The effect of inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase on TRH degradation in rat serum

COMMUNICATIONS

BIOCHEMICALANDBIOPHYSICALRESEARCH

Vol. 132, No. 2, 1985

Pages 787-794

October 30, 1985

THE EFFECT OF INHIBITORS OF PROLYL ENDOPEPTIDASE AND PYROGCUTAMYL PEPTIDE HYDROLASE ON TRH DEGRADATION IN RAT SERUM Theodore

C. Friedman

Department of Pharmacology, of the City University Received

September

20,

and Sherwin

Wilk

Mount Sinai School of Medicine of New York, NY 10029

1985

The identity of the enzymes catalyzing the degradation of thyrotropin releasing hormone (TRH) In rat serum was investigated by the use of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase. These inhibitors did not protect TRH from degradation. but o-phenanthroline afforded significant protection. The participation of "thyroliberinase", a metalloenzyme which cleaves TRH at the pyroglutamyl-His bond was implied. A coupled assay using the chromogenic substrate pyroglutamyl-His-Pro-2-naphthylamide and excess diaminopeptidase IV was developed to specifically quantitate "thyroliberinase" activity. Rat serum catalyzed the degradation of 67.5 nmoles substrate/ml serum /h. The data indicate that TRH is degraded in rat serum predominantly by "thyroliberinase" and that prolyl endopeptidase and pyroglutamyl peptide hydrolase do not contribute significantly to this 0 1985 Academic Press, Inc. process.

Early

reports

metabolite of

breakdown

(7).

Knigge of

found.

deamidation known

TRH

of

(TRH-free Schock

(3)

tripeptide

although

By

contrast,

in

TRH in

deamidation is

also peptides

pyroglutamyl

peptide

a distinct

either

of TRH is

are

enzyme

or

by

with

was

a high

indicated

the

major

deamidation

recent

studies

of

hydrolyzed 3.4.11.8) specificity

initial TRH - free for

found

(4-6).

by

the

(9,10), for

acid

It

is

now

3.4.21.26)

(8).

Although enzyme

appears

pGlu-His

the were

thiol-dependent serum

the

in

significant

(E.C. bond

(1.2).

step

evidence

pGlu-His

deamidated

formed

endopeptldase

the

the

product

of

was not

by prolyl

that

as the

amounts

human serum

cleavage

(E.C.

serum

significant

catalyzed

readily

hydrolase

acid)

more

rat

degraded

TRH in

supported

this

of

that

and

pyroglutamyl

contain

degradation

pGlu-His-Pro-OH

Studies

not

on the

bond

to

also

of

TRH.

Abbreviations used: TRH, thyrotropin releasing hormone; pGlu, py.roglutamyl; 2, N-benzyloxycarbonyl; PDMK, pyroglutamyl diazomethyl ketone; 2-NA. 2-naphthylamide; t-Boc, tert-butyloxycarbonyl; DAP IV, diaminopeptidase IV; DFP, diisopropylfluorophosphate. Synonyms of pyroglutamyl peptide hydrolase are pyroglutamyl aminopeptidase, and pyrrolidonyl peptidase. 0006-291X/85 787

$1.50

Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 132, No. 2, 1985

This

serum

appears

enzyme

BIOCHEMICAL

termed

"thyroliberinase"

to be a metalloenzyme

We have

recently

directed

inhibitors

(14-16).

Prolyl

peptide

irreversibly

by pyroglutamyl

Z-Pro-Prolinal pyroglutamyl

the

prolyl

hydrolase

and PDMK have peptide

synthesis

has

and

endopeptidase

endopeptidase

Pyroglutamyl

(11)

RESEARCH COMMUNICATIONS

been

partially

purified

and

(12.13).

described of

AND BIOPHYSICAL

and

inhibited

noncompetitively

is

inhibited

competitively ketone

been used to clarify and

of

pyroglutamyl

is

diazomethyl

hydrolase

properties

"thyroliberinase"

active-site

peptide

hydrolase

by Z-Pro-Prolinal

(14).

by 5-oxoprolinal

(PDMK) the

potent

(16).

