Vol.
92, No.
January
2, 1980
29,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 725732
1980
B-ENDORPHIN BIOTRANSFORMATION
IN BRAIN:
FORMATION OF y-ENDORPHIN
BY A SYNAPTOSOMAL PLASMA MEMBRANE ASSOCIATED ENDOPEPTIDASE
DISTINCT
J.P.H.
Burbach,
Rudolf
University
Received
J.G.
FROM CATHEPSIN D
Loeber+,
J. Verhoef
and E.R.
de Kloet
Magnus Institute
for Pharmacology,
Medical
Faculty,
of Utrecht,
Vondellaan
6, Utrecht,
The Netherlands
November
26,
1979
SUMMARY
cSPM preparations
of rat brain contain
a peptidase
activity
which
generates
y-endorphin
from B-endorphin.
Some properties
of this enzyme were
of
studied
and compared with those of cathepsin
D. Maximal accumulation
y-endorphin
upon digestion
of B-endorphin
with a cSPM preparation
was found
at neutral
pH values.
The activity
of cathepsin
D, forming
y-endorphin
and
B-LPH 78-91 was limited
to acidic
pH values.
The SPM associated
peptidase
was not
inhibited
by the specific
cathepsin
D inhibitor
pepstatin.
The peptidase
activity
remained
associated
with SPM preparations,
which were purified
extensively
by sucrose density
centrifugation.
It is concluded
that the SPM
associated
peptidase
which generates
y-endorphin
from B-endorphin
is distinct
from cathepsin
D. Such an enzyme may have a physiological
function
in the
formation
of B-endorphin
fragments
in the brain.
Recent
precursor
studies
for
(3).
shown that
neuropeptides,
al adaptation
displayed
have
(1,2).
which
In
distinct
a number
activities
y-Endorphin
(B-LPH
61-77)
opposite
to those
of psychostimulant
between
(B-LPH
drugs
(4,5).
of y-type
drugs
y- and cc-type
adaptive
behaviour
+Present
address
61-91)
are involved
in the
regulation
of behavioural
were
and,
not
paradigms,
mediated
in particular,
62-77)
displayed
a-Endorphin
(E-LPH
endorphins:
its
(1,6).
endorphins
(1,7).
(B-LPH
which
des-tyrosine-y-endorphin
of neuroleptic
B-endorphin
Studies
Therefore,
plays
activities
exerted
receptor
those
effects
that
in the
the preferential
sites
peptide
was comparable
role
fragments
resembling
was suggested
a significant
demonstrating
B-endorphin
by opiate
61-76)
as a
of behaviour-
the non-opioid
activity
it
may serve
to that
a balance
control
of
formation
of
: National
Institute
of Public
Health,
P.O.B.
1, Bilthoven,
The Netherlands.
Abbreviations:
H-LPH = B-lipotropin,
(c)SPM = (crude)
synaptosomal
plasma
membrane, HPLC = high-pressure
liquid
chromatography,
tr = retention
time.
The amino acid residue
numbers correspond
to the primary
structure
of human
B-LPH.
OOOS-291X/80/020725-08$Ol.Of'/O
125
Copyright
(4 1980 by Academic
Press, Inc.
All rights of reproduction
VI any form reserved.
Vol.
92, No.
either
2, 1980
y-endorphin
a-endorphin
this
BIOCHEMICAL
concept
proteinase
$-endorphin,
62-76)
from
S-endorphin
(8).
Recently
it
was reported,
that
cleaving
specifically
of lysosomal
generates
origin,
y-endorphin
with
of cathepsin
a specific
D with
cathepsin
generating
This
cathepsin
to pH optimum
D inhibitor
supported
D, an acid
the Leu 77 -Phe78
prompted
peptidase
COMMUNICATIONS
and des-tyrosine
by a cSPM preparation
(9,lO).
respect
RESEARCH
or a-endorphin
(B-LPH
of the y-endorphin
pepstatin,
BIOPHYSICAL
and des-tyrosine-y-endorphin
properties
those
AND
in the
us to compare
bond of
the
SPM preparation
and susceptibility
to
(II).
