N,N′-dicyclohexylcarbodiimide is a specific, reversible inhibitor of proline-β-naphthylamidase

Vol. 168, No. 3, 1990 May 16, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1001-1006

N,N'-DICYCLOHEXYLCARBODIIIYIDE IS A SPECIFIC, REVERSIBLE INHIBIMR OF PROLINE-,9-NAPHTHYLAMIDASE Takayuki

TAKAHASHI and Kenji

TAKAHASHI*

Department of Biophysics and Biochemistry, The University of Tokyo, Bunkyo-ku,

Faculty Tokyo 113,

of Science, Japan

Received March 30, 1990 SUMMARY: N,N'-Dicyclohexylcarbodiimide(DCCD) was found to inhibit the activity of proline-B-naphthylamidase purified from porcine intestinal mucosa. The inhibition is rapid and reversible, and it is not due to the dissociation of the enzyme subunits. The mode of the inhibition by DCCD is noncompetitive with respect to each of the two substrates tested. K. values of DCCD for the enzyme were determined to be 1.9 pM with proline-@-naphthylamide and 12 pM for L-leucine ethyl ester. To our knowledge, this is the first time that DCCDwas foundtobe a potent, reversible inhibitor for an enzyme. 01990 Academic Press,Inc. Mammalian

tissues

of hydrolyzing have in

synthetic

recently the

found

mucosa

homogeneity

inhibited these

carbodiimide DCCD is

intestine

having

protease

of

enzymes

capable

aminopeptidases

(1).

known

The

consisting

a molecular or an esterase protease

another

potent

that inhibitor

as a carboxyl-modifying

to

enzyme

apparent

proline-j3-

three of

because

inhibitors found

it

of

weight

We

activity

purified

(2).

we now have

(DCCD) is

generally

and

a glycoprotein

by known serine inhibitors,

of

characterization

each

a serine

variety

substrates

pig

is

polypeptides,

a large

proline-fl-naphthylamide-hydrolyzing

of

for

naphthylamidase

either

contain

58,000. the

(2).

identical It

is

enzyme

is

In addition

to

N,N'-dicyclohexylof

this

enzyme.

reagent,

and it

*To whom correspondence should he addressed at Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo ) 7-3-l Hongo, Tokyo 113, Japan. Abbreviations: DCCD, N,N'-dicyclohexylcarbodiimide; EDC, l-ethyl3-(3-dimethylaminopropyl) carbodiimide; DCU, N,N'-dicyclohexylurea; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide. 0006-291x/90$1.50 1001

Copyright 0 1990 by Academic Press, Inc. All. rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 168, No. 3, 1990

has

been

widely

translocation chain

to

in a variety

(3-9).

of

Unexpectedly,

naphthylamidase respect

used

substrates,

study

the

enzyme

complexes

the

by DCCD is

to its

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mechanism of the

inhibition

reversible and the

of

of

proton

respiratory proline-

B -

and noncompetitive results

are

reported

with in

this

communication. MATERIALS AND METHODS

Chemicals: N,N'-Dicyclohexylcarbodiimide (DCCD) was the product of Nakarai Chemicals, Japan. I-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and pig kidney leucyl aminopeptidase were N,N'-Dicyclohexylurea (DCU), dicycloobtained from Sigma. and cyclohexylamine hydrochloride were obtained from hexylamine, Wako Pure Chemical Industries, Japan. The sources of other materials used in this work were as described previously (2). Proline-p-naphthylamidase: Proline-gnaphthylamidase was purified to homogeneity from pig intestinal mucosa according to the procedures as described (2). The purified enzyme preparation had a specific activity of 0.91 pmol/min/mg protein when assayed using proline-@-naphthylamide as substrate, and it was used in this study. Enzyme assays: Proline-(3-naphthylamidase activity was assayed in toward proline+-naphthylamide was two ways. The enzyme activity assayed at pH 8.0 as described previously (2). The esterase activity of the enzyme was assayed at pH 8.0 using L-leucine ethyl ester as the substrate according to Roberts (10). Treatment of the enzyme with the compounds containing cyclohexyl DCCD and EDC were always freshly prepared before use. group(s): DCCD, DCU and N,N'-dicyclohexylamine, and cyclohexylamine HCl were dissolved in DMSO, ethanol and distilled water, respectively. For the treatment, the DMSO or ethanol content of the reaction mixtures was fixed at 0.5% (v/v) at which the enzyme activity was not affected. Polyacrylamide gel electrophoresis and gel filtration: Polyacrylamide (7.5%) gel electrophoresis (PAGE) in the presence or absence of sodium dodecyl sulfate (SDS) was performed according to the method of Laemmli (11). Gel filtration was performed by FPLC (Pharmacia) on a TSK 3000SW column (0.75 x 60 cm, TOSO) using 0.1 M Tris-HCl (pH8.0) containing 0.2 M NaCl or in 0.1 M Tris-HCl (pH 8.0) containing 0.2 M NaCl and 10 ,uM DCCD. The standard protein markers were pig kidney leucyl aminopeptidase (325 kDa), catalase (240 kDa) and bovine serum albumin (68 kDa). RESULTS AND DISCUSSION

