A new feature of angiotensin-converting enzyme in the brain: Hydrolysis of substance P

BIOCHEMICAL

Vol. 116, No. 2, 1983 October

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 735-742

31, 1983

A NEW FEATURE OF ANGIOTENSIN-CONVERTING ENZYME IN THE BRAIN: HYDROLYSIS OF SUBSTANCE P Hideyoshi Department

Yokosawa, Shogo Endo, Yasuhiko and Shin-ichi Ishii

Ogura

of Biochemistry, Faculty of Pharmaceutical Hokkaido University, Sapporo 060, Japan

Sciences,

Received September 20, 1983

SUMMARY: Highly purified rat brain angiotensin-converting enzyme hydrolyzes substance P which contains a C-terminal amino acid The hydrolysis of substance P with an amidated carboxyl group. verified by amino-group fluorometry and by high-performance liquid chromatography is inhibited by captopril, but not by phosphoramidon. The presence of sodium chloride is essential for the hydrolysis. The analyses of cleavage products indicate that the enzyme hydrolyzes substance P between Phe7-Phe8 and Phe8-Gly9 by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action.

Angiotensin-converting dipeptidyl

enzyme

carboxypeptidase

angiotensin

II

dipetide,

from

His-Leu

reported

which

(1,Z).

studies

correlation

exist

components

whereas

of the

brain,

Abbreviations: performance

releasing

regional

the

between

brain

brain the

e.g,

the

the

The purified

rat

of

been (3).

of ACE activity that

a positive

of ACE and those

system

(15)

cannot

in

of other

some areas

be found

in

of the

other

areas

nigra.

communication

of ACE from

has first

indicated

contents

a

C-terminal

by Yang and Neff

(3-14)

substantia

is

generation

distribution

such a correlation

In a previous

neuropeptides.

the

of renin-angiotensin

brain,

purification

in

in

I,

the

The ACE activity

on the

or immunoreactivity

catalyzes

angiotensin

to be present

Subsequent

(ACE, EC 3.4.15.1)

(16), brain enzyme

ACE, angiotensin-converting liquid chromatography.

135

we have

and its

reported

inhibition

was strongly enzyme;

the by various

inhibited HPLC,

by high-

0006-291 X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

Vol. 116, No. 2, 1983

bradykinin,

enkephalin

bility

of

tion

between

prompted brain

the

ACE toward

peptides

other

In the brain,

than

course

we found

hydrolyzed

group.

also

of ACE in the

MATERIALS

The suscepti-

and the

plays

in

correla-

neuropeptides a role

endogenous

participates

in the

formation

the metabolism

of

of neuro-

angiotensin. of

further

studies

on the

of ACE in the

role

of ACE in

brain;

it

the

effectively

P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leuthis

Our finding

only

through

a new feature

although

P.

neuropeptides

ACE not

system

substance

Met-NH2),

and substance

various

that

renin-angiotensin but

RESEARCH COMMUNICATIONS

of ACE and of cognate

us to propose

II

AND BIOPHYSICAL

analogue

distribution

angiotensin

role

BIOCHEMICAL

peptide

fits

lacked

well

with

the

a free

terminal

carboxyl

above

proposition

for

the

brain.

AND METHODS

Rat brain ACE was purified as previously described (16). ACE-I preparation was mainly used in this communication, because almost the same results were obtained with ACE-II preparation. Substance P, (Tyr8)-substance P, and phosphoramidon were purchased from the Peptide Institute, Osaka. Substance P octapeptide, fragment (4-ll), and Phe-Phe were obtained from Sigma Chemical Co., St. Louis, MO. Fluorescamine was a product of F. Hoffmann-La Roche Inc., Diagnostica. Captopril was kindly provided by Dr. A. Awaya of Mitui Pharmaceuticals, Inc., Tokyo. The reaction mixture (100 ~1) contained 0.1 M borate buffer (pH 8.2), 0.3 M NaCl, 50 UM substance P, and 33 ng ACE and was incubated at 37'C. The extent of hydrolysis was monitored by measuring fluorescence generated by the reaction of amino groups with fluorescamine (17) and by HPLC as previously described (18). The chromatographic equipment consisted,of a TRI ROTAR pump, a VL-611 sample injector, a GP-A30 solvent programer, and a UVI-DEC-100 spectrophotometer (JAPAN Spectroscopic Co. Ltd., Tokyo). The column used was TSK-LS-410-ODSSIL (4 x 300 mm)(Toyo Soda Kogyo, Ltd., Tokyo). Cleavage sites of substance P were determined by the following two procedures: (a) amino acid analyses on a JEOL JLC-6AH amino acid analyzer of the cleavage products separated by HPLC, and (b) identification of dansylated derivatives of N-terminal amino acid residues newly formed as a result of hydrolysis, as previously described (16). RESULTS Hydrolysis

of

fluorometrically. fluorescence

substance

P with

As shown in (amino

groups)