MATERIALS AND METHODS
Synthetic
human B-endorphin
and related
peptides
were kindly
supplied
by
Dr. H.M. Greven and Dr. J. van Nispen (Organon International
B.V., Oss, The
Netherlands).
Pepstatin
A was obtained
from the Peptide
Institute
Inc. (Osaka,
Japan) and cathepsin
D (EC 2.4.23.5)
from bovine
spleen was a commercial
preparation
from Sigma Chemical Co. (St. Louis,
USA).
A cSPM preparation
from rat forebrain
was obtained
as described
previously (12). When indicated
membranes were further
purified
by sucrose density
centrifugation
according
to Zwiers et al. (13). Briefly,
the &PM suspension
was layered
over a sucrose density
gradient
consisting
of 1.0 M and 0.4 M
sucrose.
Centrifugation
was carried
out at 100,000 gmax for 80 minutes
and
synaptosomal
plasma membranes were collected
from the 1.0-0.4
M interface.
The membranes were washed and stored at -2O'C.
S-Endorphin
(2~10~~ M) was incubated
with the SPM preparations
(3.5 mg
protein/ml)
at 37'C for 30 min in 1 ml saline
buffered
with either
25 mM
sodium phosphate
(pH 7.4, 6.7 and 5.9) or 25 mM sodium acetate
(pH 5.0 and
4.0).
In experiments
concerning
enzyme inhibition,
pepstatin
dissolved
in
ethanol
was included
in the incubation
mixture
at a concentration
of loo4 M.
In the absence of pepstatin
equivalent
amounts of ethanol
were added to the
incubation
mixtures.
The reaction
was stopped by heating
the suspension
at
95'C for 10 min and the membranes were removed by centrifugation.
The supernatant was fractionated
by HPLC.
Cathepsin
D (5 rig/ml;
12.5 U/mg) was incubated
with B-endor hin (2~10~~ M)
at pH 6.7 or pH 4.0 in the absence or presence
of pepstatin
(10 4 M) at 37OC
for 60 min. The reaction
was terminated
by heating
and the digest
was subsequently
fractionated
by HPLC.
HPLC fractionation
of the digests
was carried
out essentially
as described
previously
(14). A nBondapak Cl8 reversed-phase
column was eluted with a
concave gradient
of 30% to 75% acidified
methanol
(1.5 ml acetic
acid/l
methanol)
in 10 mM ammonium acetate
pH 4.15 at a flow rate of 2 ml/min.
The
column was calibrated
with a series
of synthetic
peptides
related
to Bendorphin
as indicated
in fig.
I and fig.
3.
y-Endorphin
present
in the digests
was identified
and quantitated
by
combined application
of HPLC and radioimmunoassay.
Fractions
of the HPLC
eluates
containing
y-endorphin
were collected.
Methanol
was evaporated
in
vacua at 60°C and aliquots
of the residual
solution
were subjected
to a
radioimmunoassay
system for y-endorphin
as described
elsewhere
(14).
726
Vol.
92, No.
BIOCHEMICAL
2, 1980
AND
BtOPHYSICAL
RESEARCH
COMMUNICATIONS
KWJLTS
Digestion
the
other
35.4
of
accumulation
of
peptides
min)
and
Figure
B-endorphin
a small
y-endorphin
a-endorphin
by
a cSPM
number
(tr
(t r
=
of
36.8
= 26.8
preparation
peptide
min),
min)
of
rat
fragments
brain
(fig.
des-tyrosine-y-endorphin
were
detectable
resulted
1A).
in
Among
(tr
by
UV absorbance
1:
HPLC separation
of peptides
accumulated
during
digestion
of B-endorphin
with
SPM preparations.
B-Endorphin
was incubated
with
a cSPM fraction
in
the absence
(A) or presence
(B) of pepstatin
at pH 6.7.
The HPLC
fractionation
of a B-endorphin
digest
obtained
with
a purified
SPM
preparation
is shown (C),
Experimental
details
are described
in the text.
The elution
positions
of a series
of synthetic
B-endorphin
fragments
and
pepstatin
are indicated
at the top.
X and Y represent
organic
contaminants
of the mobile
phase.
727
=
at
Vol.
92, No.