Figure

1

shows

naphthylamidase EDC had no effect

the

activity. on it.

effects

of

DCCD inhibited The

inhibition 1002

DCCD and the

EDC on proline-genzyme

activity,

by DCCD was consistently

but

BIOCHEMICAL

Vol. 168, No. 3, 1990

0

AND BIOPHYSICAL

4

8

12

16

RESEARCH COMMUNICATIONS

20

INHIBITOR (~JM) Fig.1. Effects of DCCD and EDC on proline-p-naphthylamidase activity. Proline-fl-naphthylamidase, was preincubated at 3 WI 37OC in 0.5 ml of 0.1 M Tris-HCl (pH 8.0) with DCCD (0) or EDC (0) at the indicated concentrations. After incubation for 15 min, the enzyme activities were assayed at 37OC with proline-j3naphthylamide. The activity without inhibitor was 0.0027 pmol/min and was taken as 100%. Essentially the same results were obtained in the experiment where the preincubation was omitted.

observed

when

adjusted

to

and

assayed

pH 8.0

M sodium

0.1

virtually

the

phosphate),

dialyzed

of

activity

fully

restored

in

was restored when

3000SW

equilibrated

were

enzyme

M NaCl.

before

is of

no change

only

a protein

the

monomer

3 identical in the

and

inhibition

being

after

gave

activity

removed

by

band with

patterns

a molecular

polypeptide

that is

1003

gel

of TSK

(pH

8.0)

of

the

conditions Both

results: weight

i.e., of

of

(2).

the we

58,000,

g-naphthylamidase,

compose

patterns

the

a HPLC column

same

the

was also

DCCD treatment. the

was

18 h,

M Tris-HCl

0.1

with min

10

and nonreducing the

proteins

preincubated

4OC for

electrophoretic

reducing

electrophoretic

at

using

with

by dialysis

20°C for

was

sample

eluted

subunits,

at

The enzyme

inhibitor

The

under

enzyme

(pH 8.0)

(90%).

the

and

and untreated

detected

the

was tested

(pH 8.0)

DCCD-treated

SDS-PAGE

compared

treated

which

the

0.2

in

of

When the

M Tris-HCl

0.1

of

protein

extent

DCCD inhibition

M Tris-HCl

0.1

filtration

containing

previously

M triethanolamine-HCl,

0.1

the

techniques.

against

enzyme

buffers

same.

filtration

DCCD (40 ,uM)

different

three

M Tris-HCl,

(0.1

The reversibility and gel

in

an

In addition,

was observed

for

the

Vol.

168, No. 3, 1990

DCCD-treated findings the

BlOCHEMiCAL

and untreated

support

the

noncovalent

protein.

with

10

(pH 8.0) with

the

DCCD,

that

Since

DCCD is

It

generally

interest

Only

in

magnitude

the

compounds

DCU inhibited higher

see

the

to

DCU in whether

containing enzyme,

concentration

compound.

20°C for

in 0.1

the

elution This

30 min

activity

was eluted

position

was

indicated

dissociation.

be unstable aqueous

a gel

M Tris-HCl

result

and is

solution,

an approximately

was of

could

tested

group(s)

DCU was

readily

it

formed

We therefore

of

(Table 2 orders

needed

Concentration

to

Remaining activity

W) 50

(%I 60.5

200

36.1

N,N'-Dicyclohexylamine

200

92.0

Cyclohexylamine

200

76.8

N,N'-Dicyclohexylurea

The

procedures

(DCU)

employed were the same as in Fig. 1004

1.

be

DCU and

Table I. Effects of N,N'-dicyclohexylurea, N,N'-dicyclohexylamine, and cyclohexylamine on proline-@-naphthylamidase activity Compound

of

by FPLC over

cyclohexyl but

from

total

DCU thus

inhibition.

results

DCCD, and it

enzyme.

thought

These

polypeptides

the

caused by subunit

stable to

that

SDS.

by DCCD would

at

was fractionated

untreated

was not

particular

a few other

subunit

17% of

was found

of the

to an inert,

involved

the

incubated

only

the

inhibition

0.2 M NaCl and 10pM

converted

directly

with

the

of

which

inhibition

of TSK 3000SW equilibrated

same buffer.

inhibition

protein

that

enzyme

containing

the

the

was first

at

column

same as that

enzyme

of

The enzyme

in PAGE without

the

dissociation

The treated

filtration

that

possibility

from

PM

remained.

the

the

resulted

the

proteins

interaction

We examined have

idea

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I). of

cause

Vol.

168, No. 3, 1990

inhibition the

BIOCHEMICAL

comparable

inhibition

converted acts

DCCD is

as a specific

Because naphthylamidase,

its

Figs.

respect

ethyl

ester.

