β-Endorphin: Structure-activity relationships in the guinea pig ileum and opiate receptor binding assays

Vol. 74, No. 2, 1977

BIOCHEMICAL

/3-ENDORPHIN:

STRUCTURE-ACTIVITY

GUINEA

PIG Byron

ILEUM A.

Ping Hormone

OPIATE

RECEPTOR

David

Law*,

Horace

H.

and San

*Department Francisco,

Laboratory California,

of November

AND

29.

RESEARCH COMMUNICATIONS

RELATIONSHIPS

Doneen,

Yee

Research

Received

AND BIOPHYSICAL

Chung,

BINDING

Donald

Lob*

IN

THE

ASSAYS

Yamashiro,

and

Choh

Hao

Li

of Pharmacology, California 94143

University

1976

SUMMARY: The opiate activities of some derivatives and enzymatic digests of camel and human P-endorphin were determined in the guinea pig ileum and rat Derivatives of (3-endorphins altered within brain opiate receptor binding assays. the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of Ph-endorphin produced only Chymotryptic digestion greatly weakened the opiate small reductions in activity. activities of Ph-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of P-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of P-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack. Several of

brain

have

have

been

the

isolation

the

camel

been

purified and

have

human

which

isolated. from

also

and

and

potent

been

identified

and

opiate in

pituitary -(61-91), Abbreviations: P-endorphin; P,-LPH, sponding

f3-LPH

and (8,

respectively.

correspond Each

Met-enkephalin, /3h-endorphin, sheep p-lipotropin; to residues (61-65),

Copyrighr 0 I977 by Academic All rights of reproduction in any

brain

the

of these

pituitary

to

(1,2).

Li

been

peptides

656

the

opiate and

and

peptide

of the

within

possess

, of the

reported from

untriakontapig

(4),

sheep the

(5),

pig

of Met-enkephalin, the

-(61-76),

enkephalin; PC -MSH,

(3)

from

sequences

contained

Chung

(P-endorphin)

characterized acid

receptors

Leu-enkephalin,

homologous

p-LPH-(61-65),

methionine human P-endorphin; p-LPH-(61-65),-(61-76), -(61-76), etc.

Press, Inc. form reserved.

Met-

glands

amino are

named

The

have The

for

acid

activities.

P-endorphins

9)and

ligands

amino

y-Endorphins (7).

y-endorphins,

bovine

of a thirty-one

neurohypophysis-hypothalamus a-

endogenous pentapeptides,

porcine

with

(I-

(5,6).

are Two

structure

pituitary

peptides and

peptides

substantial

sequence

of

-(61-77),

and

morphine-like

PC-endorphin, camel camel (3-melanotropin; etc., that peptide corresequence of /3-lipotropin.

Vol. 74, No. 2, 1977

activities

when

specific

binding

widely

to

--in

brain

in

action

mice

enkephalins

Phe4

assays.

Met5

or

(14-

17).

We

Results

the

guinea

when

measured weak

rats

injected

Leu5

residues

have by

guinea

of these

studies

are

2,6,7). --in

vivo. analgesic

centrally

(12)

or

have the

for

the ileum

reported

and

intravenously

vitro

opiate

and

as

display Leuafter

(3-endorphin

the

first

and action

shown --in

or they

Met-

contrast,

for

assay,

However,

In

pig

Materials

ileum

(10-13).

analyzed

of /3-endorphin

pig

transient

relationships

and

relationships

in

(1,

or

when

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

receptors

display

Structure-activity

W-l,

as

activities

a-endorphin

injection analgesic

vitro

opiate

opiate and

intracranial potent

measured

different

enkephalins

BIOCHEMICAL

has (13).

importance

time

the

rat

brain

of the

activities

of

structure-activity opiate

receptor

herein.

