. Biophys. Res. Commun., 53, 1304-1309. Li, C.H., Barnafi, L., Chretien, M. and Chung, D. (1965). Nature, 208, 1093-1094. Gilardeau, C. and Chretien, M. (1970). Can. J. Biochem., 48, 1017-1021. Chretien, M. and Gilardeau, C. (1970). Can. J. Biochem., 48, 511-516. Polypeptide Hormones: Molecular and Cellular Aspects, Ciba Found. Elsevier, New York (1976). Syw., Queen, G., Pinsky, C. and LaBella, F. (1976). Biochem. Biophys. Res. Commun., 72, 1021-1027. LaBella, F., Queen, G. and Pinsky, C. (in press). Canad. J. Physiol. Pharmacol. LaBella, F,, Dular, R., Queen, G. and Vivian, S. (1975). Endocrinology, 96, 1559-1565. Moon, H.D., Li, C.H. and Jennings, B. (1973). Anat. Rec., 175, 529-538. nessy, C., Herlant, M. and Chretien, M. (1973). C.R. Acad. Sci. (Paris) 338, 276-335. Furth, J., Chretien, M., Moy, P. and Granman, J. (1975). Amer. J. Pathol., 78, 20. Birk, Y. and Li, C.H. (1964). J. Biol. Chem., 239, 1048-1052. Lie., M., Gilardeau, C. and Chretien, M. (1972). Acta Endocrinol., 69, 507-510. Chretien, M., Lis, M., Gilardeau, C. and Benjannet, S. (1976). Can. J. Biochem., 54, 566-570. Simantov, R. and Snyder, S.H. (1976). Proc. Natl. Acad. Sci., 73, 2525-2519.

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