1.9 PM for

with

the

DCCD might

unlikely.

be

The results

for

inhibition

that

ascribed

to

indicate

inhibitor was further

inhibition

DCU

that

constants

proline-f3-naphthylamide

DCCD

proline-gin detail.

As

by DCCD is

noncompetitive

as well

as L-leucine

(Ki)

and

of studied

proline-p-naphthylamide

The inhibition

possibility

proline-p-naphthylamidase.

a reversible

2 and 3, the to

RESEARCH COMMUNICATIONS

by DCCD. Thus,

inhibitor

DCCD is

with

that

observed

from

shown in

to

AND BIOPHYSICAL

12

were

calculated

JIM for

to

be

L-leucine

ethyl

widely

used in

ester. Carbodiimides, biochemical inhibitors (3,4), (61,

studies. of

c oxidase

Ca2+-ATPase and

7 =

The

DCCD, are reagents compounds

ion-translocation

cytochrome

(8),

02

in particular

from

Na+-H+

proteins, (5),

from

the

rat

5

10

03

be

(71,

potent

F1,Fo-ATPase

K+-H+

renal

-i--

( mM I-’

to

including

reticulum

80.

0

known

mitochondrial

sarcoplasmic

exchanger

are

exchanger

Na++K+-ATPase brush

border

40,

J

0

0.1 (mM)-’

Fig.2. Analysis of inhibition of proline-fl-naphthylamidehydrolyzing activity by DCCD. Proline-fl-naphthylamidase activity assayed at 37OC at various concentrations of substrate was proline-B-naphthylamide in the presence of various concentrations of DCCD. DCCD concentrations were: 0 (o), 4 (.), 6 (A), 8 (A), The figure is a double-reciprocal plot of the and 1 0 (x) @I. data. Fig.3. Analysis of inhibition of L-leucine ethyl esterhydrolyzing activity by DCCD. Proline-j3-naphthylamidase activity at 37OC at various concentrations of substrate Lwas assayed leucine ethyl ester in the presence of various concentrations of 1.96 (0). 3.92 (A), and DCCD. DCCD concentrations were: 0 Co), The figure is a double-reciprocal plot of the data. 7.84 (A) @I.

1005

0.2

Vol.

membrane

(9). In

(12,13).

manner these

DCCD also

all

enzymes

the

demonstrated

the

proteins.

of In

CAMP-dependent

inhibitions

pHs and are

modification

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibits

cases,

at acidic

covalent of

BiOCHEMICAL

168, No. 3, 1990

occur

in

irreversible. that

the

Detailed

by DCCD presented

and reversible.

The mode of the

inhibition

the

indicating

DCCD combines

substrates,

a site

other

interferes

with

information are

not

potent,

than

its

of

the

characteristic

feature

reaction.

of

current

proline-?-

study

is

site

with

the

and,

consequently,

However,

there

site

other

enzymes

on which

inhibitor.

Thus,

the

rapid

with

interaction

of

groups

is noncompetitive

on the any

reversible

the

substrate-binding

available aware

that

in

on

due to the

carboxyl

inhibition

naphthylamidase

studies are

important the

kinase

a time-dependent

inhibitions

catalytically

contrast,

protein

of

the

enzyme

is

at

yet

no

protein.

DCCD acts

DCCD inhibition

We as

a

is

a

proline-P-naphthylamidase.

REFWENCES 1. 2. 3. 4.

McDonald, J.K., and Barrett, A.L. (1986) in Mammalian Proteinases (McDonald, J.K., and Barrett, A.J., eds) Vol. 2, Academic Press, Orlando, FL PP- 23-100, Takahashi, T., Ikai, A., and Takahashi, K. (1989) J. Biol. 11565-11571 Chem. 264, Beechey, R-B., Robertson, A.M., Holloway, C-T., and Knight, 1-G. (1967) Biochemistry 6, 3867-3879 Liibben, M., and Schafer, G. (1989) J.Bacteriol. 171, 61066116

5. 6. 7. 8.

Casey, R.P., Thelen, M., and Azzi, A. (1979) Biochem. Biophys. Res. Commun. 87, 1044-1051 Martin, W.H., Beavis, A.D., and Garlid, K.D. (1983) J. Biol. Chem. 259, 2062-2065 Pick, U., and Racker, E. (1979) Biochemistry 18, 108-113 Pedemonte, C-H., and Kaplan, S.H. (1986) J. Biol. Chem. 261, 3632-3639

10. 11. 12.

Kinsella, J.L., Wehrle, J., Wilkins, N., and Sacktor, B. (1987) J. Biol. Chem. 262, 7092-7097 Roberts, P.S. (1958) J. Biol. Chem. 232, 285-291 Laemmli, U.K. (1970) Nature 227, 680-685 Buechler, J.A., and Taylor, S.S. (1988) Biochemistry

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