Fig.

the

1, the

was proportional 736

ACE was measured generation to time

of and was

Vol. 116, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

0

30 TIME

RESEARCH COMMUNICATIONS

60 (min)

1. Hydrolysis of substance P with ACE in the presence (0) ii%%a sence (0) of 1 UM captopril, as monitored by measuring fluorescence generated by the reaction of amino groups with The vertical coordinate indicates (Ft-Fo)/Fo, f luorescamine. where Ft represents fluorescence measured at a given reaction time and Fo indicates the initial fluorescence.

completely

inhibited

by 1 UM captopril.

Hydrolysis

P analogues,

(Tyr8)-substance

P and substance

gave almost

the same results.

A concentration

10 nM of captopril ramidon tory

was required

at a concentration

activity.

of

The presence

for

P octapeptide, of approximately

50% inhibition.

10 PM exhibited of

of substance

Phospho -

negligible

sodium chloride

was essential

for

the hydrolysis

of substance

P, in a manner similar

for

the hydrolysis

of a typical

synthetic

His-Leu

(16).

The rate

P was comparable (Arg6)

treated Five

in the peptide

to those with

major

reaction

Phe and substance

increase

of

the

generation

of

substance

P, respectively.

fluorescence

As shown in Fig.

from substance

(16).

P and ACE (Fig. with

2).

standards

by HPLC The Phe-

The area of peak 5, substance

of time described

Z(d),

hippuryl-

peaks were detectable

in peaks 4 and 5 coeluted

as a function

to that

and Leu-enkephalin-

same enzyme preparation

of peaks 1, 2 and 4 increased (c) 1 *

substrate,

from bradykinin

peptide-containing mixture

materials

P, decreased

of amino group

inhibi-

in an inverse above

(Fig.

as a function captopril 737

manner to the l),

of time

while (Fig.

(1 PM) completely

those 2(a)-

Vol. 116, No. 2, 1983

BIOCHEMICAL

, 1

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

4

2

8

10

TIME

12

(min)

gfiizts

2. High-performance liquid chromatography analysis of of ACE digestion of substance P. The reaction mixture was incubated at 37’C for 0 (a), 15 (b), and 30 min (c) in the absence of captopril and for 30 min (d) in the presence of 1 PM captopril, and 40 ul-aliquots were analyzed by HPLC using a TSKLS-140-ODS-SIL column (4 x 300 mm). The column was equilibrated Elut ion with acetonitrile/50 mM H3P04, pH 1.9, (5 : 95, V/V). was carried out at room temperature with a 16 min linear gradient of S-65% acetonitrile in 50 mM H3P04, pH 1.9, at a flow rate of 1 ml/min. The absorbance at 220 nm was monitored.

inhibited

the hydrolysis

of substance

compositions

of cleavage

products

in Table

The results

allowed

I.

ed products. and (9-11)

The detection indicates

Phe’ and Phe8-Gly’ latter

the

amount of peptide

to that

Phe, appears carboxypeptidase

of

substance

activity

(l-7),

substance

fragment

than (9-11)

from fragment

of ACE. 738

Although

isolat(1-8)

P at Phe”-

endopeptidase-like

P hydrolyzed.

to be generated

of these

fragments

ACE can cleave

Amino acid

by HPLC are shown

the assignment

of peptide

bonds by its

ACE.

separated

bond seems to be more susceptible

because rable

that

P with

action. the

former

was almost

Fragment (l-8)

The one, compa-

(7-8))

Phe-

by the dipeptidyl

fragment

(8-11)

has

BIOCHEMICAL

Vol. 116, No. 2, 1983

Amino

Acid

Compositions

Peak

14 0 12 0

aThe extent b Determined

27 0 30 0

15 0 13 0

the

11) is completely endopeptidase

action

newly

of

and,

carboxypeptidase substance

P was 66%. of substance

P hydrolyzed.

of fragment supports

substance

(9-

the

P.

of hydrolysis

of

to be in the following the

successive

of fragment

action,

8 89 2 86

(7-8) Complete

as the result

possibly,

Yield (%jb

(l-8)

the generation

indicating

Pa

(l-7) (9-11)

by 1 PM captopril

formed

Substance

Fragment Identified

0 28 0 0

ACE were determined

(l-8)

toward

0 36 0 0

of ACE toward

Phe,Gly>Gln>>Lys,

dipeptidyl

2 35 0 0

that

inhibited

P with

of fragment

Products

of substance of the amount

fact

activity

N-termini

order;

29 13 1 0 21 23 0 100

of hydrolysis on the basis

not been detected,

substance

TABLE I Cleavage

of

RESEARCH COMMUNICATIONS

Amino Acid (No1 %) Pro Lys Glu Phe Gly Leu Met

Arg 1 2 3 4 5

AND BIOPHYSICAL

(l-7)

as well

P as described

cleavages by the

as the

endopeptidase

above.