roles in

In the of prolyl

the

degradation

(15) present

and study

endopeptidase, of

TRH in

serum. MATERIALS

AND METHODS

pGlu-2NA was obtained from the United States Biochemical Corporation (Cleveland, OH). pGlu-Hls-Pro-2NA was obtained from Bachem, Inc. (Bubendorf, Switzerland). PDMK (16) and Z-Pro-Prolinal (14) were synthesized as described previously. TRH, Pro-2NA, t-Boc-His, o-phenanthroline, dithiothreitol and thimerosal were purchased from Sigma Chemical Co. (St. Louis, MO). N,N'-Dicycloheylcarbodiimide was obtained from Aldrich Chemical Corp. (Milwaukee, WI). I-125-TRH was obtained from New Trasylol was obtained from Mobay Chemical Corp. (New England Nuclear (Boston, MA). York, NY). Pentex bovine serum albumin was obtained from Miles Laboratories (Kanakee, IL). Silica-coated plates (Polygram Sil G/UV 254, 40 mm x 80 mm) for thinlayer chromatography were purchased from Brinkmann Instruments (Westbury, NY). Rabbit sera and diisopropylfluorophosphate were obtained from Calbiochem-Boehring (San Diego, CA). Bovine brains were obtained from a local abbatoir. TRH antisera was a generous gift from Dr. Piers Emson of the MRC Neuropharmacology Unit, Cambridge, UK and was the same antisera as previously characterized (17). His-Pro-2NA was prepared by coupling t-Boc-His to Pro-2NA in the presence of N,N'-dicyclohexylcarbodiimide. The t-Boc group was removed by treatment with trifluoroacetic acid and the trifluoroacetate salt of His-Pro-2NA was obtained by removal of the trifluoroacetic acid by evaporation followed by precipitation with ether. DAP IV was purified to apparent homogeneity from rabbit kidney cortex essenThe purified enzyme liberated 500 tially as described by Yoshimoto and Walter (18). pmol 2NA per mg protein per h from the substrate Gly-Pro-2NA. Pyroglutamyl peptide It co-purifies with prolyl hydrolase was partially purified from bovine brain. endopeptidase up to the Sephadex G-100 chromatography step (19). Pyroglutamyl peptide hydrolase (MW 24,000-30.000) is then resolved from prolyl endopeptidase (MW 66,000) by the Sephadex G-100 column. At this stage bovine brain pyroglutamyl peptide hydrolase was purified 220 fold from the original supernatant with a specific activity of 66 units/mg protein (one unit is defined as the amount of enzyme releasing 1 nmol 2NA per h from pGlu-PNA). Male Sprague-Dawley rats weighing about 250 g were used for all experiments. They were fed a commercial Purina laboratory chow diet. Animals were decapitated and the blood collected, allowed to clot and then centrifuged at 2000 g for 20 min. The serum was removed and assayed imnediately for enzyme activity. Determination

of TRH Deqradation:

The incubation mixture (final volume 250 Pl) contained 10 ~1 inhibitor (10m5 M) or buffer, 50 ~1 of serum and 50 mM Tris-HCl buffer (pH 7.5). After a 10 min pre788

Vol. 132, No. 2, 1985

BIOCHEMICALAND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

incubation, 20 ~1 TRH (100 ng) was added. Incubations were carried out for 2 h at 37°C and were stopped with 250 ~1 methanol. Control tubes received TRH after the incubation. The tubes were centrifuged at 1000 g for 10 min, 50 ~1 supernatant was removed and evaporated to dryness under a stream of nitrogen. The samples were reconstituted in 500 ~1 of RIA buffer (0.1 mN thimerosal, 5% solution of trasylol containing 50,000 Kallikrein Inactivator Units/100 ml buffer, 0.2% bovine serum albumin in phosphate-buffered saline) and frozen at -2O'C until the RIA was performed. The RIA procedure for TRH was based on the method of Jeffcoate et al. (17). The degradation of TRH was assayed in duplicate and the RIA for each of the two tubes was run in triplicate. The amount of TRH remaining after incubation was compared to control tubes with no incubation to determine percentage degradation. The antisera does not cross-react with TRH-free acid. Z-Pro-Prolinal and PDMK do not affect the RIA for TRH. Determination

of Enzymatic

Activities

with

Chromoqenic

Substrates:

The activities of all enzymes were measured spectrophotometrically by determining the release of 2NA from the appropriate chromogenic substrate, according to the method of Bratton and Marshall (20) as modified by Goldbarg and Rutenburg (21). All assays were run in duplicate. Protein was measured by the method of Lowry et al. (22) * pGlu-PNA was used to determine pyroglutamyl peptide hydrolase activity as described earlier (23). "Thyroliberinase" activity was determined with pGlu-His-Pro-2NA in a coupled assay with excess DAP IV in the presence of Z-Pro-Prolinal, a specific inhibitor of prolyl endopeptidase (14). The assay is based on the following reaction sequence: 1.

pGlu-His-Pro-2NA

2.