2, 1980
BIOCHEMICAL
AND
5
Figure
HPLC fractionation
fragments
y-endorphin
upon
around
2).
RESEARCH
COMMUNICATIONS
8
PH-
2:
B-endorphin
dependent.
7
6
pH dependency of the accumulation
endorphin with a cSPM preparation.
fractionation
by radioimmunoassay
210 nm during
BKIPHYSICAL
The largest
pH 7, while
of the digest.
has been
incubation
of y-endorphin
upon incubation
of By-Endorphin
was quantitated
after HPLC
as described in the text.
reported
previously
of B-endorphin
amounts
at acidic
with
of y-endorphin
pH values
At pH 4.0 no accumulation
The identification
only
(8).
The accumulation
the cSPM preparation
were
small
of y-endorphin
of these
detected
amounts
was pH
at pH values
were
was detectable
of
present
(fig.
in the HPLC
profile.
The fractionation
profile
endorphin
in the presence
contained
identical
compared
did
not
inhibit
of pepstatin
amounts
to the digest
of a digest
obtained
is depicted
of y-endorphin
in fig.
activity
of B-endorphin
with
in fig.
and of residual
1A. This
the peptidase
by incubation
clearly
which
IB. The digest
B-endorphin
demonstrates
converts
of B-
that
B-endorphin
when
pepstatin
to
y-endorphin.
Incubation
formation
of y-endorphin
B-LPH 78-91
of cathepsin
observed
(fig.
(fig.
3A).
D completely
cathepsin
D at pH 4.0
and the complementary
C-terminal
-4 M pepstatin
The presence
of 10
(fig.
3B).
At pH 6.7 no activity
3C).
128
resulted
in
B-endorphin
inhibited
the
fragment
the activity
of cathepsin
D was
Vol.
92, No.
Figure
BIOCHEMICAL
2, 1980
AND
BKIPHYSICAL
RESEARCH
COMMUNICATIONS
time (mlnl
3:
HPLC separation
of peptides formed by digestion
of 8-endorphin
with
cathepsin D in the absence (A) or presence of pepstatin
at pH 4.0 (B) and at
pH 6.7 (C). The elution
positions
of a number of synthetic
B-endorphin
fragments, pepstatin
and organic contaminants
of the mobile phase (X and Y) are
indicated
at the top. The incubation
conditions
are described in the text.
During
digestion
density
gradients
of 8-endorphin
y-endorphin
such as des-tyrosine-y-endorphin
y-endorphin
generating
activity
by a SPM which
accumulated
among other
and u-endorphin
was contained
729
was purified
(fig.
in the
over
B-endorphin
IC).
SPM.
sucrose
fragments
Apparently
the
Vol.
92, No.
2, 1980
BIOCHEMICAL
AND
BIOWYSICAL
RESEARCH
COMMUNICATIONS
DISCUSSION
The present
brain
study
is mediated
of this
by pepstatin
of cathepsin
y-endorphin
acidic
the
from
although
used
(15).
(IO).
In our
to acidic
conditions.
but no activity
prolonged
amounts
proteolytic
mechanisms
the final
the present
clearly
study
cleaves
Cathepsin
shock of the lysosomal
generating
extensive
et al.
washing
with
washed
was
at pH 6.7
Moreover,
pH 7, whereas
process.
with
pepstatin
Although
of y-endorphin
of y-endorphin
in the digests,
SPM associated
peptidase
pH values.
which
associated
This
was extended
In experiments
proteolytic
is
liberated
In our membrane
remained
of
bond of b-LPH
D was detectable
the
-Phe78
extract
B-endorphin
the formation
that
77
D was limited
spleen,
around
amount
enzyme,
in 0.9% NaCl.
In experiments
in this
following
fraction.
the Leu
reaction
by pepstatin.
on
at pH 3.2
enzyme concentrations.
at neutral
a soluble
optimal
same peptide
bovine
to
depending
of cathepsin
D from
demonstrates
enzyme activity
of this
accumulated
S-endorphin
D is
the range
accumulated
effect
limited
D in a crude
the
inhibited
of y-endorphin
may have affected
activity
from
to generate
is
cleaves
to cleave
at high
was completely
it
Cathepsin
of cathepsin
incubation
D acts
pH value
the activity
cathepsin
have any inhibitory
subsequent
distinct
reported
have been reported
(9).
while
experiments
Using
D action
not
to inhibition
are
however,
cathepsin
was found
to pH 7.0
did
values
readily
of pH 3.5-4.5,
SPM maximal
resistance
has been
pH range,
and at this
pituitary
at pH 4.0,
Its
Human pituitary
at a pH optimum
cathepsin
in
to y-endorphin.
