Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 14, 1990

Pages 620-626

LEUKOTRIENE A4 HYDROLASE

Michiko

Minami,

Haruhiko

Nobuya

IS A ZINC-CONTAINING A M I N O P E P T I D A S E *

Ohishi,

Bito, Hiroo

Wada, and

Hiroyuki

Yousuke

Takao

Mutoh,

Seyama,

Takashi

Izumi,

SHiroyuki

Toh

Shimizu +

Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, Tokyo $Protein

Engineering

113, Japan

Research

Suita, Osaka

Institute,

6-2-3

Furuedal,

565, Japan

Received October 26, 1990

SUMMARY: A comparison of amino acid sequences revealed that leukotriene A 4 (LTA 4) hydrolase is homologous to various types of aminopeptidases. Consistently with the finding, the purified LTA 4 hydrolases from both human and guinea pig sources contained equimolar zinc ion, as determined by atomic absorption spectrometry. The enzyme had a significant amount of aminopeptidase activity toward synthetic peptide substrates. Both LTA 4 hydrolase and aminopeptidase activities were inhibited by o-phenanthroline, p-chloromercuribenzoic acid, and Leu-thiol with similar IC50 values. Co-purification as well as co-immunoprecipitation of both enzyme activities with an affinity-purified antibody against LTA 4 hydrolase strongly suggest that the two enzyme activities reside in a single protein. ~ 1990 Academic Press, Inc.

Leukotrienes involved

in inflammation

is converted

by

LTB4,

by

have

shown

that

and

tissues

of the

observed

(LTs)

and

LTA 4 hydrolase

epithelial

disorders

Biochemical

and

of the

Although

small

active

(1-5).

compounds

Arachidonic

is further

distributed the

intestine

most (ii),

acid

transformed

immunohistochemica/

is ubiquitously

pig (i0, ii).

cells

of biologically

to LTA 4 (6), which

(7-9).

guinea

a family

immunological

5-1ipoxygenase

LTA 4 hydrolase

in

constitute

in

dense these

studies

various staining cells

to

cells was

lack

*

This w o r k was supported in part b y a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.

+

To w h o m correspondence and reprint requests should be addressed. Fax 81-3-5689-2704.

Abbreviations used: LT, leukotriene; PCMB, p-chloromercuribenzoic acid. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

620

5-

Vol. 173, No. 2, 1990

lipoxygenase

(12).

(13), endothelial such

a high

would

have

transfer

to

sequence

be

considered.

several

the

We

now

enzyme

peptides.

biosynthesizing

tissues

(14),

B-lymphocytes

is present

in

vitro

LTA 4

in

human

obtained

systems

(13-16), may

no

for

etc.

Why

5-1ipoxygenase

the

thereby

play

erythrocytes (15)

in cells with

was

hydrolase

including

of LTA 4 hydrolase describe actually has

LTA 4 hydrolase

lipids and

noted

Evidence

groups

LTA 4 hydrolase

that

also

intercellular providing

another

one

physiological

cells.

homology

purified

was

activity

Alternatively,

(17-19).

finding

cells of vascular

Recently,

Thus,

similar

of LTA 4 in various

role in these

and

A

LTA 4 hydrolase

explanation.

dases

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The

systems

toward

several

lines

contains

demonstrated

two

intrinsically of the

different

types

significance

to be reevaluated

EXPERIMENTAL

domain

of evidence

amount

physiological has

have

to the zinc-binding

a significant

acts

ours

an

a

of aminopepti-

indicating

that

the

equimolar

zinc

ion

aminopeptidase of

partial

cellular

activity. compounds,

of LTA 4 hydrolase

and

LT

in this context.

PROCEDURES

Materfals.---LTA4 w a s prepared as described (8). Recombinant h u m a n L T A 4 hydrolase w a s obtained using an expression vector (pEX 85), and purified by our methods (20). Native L T A 4 hydrolase was purified to homogeneity from the guinea pig small intestine, essentially b y the method b y Bite et al. (21). All other chemicals and reagents were of analytical grade. Determination of the e n z y m e activities---The L T A 4 hydrolase activity was measured, as described previously (8). The aminopeptidase activity was determined according to Pfleinderer (22). W h e n nitroanilide derivatives w e r e used as substrates, the standard reaction mixture contained 50 m M Tris-HC1 buffer (pH 7.2), e n z y m e a n d substrate in a total volume of 0.525 ml. After incubation at 25oc for 30 min, the absorbance at 405 n m w a s measured using a Shimadzu UV-730 spectrophotometer. In case of naphthylamide derivatives as substrates, the reaction mixture (0.I ml of the total volume) containing 50 m M Tris-HCl buffer (pH 7.2), e n z y m e and substrate w a s incubated at 25oc for 5 min. The reaction w a s terminated by the addition of 0.2 ml of 2 % HCI in ethanol and 0.2 ml of 0.02 % (w/v) p-dimethylaminosynnamaldehyde in ethanol. After leaving the samples at room temperature for 30 min, the absorbance at 540 n m w a s measured. The carboxypeptidase was assayed by the method of Folk (23). T h e incubation mixture contained 25 m M Tris-HCl buffer (pH 7.6)/0.1 M NaCI, substrate (hippuryl-Gly-Gly or hippuryl-L-Arg) and e n z y m e in a total volume of 0.62 ml. The increase in the absorbance at 254 n m was continuously monitored using a Shimadzu spectrophotometer U V 730. In all cases, one unit of the e n z y m e activity was defined as the activity that hydrolyzes 1 ~ m o l of the substrates at 37oc (LTA 4 hydrolase) or 25°C (aminopeptidase) for 1 min. T h e specific activity was expressed as units/mg of protein. The protein concentration was determined b y the method of L o w r y et a/. (24) with bovine s e r u m albumin as a standard.