His-Pro-2NA

"thyroliberinasek DAP IV

pGlu + His-Pro-2NA *

His-Pro

+ 2NA

Z-Pro-Prolinal blocks the cleavage of the Pro-2NA bond by prolyl endopeptidase. The incubation mixture (250 Pl final volume) contained 10 ~1 Z-Pro-Prolinal (final concentration lob5 M), 10 ~1 DAP IV, 50 ~1 serum and 50 mM Tris-HCl buffer (pH 7.5). Pyroglutamyl peptide hydrolase which also cleaves this substrate can be blocked by including PDMK (10e5 M) in the .incubation mixture. Tubes were preincubated for 10 min at 37'C and 10 Pl pGlu-His-Pro-2NA (10 mM in DMSO) was then added. The reaction proceeded for 2 h and was stopped by 250 ~1 10% TCA. Free 2NA was quantiated as described. The pGlu-2NA-cleaving activity in serum was measured as described earlier (23). The incubation mixture (final volume 250 ~1) contained 50 mM Tris-HCl buffer (pH 7.5) and 50 ~1 serum. In tubes containing DTT and EDTA, 20 ~1 20 mM DTT and 20 ~1 20 mM EDTA (pH 7.2) was added. Tubes were preincubated for 10 min at 37'C. pGlu-2NA was then added and the reaction allowed to proceed for 2 h at 37OC. Incubations were stopped by adding 250 ~1 10% TCA and free 2NA was quantiated as described. Identification pGlu-His-Pro-2NA:

of the

"Thyroliberinase"

Catalyzed

Cleavage

Products

of

Rat serum was prepared as described above. DFP was added to inhibit endogenous DAP IV and prolyl endopeptidase. The incubation mixture (250 ~1 final volume) contained 10 Pl DFP (final concentration 0.66 mM), 10 ~1 PDMK (final concentration lo-5 M). 50 Pl serum and 50 mM Tris-HCl buffer (pH 7.5). The tubes were preincubated for 30 min at 37°C and 10 ~1 pGlu-His-Pro-2NA (50 mM in DMSO) was then added. The reaction proceeded for 17 h at which time the incubation mixtures as well as equivalent reaction mixtures which were not incubated were spotted on silica G thin-layer chromatography plates. Authentic His-Pro-2NA as well as His-Pro-2NA generated by incubation of pGlu-His-Pro-2NA with partially purified bovine brain pyroglutamyl peptide hydrolase were also spotted. The plates were developed with 789

Vol. 132, No. 2. 1985

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

both solvent system I (chloroform: methanol: 25% ammonia: system II (1-propanol: water: ammonia; 7:3:1) and were visualized let lamp and by spraying with the Pauly reagent (24).

5:3:1) and solvent under a ultravio-

RESULTS Radioimmunoassay tion

products

was

did

not

approximately

tration

rat

with

Prolinal

50

(lo-*

tamyl

rat

are thought

at

hydrolase.

TRH-like

serum,

serum.

the

with

TRH

chelator,

This

indicates

that

do

not

in

serum,

in

of

used

serum.

the

"thyroliberinase"

(11).

a coupled

of

TRH in

to

the

previously

added

of

recovery. 1,

after

addition

the

2 h of of

TRH

Z-ProHowever,

from

endopeptidase The

degrada-

and

pyroglu-

degradation

of

by o-phenanthroline. described

TRH

a concen-

combination.

TRH.

inhibited

further

study

pGlu-2NA

assay

rat

serum

using

in

the

was used

activity

in

+ PDHK

role

TRH

These

"thyroliberi-

of

to assay serum

was

pGlu-His-Pro-2NA

enzymes

degrading

pyroglutamyl reported

as the

peptide to

prefer

substrate

1

the

presence

of

X TRH REMAINING

INHIBITOR None Z-Pro-Prolinal PDMK Z-Pro-Prolinal o-phenanthroline

by

most

is

Table Degradation

Table

in

prolyl

affect

degrada-

(11-13).

were

bond

or

degrade

action

in

of

PDMK. at

not

affected

protected

significantly

the

and

indicated not

the

Recovery

did

singly

by an enzyme which

substrates

peptides

either

since

(17).

experiment,

was

H)

degradation

Z-Pro-Prolinal

degradation

(lo-'

its

antiserum

As

be a metalloenzyme

Since

the

readily.

a metal

with

pGlu-His

study

inhibitors,

mM),

consistent to

the

PDMK

hydrolase

Chromogenic TRH

react

degraded

to be catalyzed

findings nase"