SPM preparations
proteinase,
different
as substrate
anterior
even during
with
(9,lO).
bond of B-LPH and B-endorphin
cleaved
B-endorphin
such as pH dependency,
acid
B-endorphin
hemoglobin
porcine
biotransformation
D.
pH values,
using
S-endorphin
converts
association
D, a lysosomal
substrate
which
enzyme,
and intimate
Cathepsin
that
by a peptidase
Some properties
those
demonstrates
preparations
with
membranes
730
the y-endorphin
the membranes
may be compatible
brain
upon hypo-osmotic
with data
125
and
I-labeled
after
of Austen
S-endorphin
Vol.
92, No.
these
BIOCHEMICAL
2, 1980
authors
have found
In addition,
we observed
purification
of the
described
but
lead
from
is mediated
that
by a peptidase
with
complementary
together
with
y-endorphin
observation
suggests
peptidase
and that
biotransformation
that
these
decapitation
endorphin
fragments
E-endorphin
data
provided
by rapid
des-tyrosine
of B-endorphin.
in the
in vitro
des-tyrosine-y-endorphin
enzymatic
conversion
an endo-
limiting
(17).
produced
step
in the
peptides
action
at these
731
or to a-endorphin
plasma
sites
that
of the
membranes
These
and their
cleavage
may play
converted
In view
B-
(19).
by selective
study
intact
in CSF no
a-endorphin
is rapidly
(unpublished).
of B-endorphin
while
We have observed
y-endorphin
in the
be demonstrated
formed
or
Moreover,
CSF (18)
in this
in synaptosomal
presence
irradiation
methods.
y-endorphin,
neuropeptides.
system
Their
of peptidases
could
described
seemed unlikely
due to
by microwave
extraction
activity
fragments
artificially
inactivation
that
It
procedure.
either
endogenous
sites
we have
This
des-tyrosine
in human lumbar
activity
receptor
D
S-LPH 78-91,
enzyme is
rate
gland
by aminopeptidase
carboxypeptidase
of neuropeptide
heat
evidence
of these
on the pH of the
presumed
rats
The endopeptidase
generation
by cathepsin
Previously
generating
extraction
peptidase
are
formation
(unpublished).
and their
were
are present
fragments
The here
of B-endorphin,
the initial,
by different
suggestive
activity
membranes.
properties.
and pituitary
of the
converting
even after
in the brain.
and the
and subsequently
was preserved
such proteolytic
digests
a-endorphin
sacrifice
pH (16).
the SPM associated
fragment
in
fragments
followed
distinct
COMMUNICATIONS
at neutral
not due to contamination
involved
brain
degradation
upon
tissues
in rat
S-endorphin
post-mortem
persisted
is
RESEARCH
plasma
that
the y-endorphin
y-endorphin,
have been found
that
in S-endorphin
of h-endorphin
Recently
indicating
C-terminal
that
it
activity
us to conclude
is
the
peptidase
of synaptosomal
S-endorphin
detected
BIOPHYSICAL
[ 1251]-y-endorphin
of
the
component
experiments
of y-endorphin
formation
cSPM fraction
may be an intrinsic
AND
a key role
depending
to
by a
localization
(20-24)
may be of physiological
Vol.
92, No.
2, 1980
importance,
Further
y-endorphin
generating
homeostasis
in brain.
BIOCHEMICAL
studies
are
AND
required
endopeptidase
BIOPHYSICAL
to establish
as a regulatory
RESEARCH
COMMUNICATIONS
the significance
factor
of the
in endorphin
REFERENCES
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(van Ree,
J.M.,
and Terenius,
L., eds.) pp. 113-122,
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Biomed.
Press, Amsterdam.
2. Burbach,
J.P.H.,
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732