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