~1

(1

peptide

appears

is

or

us to

in any degradation

TRH

M),

o-phenanthroline by

than

serum,

incubation

cross

100% and

much higher In

tion

of TRH allowed

13.7 10.8 10.4 8.8 78.5

various

inhibitors

+ SEH

+ 4.9 + 4.6 + 4.0 + 3.2 + 11.5*

Degradation of TRH was determined by RIA as described in l4aterials and Methods and was compared to controls in which TRH was added at the conclusion of the The final concentration in the assay was 10e5 H for PDMK and incubation. Inhibitors were Z-Pro-Prolinal and 1 IIIM for o-phenanthroline in all experiments. Data are mean values of 4 to 6 preincubated 10 min before addition of substrate. The level of significance was found by comparing the determinations + SEH. percentage of degradation in the presence of inhibitor to the percentage of degradation in the absence of inhibitor by the two-tailed Student's T-test. * p < 0.005

and

Vol. 132, No. 2, 1985 excess

DAP IV was developed

recognize

of 2NA from

dependent

enzyme

upon

capable hydrolase

2NA by

DAP IV

residue

from position inhibit

Pro-2NA

bond

bution

of

The

the

action

did

COMMUNICATIONS

"thyroliberinaseti

would

from

also

incubation

be 67.5

+ 4.7

nmol/ml

exceeded

the

was virtually

DTT.

Omission

activity not

of

since

significantly

reduce

hydrolase

was

responsible

Moreover,

when

not

pGlu-2NA

o-phenanthroline

but

the

was used

not

lo-'

otherwise

H, will cleave

mixture,

the

the

contri-

Incubation

of

partially

this

serum,

pGlu-2NA

the

puri-

substrate

when

DAP

peptide

large

as substrate,

only

that

serum was

of

2).

of

slightly

2NA from The

former

of

enzy-

DAP IV (25).

activity

PDMK

peptide

pGlu-His-Pro-2NA

was not

EDTA

reduced

pyroglutamyl

enzymatic

activity

release (Table

amounts

cleavage

in rat

of o-phenanthroline,

assay

indicating

by PDMK and this

penulti-

of pyroglutamyl

by a combination

the

of

Similarly,

rat

2NA from

relatively

for

in the

pGlu-His-Pro-2NA In

N=4.

activity

dipeptide

2NA.

with

DAP IV from

contains

residue

A combination free

His-Pro-

an N-terimal

2NA from

by an

pyroglutamyl

eliminated,

2NA.

release

of

inhibited

exogenous serum

not

serum/h;

totally

of 2NA from

could

be

release

assayed

release

removal

incubation

will

released

of "thyroliberinase"

the

1)

(either

which

in

mixture.

DAP IV readily

(reaction

at a concentration

not

did

endopeptidase

residue

and a proline

hydrolase did

bond

IV cleaves

included

hydrolase

the

by the

endopeptidase

PDMK is

of prolyl

pGlu-His

Z-Pro-Prolinal

DAP IV only

absence

pyroglutamyl

N-terminus

of prolyl

peptide

the

Diaminopeptidase

peptide

with

the

followed

use of

pyroglutamyl

pGlu-His-Pro-2NA activity

of

N-terminal

a free

If

and excess

to

the

with

(25).

The activity

matic

RESEARCH

that

in

cleavage

2).

directly.

IV was omitted hydrolase

initial

(reaction

pyroglutamyl

found

anticipation

or "thyroliberinase")

pGlu-His-Pro-2NA

and

the

a peptide

totally

fied

the

BIOPHYSICAL

pGlu-His-Pro-2NA

of removing

peptide

mate

with

AND

pGlu-His-Pro-ZNA.

The release is

BIOCHEMICAL

in

serum.

was inhibited

stimulated

by

by DTT (Table

2). The under thin-layer system

cleavage conditions

products

of

favoring

0.72

in

expression Unreacted

chromatography. I and

pGlu-His-Pro-2NA

solvent

system

of

formed

by incubation

"thyroliberinase"

with

were

has an Rf of 0.62

II.

of

791

pGlu-His-Pro-2NA

serum

determined

pGlu-His-Pro-2NA Incubation

rat

in

by

solvent with

rat

Vol. 132, No. 2, 1985

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

Table 2 of inhibitors and activators on the pyroglutamyl-peptide hydrolyzing activities of rat serum

The effect

Specific ADDITION OR OMMISSION

Activitv

(nmoles/ml/h)

pGlu-His-Pro-2NA(1)