621

Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Determination of metals b.V atomic absorption spectroscop.v---Several lots of the purified e n z y m e were applied to a gel filtration column (Superose 12, 1 x 30 cm, Pharmacia), which was equilibrated with 20 m M Tris-HCl (pH 8)/0.15 M NaCI/2 m M E D T A in order to remove various divalent cations nonspecifically adsorbed to the enzyme. The protein concentration was adjusted to 1-2 mg/ml. The zinc and iron contents were measured with a Hitachi Atomic Absorption Spectrometer Model Z-6100. Immunoprecipitation---Affinity-purified antibody against h u m a n recombinant L T A 4 hydrolase was obtained, as described previously (9). Western blot analysis revealed a single protein band with an Mr of about 68,000. Protein A Sepharose (50 /xl) and antibody (7.7-61/xg) were mixed for 2h at 4°C in 50 m M Tris-HCl buffer/500 m M NaCI (pH 7.2). The purified e n z y m e (14 /~ g) was added, and mixed well for further 15 min, followed by centrifugation at 8,000 x g for 1 min at room temperature. L T A 4 hydrolase and aminopeptidase activities in the supernatant fraction were determined by the methods described above.

RESULTS Zinc

content of LTA 4 hydrolase---The zinc content of the e n z y m e

mined by atomic absorption spectrometry,

after passing the e n z y m e

gel filtration column equilibrated with EDTA-containing ent lots of h u m a n enzyme.

The e n z y m e

thereby

was

purified from the guinea

determined.

indicating

through

Three

pig small intestine

a

differ-

contained

As a control, iron content in each lot of the

Essentially, no iron

that

nonspecificaily b o u n d the n u m b e r

buffer.

deter-

recombinant L T A 4 hydrolase contained 0.7 - 1 mol of zinc/mol

0.99 mol of zinc/tool of enzyme. enzyme

was

EDTA-treatment

was

removed

to the protein surface.

detected various

These

of zinc ligands predicted from the primary

in any divalent

sample, cations,

results agree well with structure of the L T A 4

hydrolase molecule.

Aminopeptidase activity of LTA 4 hydrolase---The aminopeptidase measured

using various synthetic substrates.

Km

nitroanilide were 0.5, and 0.67 mM, respectively, to those of hog kidney aminopeptidase

M

activity was

values for Leu-, and Ala-pwhich

(0.5 mM)

are a/most comparable

(25), although V m a x

va/ues

(1.4 and 0.7 units/mg of protein, respectively) were about 1/3 to 1/6 of those of the purified aminopeptidase M.

W h e n compared

of naphthylamide

derivatives

derivative proved

to be the best substrate,

derivative.

Essentially

thylamide) with

conjugated

with

with the rate of hydrolysis

various followed

no activity was detected

amino by

acids, the

the Arg-

and

AlaPro-

(less than 1/30 of Ala-naph-

Lys-, Set-, Glu-, G!y- , Asp-, Val-, Leu-, or Met-derivative.

622

Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A small but significant amount of the e n z y m e was

observed

in the purified L T A 4 hydrolase preparation

No carboxypeptidase hydrolase.

activity catalyzing L T D 4 to LTE 4 (0.26 nmol/min rag).

activity was detected in the purified preparation of L T A 4

Although L T A 4 hydrolase undergoes

13), the aminopeptidase

a suicide-type inactivation

reaction of the e n z y m e

with Leu-p-nitroanilide as a substrate.

proceeded

linearly for 30 rain

However, w h e n the e n z y m e

cubated with 2.4 /x M L T A 4 for 5 min, there were

(9,

was

prein-

no detectable amounts

of the

arninopeptidase and L T A 4 hydrolase activities, hence indicating that the e n z y m e was inactivated.

The results suggest that peptide substrates

have no effects

on inactivating the enzyme, but that L T A 4 inactivates the site(s) essential for hydrolysis

of both

L T A 4 and

kidney aminopeptidase

peptides.

A

commercial

preparation

of

hog

M had no L T A 4 hydrolase activity.

Evidence that a single e n z y m e catalyzed both reactions---The h u m a n recombinant

e n z y m e was purified successively by Mono chromatographies.

In

(Leu-p-nitroanilide

all cases,

the

as a substrate)

Q, Mono

LTA 4

P and

hydrolase

activities were

by

the

control p r e i m m u n e

addition

of the

affinity-purified

IgG had no effect.

binding reagent) and P C M B activities with m u c h

Furthermore,

the same

were

aminopeptidase (Fig. IA and

dose-dependently

antibody

IC50 values.

o-phenanthroline

0.01 and

were

ICs0 values 0.7 m M

pre-

(a zinc-

of o-phenanthroline

and 0.3 raM, respectively,

0.02 /xM, respectively.

results indicate that L T A 4 hydrolase requires

single

B).

(Fig. 2), while

L-Leu-thiol

/xM) inhibited 75 % of L T A 4 hydrolase and 90 % of aminopeptidase.

activity, and

12 column

inhibited both L T A 4 hydrolase and aminopeptidase

for L T A 4 hydrolase and aminopeptidase while those of P C M B

and

coeluted

Both L T A 4 hydrolase and aminopeptidase activities were cipitated

Superose

that aminopeptidase

and

(2.5

All these

a divalent Zn for its catalytic

L T A 4 hydrolase

activities reside

in a

protein.

DISCUSSION Arachidonate 5-1ipoxygenase and L T A 4 hydrolase are two enzymes which in association produces

biologically active 623

LTB 4

(1-5).

Since

an

intermediate,

Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

I

o