Methods

PC-Endorphin (18), Ph-endorphin (19) and (3,-MSH (20) were and p,-LPH were purified as thesized by the solid-phase method. p,was one of the tryptic fragments (T-17) described (8,9). Ph-LPH-(61-69) in the amino acid sequence determination of Ph-LPH (9). Met-enkephalin purchased from Bachem Laboratories (Marina de1 Reyes, Ca. ).

synpreviously utilized was

Acetone-treated Ph-endorphin was prepared by reaction of 12.8 mg of the peptide in 50% aqueous acetone (20 ml) for 21 hr at 24’. Partition chromatography on Sephadex G-50 in 1-butanol-pyridine-0.6 M NH40Ac (5:3:10) gave essentially one peak with Rf 0.48 (Rf of Ph-endorphin, 0.37) and 9.1 mg of product. On paper electrophoresis (pH 3.7, 400 V, 4 hr) the substance behaved as a more acidic material (Rf 0.48 relative to lysine as compared to Rf 0. 54 for P-endorphin). A sample heated in 0. 1% acetic acid for 10 min at 100” liberated acetone. All these properties parallel the behavior of acetone-oxytocin whose structure (21) consists of an isopropylidene bridge between the N-terminal amino group and the nitrogen of the peptide bond between sequence positions 1 and 2. (Anisylated-Glu31)-(3h-endorphin synthesis of h-endorphin (19) and was on Sephadex G-50 in 1-butanol-pyridine-0. gave a Rf of 0.53. Amino acid analysis, peptide mapping indicated an anisylated C-terminus.

endorphin (Eastman

(Homoserine5)-(3h-endorphin in 0. 5 ml 70% After Organic).

formic stirring

acid

was isolated as a by-product in the purified by partition chromatography 1% acetic acid (5:3:11) in which it ultraviolet spectral analysis and glutamic acid residue (22) at the

further

was produced by dissolving 10 mg phfollowed by addition of 20 mg CNBr at 24” for 24 hr in the dark, the solution

657

Vol. 74, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was diluted with a large volume of water and lyophilized. Peptide mapping and end-group analysis showed that no cleavage occurred. The Met-Thr bond has previously been shown to be resistant to CNBr cleavage in 70% formic acid (23). The amino acid analysis indicated that Met5 was converted to homoFormic acid treated-Ph-endorphin was produced as above serine by CNBr. except no CNBr was added. Carboxypeptidase A digestion of ph -endorphin 1.6 mg of Ph-endorphin in 0. 1 ml, pH 8.5, Tris-HCl carboxypeptidase A (Worthington). The suspension 8 hr and the peptide recovered by lyophilization.

was done buffer and was incubated

by dissolving adding 0. 05 mg at 37 a for

Tryptic digestion of Ph-endorphin was performed by dissolving Ph-endorphin in 0.2 ml 0.2 M NH4 OAc buffer (pH 8. 0). Trypsin (Serva), was added and the solution incubated at 37 ’ for 8 hr. The reaction was by addition of 1. 8 ml 0. 05 N HCl. Chymotryptic digestion of Ph-endorphin done as above except that chymotrypsin (Calbiochem) was used.