None -DAP IV +o-phenanthroline +PDMK +DTT/EDTA +DTT/EDTA/o-phenanthroline

75.2 55.8 50.0 67.5 41.0 1.2

DGIu-~NA(~)

+ 5.0 + 6.1" + 12.9* + 4.7 f 11.8* + 0.7"

27.1 + 2.9 6.0 30.6 26.2 2.5

+ + + +

2.6* 3.8 4.7 2.5*

1) Activity with pGlu-His-Pro-2NA was measured in the coupled assay as described in Materials and Methods. 2) Activity with pGlu-2NA was measured as described in Materials and Methods. The final concentration in the assay was 1 mM for o-phenanthroline, 10m5 M for PDHK and Z-Pro-Prolinal and 1.6 ml4 for EDTA and DTT. Inhibitors were preincubated 10 min before addition of substrate. Data are mean values of at least 4 determinations t SEM. The level of significance was found by comparing the activity in the presence of inhibitors to the activity in the absence of inhibitors by the two-tailed Student's T-test. * p < 0.05

serum

gave

solvent the

rise

system product

pyroglutamyl

to

a spot

with

II.

This

product

formed

by

incubation

peptide

an

Rf of

co-migrated of

0.09

in

with

solvent

system

authentic

pGlu-His-Pro-2NA

I and

0.17

His-Pro-2NA with

and

partially

in with

purified

hydrolase.

DISCUSSION Our findings peptide that

demonstrate

hydrolase in

rat

do not

serum

lism.

That

the

participation

specificity

His action dase

could

bond of this

then

can

release

do not

the

of TRH by rat contribute

was developed

TRH-like

TRH analog by the

can also

hydrolase

2NA from

the

which

taking (11).

of excess

792

to

indicates

to TRH metabo-

by direct

suggested the

pGlu-His

account

the

apparent

We reasoned

that

"thyro-

form

DAP IV.

by pyroglutamyl

can be blocked substrate

This

cleaves

into

pGlu-His-Pro-2NA

be cleaved

serum.

by o-phenanthroline

peptides

action

and pyroglutamyl

significantly

a metalloenzyme

assay

be released

peptide

endopeptidase

of TRH was afforded

enzyme for

substrate

of pyroglutamyl which

enzymes

A coupled

cleave

of prolyl

the degradation

"thyroliberinase"

of this would

inhibitors

protection of

high

chromogen

two

significant

of TRH (11-13).

liberinase"

prevent

these

bond

that

His-Pro-2NA. Although

peptide

by PDMK. hydrolysis

the

The pGlu-

hydrolase,

Prolyl

the

endopeptiof

the

Pro-2NA

Vol.

132,

No. 2, 1985

bond was blocked tors

BIOCHEMICAL

by addition

and excess

DAP IV,

to prevent

graphic

of the

analysis

Although enzyme

does

much poorer not

able

to

Cleavage vity

serum not

was not

pGlu-His serum

reaction

detect

significant

is

amounts to

amounts

by DTT nor

further

demonstrated

reported supported

of

of

prolyl

for

pyroglutamyl

catalyzed

(11).

substrate.

That

of the

by studies

on the

pGlu-His

endopeptidase

site

in

since

this

serum. acti-

of pGlu-2NA

of TRH cleavage

degradation

of

a

We were

hydrolase

hydrolysis

this

TRH is

"thyroliberinase".

Slow

bond.

(26)

Presumably

peptide

the major

When

chromato-

by "thyroliberinase" by PDMK.

two inhibi-

thin-layer

cleavage

than

inhibited

the

TRH degradation.

endopeptidase

was apparently

has been

bond

prolyl

DAP IV,

COMMUNICATIONS

of the

2NA from

of endogenous

contribute

for

stimulated

In the presence

products

significantly

RESEARCH

serum released

measurable

substrate

"thyroliberinase"

with

the action

contains

of pGlu-2NA

BIOPHYSICAL

of Z-Pro-Prolinal.

incubation

DFP was included

AND

by

is the

TRH analogs

by

(27).

The availability of "thyroliberinase" and its

possible

of a simple will

facilitate

and sensitive studies

colormetric on the

assay

physiological

for role

the measurement of this

enzyme

regulation.

ACKNOWLEDGEMENTS This research Development Award to T.C.F.

was supported by an NIH grant NS-17392, a Research Scientist MH-00350 to S.W. and a Medical Scientist Training Grant GM-07280

REFERENCES 1.

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Wilk,

S.

and

Orlowski,

M.

(1983)

3.

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