DISCUSSION We described P was hydrolyzed contained group.

a new feature with

the purified

a C-terminal

2) and the endopeptidase for

ACE of

fluorogenic

amine

a recent

(19) .

generation

activity

substrate

The possibility

that

the

Substance

ACE, although

an amidated

it

carboxyl

carboxypeptidase

(1,

of ACE has not been reported

on the hydrolysis containing that

with

C-terminal

the hydrolysis

in our ACE preparation

hydrolysis

of a fragment,

with

brain

as dipeptidyl

report

is due to a contaminant the fact

rat

amino acid

ACE is characterized

except

of ACE in the brain.

of substance Gly-Leu-Met-NH2, 739

human kidney nitrobenzylof substance

is

P and,

ruled

out

especially,

from substance

P by the

P are

Vol. 116, No. 2, 1983

completely

BIOCHEMICAL

inhibited

by captopril,

Furthermore,

the presence

the activity

of ACE toward

that

toward

specific

inhibitor

for

His-Leu

liberated

is essential

(1,Z).

activity

toward

for to

Phosphoramidon,

metalloendopeptidase (ZO),

substance

of ACE, substance 8 bond between Phe -Gly’

fragments

sequentially,

(l-8)

releasing

susceptibility

of

been thought

and exerts

a

having

does not

inhibit

P or toward

the

hippuryl-

the

conformation important

role

various

present

free

in the brain

it

carboxyl

scissile

amidated

on their

group,

leads

activity

groups.

activity

carboxyl

The

of ACE. are

ACE may involve free

to the

an

termini

that

lacking

accessibility

even

bonds may play

can be thought

of neuropeptides

has

some circumstances

the endopeptidase with

(21))

carboxyl endopeptidase

have no terminal

neuropeptides

depending

C-terminal

P around

of good

ACE, whose substrate

under

in enhancing

cleaved

The demonstration

enhanced

in the metabolism

termini,

were further

ACE has an intrinsic

of substance

hydrolyzed

or Phe7-Phe 8 and the

P toward

a free

activity

substrates

P was initially

dipeptides.

to require

this

and (l-7)

substance

us to suppose that

also

substrate

of ACE.

P, in a manner simialr

action

the peptide

Since

inhibitor

(16).

By the

if

a specific

substance

neutral

of ACE either

RESEARCH COMMUNICATIONS

sodium chloride

carboxypeptidase

activity

at

of

most typical

dipeptidyl

AND BIOPHYSICAL

carboxyl

active

site

of

the enzyme. Substance fied

from human brain

chelating

agents

The cleavage Gly’.

P-degrading

sites

Another

(18). not

The enzyme was inhibited

either

were between

enzyme that

has been recently bound neutral

but

enzyme has been reported

reported

by captopril Gln6-Phe7,

hydrolyzes

metalloendopeptidase

Phe7-Phe8

with

susceptible 740

by metal-

or phosphoramidon.

substance

to be identical

to be puri-

and Phe8-

P in pig

caudate

a membraneto phosphoramidon

BIOCHEMICAL

Vol. 116, No. 2, 1983

The cleavage sites Glyg-Leu 10 . The molecular

were between

(7+21-

er than

that

of ACE.

brain lism

which

have

(18,22). of substance

P is distributed

weights

been

weight

P in a specific at the

high

exist

site, between

to hydrolyze

whether

Phe7-Phe8

two enzymes

in cleavage

reported

To elucidate

RESEARCH COMMUNICATIONS

Gln6-Phe7,

of the

Differences

and molecular

susceptibility, enzymes

AND BIOPHYSICAL

area

concentration,

were smallinhibitor

ACE and the

substance

ACE involves of brain, further

and

in

P in the the metabo-

where

substance

studies

are

now in progress. ACKNOWLEDGMENT the

This study was supported in part Ministry of Education of Japan.

by a research

grant

from

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AND BIOPHYSICAL

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