1. 1 mg 0.02 stopped was

Leucine aminopeptidase digested peptides were prepared as follows: PC-endorphin (0. 34 mg) and Met-enkephalin (0. 06 mg) were each dissolved in 0.1 ml pH 8. 5 Tris buffer. Leucine aminopeptidase (0. 007 mg, Worthington) was added to both samples, and the solutions incubated for 2 hr at 37’. The digests were acidified to pH 3, lyophilized, and portions removed for amino acid analysis 0 Narcotic agonist activities were measured from the depression of electrically-stimulated contractions of the guinea pig ileum prepared as Briefly, a 2-3 cm segment of ileum was mounted previously described (24). between internal and external platinum electrodes in a 25 ml bath containing Kreb’s solution (with 125 nM diphenyamine as antihistamine) at 37’ and aerated with 95% 0215% CO2. Tension changes evoked by rectilinear electrical pulses (0.4 msec duration, 0.1 Hz, 80 V) were recorded by a Grass 2 g) coupled to a model 7 Grass Isometric Pressure Transducer (tension, Polygraph. Peptides and normorphine were assayed on two ileum preparations at 3- 5 concentrations. Opioid activity was also assayed as the inhibition of stereospecific 10 Ci/mmole) binding to lysed rat f 3H]-morphine (New England Nuclear, synaptic plasma membranes prepared essentially as described (25). Synaptic plasma membranes (200 yg protein) were preincubated in 2 ml sodium-free HEPES buffer (26 mM, pH 7.5) at 25” for 10 min with peptides, morphine sulfate or dextrorphan. Next [ 3H] -morphine was added (4 nM) and the incuAssays were terminated by filtration followed by bation continued for 15 min. three 4 ml rinses with ice-cold buffer on glass fiber filters (Whatman GF/c). Filters were dried and counted by liquid scintillation spectrophotometry. Stereospecific opiate binding, the difference between [ 3H] -morphine binding in the presence of dextrorphan (1 x 10e6M) and morphine sulfate (1 x 10e6M) was 45% of the total binding. Peptides were assayed in quadruplicate; standard errors of the mean c-p. m. were always less than 6%.

the

IC50

Potencies (inhibiting

of peptides in molar concentration

both

assays - 50%)

658

were calculated as to that of PC-endorphin.

the

ratio

of

mg,

Vol. 74, No. 2, 1977

BIOCHEMICAL

Table Opiate

Activities

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

of (3, -Endorphin

and

Related

Preparation

Peptides

Relative pig

Guinea ileum

Potency:k in Brain opiate receptor binding

Pc-endorphin

1.00

1.00

Ph-endorphin

1.00

1.00

Met-

0.25

0.

0.65

---

enkephalin

Normorphine

34

Ps-LPH

< 0.01

<

0.01

Ph-LPH

<

0.01

<

0.01

Pc-MSH

<

0.01

<

0.01

Ph-LPH-(61-69) Acetone

treated

-Ph-endorphin

(anisylated-Glu31) Formic

-Ph-endorphin

acid-treated-Sh-endorphin

(5-homoserine)-Ph-endorphin

<

p h -endorphin:

Carboxypeptidase

Ph-endorphin:

Chymotryptic

Ph-endorphin:

Tryptic

digest

Leucine

aminopeptidase

PC

-endorphin:

Met-enkephalin:

digest

* Potency of PC -endorphin -endorphin standard to PC in the guinea pig ileum binding assay.

to PC-endorphin are

summarized

activities as in

of

measured Table

<

digest

0.01

0.02

0.76

0.75

1.00

1.00

< 0.01

1.00

1.00

0.13

0.03

0.71

0.61

0.97

0.93

0.01

<

is defined as 1.00. Other preparations on a molar basis. IC50 of PC-endorphin: assay; 1. 2 -3.7 x 10s7M in the rat brain

Results Opiate

digest

aminopeptidase

0.26

0.01

A digest

Leucine

0.20

I.

and the

0.01 were compared 2.5-g. 0 x 10s8 opiate receptor

Discussion

various

peptides

in

the

guinea

In

the

ileum

pig assay

CFQ

and ileum PC-

enzymatic and

and

rat

digests brain

Ph-endorphins

relative

receptor

assays were

each

M

Vol. 74, No. 2, 1977

55%

more

potent

BIOCHEMICAL

than

normorphine,

Similarly

in

the

compared

to

(3-endorphins.

was In

opiate

somewhat

less

contrast,

when

tested

SC-MSH endorphin

to

the

which

incorporates

(opiate

receptor

Whereas residue

with

opiate

the

its at

In

opiate

activities

in as

well

Ph-endorphin residues

had

residues

other

potency reduced

/3,-LPH-(61-69),

may

be

little

of

in activities which

directly

(71%)

even

P,-LPH

and

or

SC-

by

a bridge

(ileum)

and

2%

anisylated in

the

carboxyl-terminal

the

interaction to

chymotrypsin

5-

at

opiate

activities. glutamic

of Ph-endorphin homoserine

diminished

be

expected On

residues.

the

no

specificity

within

A)

or

(trypsin)

limited

been

shown

to

again

the

digestion, and

still be

to

can

Trypsin

can

1%

decline

of Met5

indicating

(4),

were

26).

Tyr’-Gly’

only

essential,

with

A has

ileum

assay,

Ph-endorphin.

having

action.

(11,26).

either

also

between

a modest

conversion

(carboxypeptidase

opiate

in

Sh-Endorphin,

showed

aromatic

enzymes

l-9),

observed

to normorphine

activity.

Chymotrypsin

LPH-(61-91)

for

endorphin these

no

at

(see

Tyr’,displayed

residue,

Carboxypeptidase porcine

of

addition,

assays.

as with

activities. from

group

of Ph-endorphin both

(34%)

to Sh-endorphin-(

activity

M

cyclized

contribute

receptors.

-6

x 10

potency

shown).

Tyr to

no

Met-enkephalin.

reduced

previously

antagonism

of Ph-endorphin

appears

potency Phe4

amino

acid

Hydrolysis

5.0

Sh-endorphin,

amino-terminal

the

showed

of

than

corresponding as

SC -MSH

not

treated

glutamic

abolished

-69))

naloxone-like

(data

active had

Met-enkephalin, and

show

assay)

its

acid

than

more

Met-enkephalin

concentration

ileum

C-terminal

assay,

Sh-LPH

failed

Acetone

its

potent

high

in

4-times

Sh-LPH-(61

at the

also

and

receptor

and

S,-

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

greater

expected

to

to

receptor than

the

be

produced

cleave

&-endOrphin

other

hand,

the

amino-terminal effects

cleave

the

digestion

Gly9’-Gln91

reduced

of the the

opiate dipeptide

of these

somewhat (61%).

activities by

vitro

nature

binding

of five

on --in

non-essential

however,

opiate

considerably

action

ph-

Interestingly, nonapeptide of trypsin

on

Vol. 74, No. 2, 1977

(3h-endorphin. or

BIOCHEMICAL

Therefore,

some

other

the

tryptic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

trypsin

digest

or

fragment,

contains

fragments

in

undigested

&-endorphin

combination,

having

opiate

activity. PC-Endorphin aminopeptidase detected

was as

by

acid

showed

Met-enkephalin, lost

all

residue

by

protected

known

to

potent

[ D-Ala21

rapidly

P-endorphin greater

only

especially

resistance,

to

(28). Met-enkephalin at

differ

This work was Acknowledgements. National Institutes of Health (GM-2907) B.A.D. is a recipient of a Fellowship Metabolism and Digestive Diseases.

this

(27),

but

The

greater vivo

and from

in

digest

contrast,

to

part

NIDA grants the National

by

bond, the

amino

of Met-enkephalin and

plasma

readily

analgesic result

of

Tyrl-Gly’

brain

less

may

in

in

that

as

digest

Therefore,

amino-terminus,

supported

enzyme

of the

from

leucine

of tyrosine

activities;

Enzymes

--in its

1,

opiate

attack.

enkephalins

to

Table

digestion.

appears

with

amounts

hydrolysis

aminopeptidase

-Met-enkepahlin compared

of

hydrolysis

trace

in

stoichiometric

exopeptidase

inactivate

shown

dimunition

of PC-endorphin from

limited

of only

As

slight

leucine

very

liberation

showed

after

terminal

by

to

analysis.

which

activity

being

indicated

amino

PC-endorphin

susceptible

the

potency in

enzymic

a grant (DA-01314 Institute

part

are

more of

from

its

digestion.

from

the

and DA-00564). of Arthritis,

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2. 3. 4. 5. 6. 7.

Hughes, Morgan, Simantov, 2515-2519.

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BIOCHEMICAL

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662