Báo cáo khoa học: Characterization of membrane-bound prolyl endopeptidase from brain ppt

Characterization of membrane-bound prolyl endopeptidase
from brain
Jofre Tenorio-Laranga
1
, Jarkko I. Vena
¨
la
¨
inen
2
, Pekka T. Ma
¨
nnisto
¨
3
and J. A. Garcı
´a-Horsman
1,3
1 Centro de Investigacio
´
n Prı
´
ncipe Felipe, Valencia, Spain
2 Department of Pharmacology and Toxicology, University of Kuopio, Finland
3 Division of Pharmacology and Toxicology, University of Helsinki, Finland
Prolyl oligopeptidase (POP; EC 3.4.21.26) is a serine
peptidase with prolyl endopeptidase (PE) activity,
cleaving short peptides at the C-terminal side of pro-
line residues, and is highly expressed in brain. Given
that several neuropeptides, such as substance P, argi-
nine–vasopressin, thyroliberin and gonadoliberin, are

putative POP substrates, the importance of this prote-
ase in several brain processes has been suggested [1].
However, the precise role of POP in the brain has yet
to be defined. Specific inhibitors of POP increase the
levels of these neuropeptides in the brain, exert anti-
amnesia effects, and reverse memory and learning defi-
cits produced by certain lesions [2]. Mammalian POP,
encoded by the gene Prep, has been purified and crys-
tallized, and its structure has been solved; it has been
considered to be soluble cytoplasmic enzyme. There
has not been any structural or sequence-derived infor-
mation that would suggest that the Prep gene product
Keywords
neuropeptides; neurotransmission; peptide
metabolism; prolyl endopeptidase; prolyl
oligopeptidase
Correspondence
J. A. Garcı
´
a-Horsman, Division of
Pharmacology and Toxicology, University of
Helsinki, Viikinkaari 5E, 00014 Helsinki,
Finland
Fax: +358 9 191 59471
Tel: +358 9 191 59459
E-mail: arturo.garcia@helsinki.fi
(Received 7 March 2008, revised 3 July
2008, accepted 4 July 2008)
doi:10.1111/j.1742-4658.2008.06587.x
Prolyl oligopeptidase (POP) is a serine protease that cleaves small peptides

at the carboxyl side of an internal proline residue. Substance P, arginine–
vasopressin, thyroliberin and gonadoliberin are proposed physiological
substrates of this protease. POP has been implicated in a variety of brain
processes, including learning, memory, and mood regulation, as well as in
pathologies such as neurodegeneration, hypertension, and psychiatric disor-
ders. Although POP has been considered to be a soluble cytoplasmic pepti-
dase, significant levels of activity have been detected in membranes and in
extracellular fluids such as serum, cerebrospinal fluid, seminal fluid, and
urine, suggesting the existence of noncytoplasmic forms. Furthermore, a
closely associated membrane prolyl endopeptidase (PE) activity has been
previously detected in synaptosomes and shown to be different from the
cytoplasmic POP activity. Here we isolated, purified and characterized this
membrane-bound PE, herein referred to as mPOP. Although, when
attached to membranes, mPOP presents certain features that distinguish it
from the classical POP, our results indicate that this protein has the same
amino acid sequence as POP except for the possible addition of a hydro-
phobic membrane anchor. The kinetic properties of detergent-soluble
mPOP are fully comparable to those of POP; however, when attached to
the membranes in its natural conformation, mPOP is significantly less
active and, moreover, it migrates anomalously in SDS ⁄ PAGE. Our results
are the first to show that membrane-bound and cytoplasmic POP are
encoded by variants of the same gene.
Abbreviations
AMC, amido-4-methylcoumarin; cPOP, cytoplasmic prolyl oligopeptidase; ER, endoplasmic reticulum; HA, hydroxylapatite; mPOP,
membrane-bound prolyl oligopeptidase; PE, prolyl endopeptidase; POP, prolyl oligopeptidase; PPP, pure pig recombinant prolyl
oligopeptidase; Z-Gly-Pro-AMC, N-carbobenzoxy-glycyl-prolyl-7-amido-4-methyl-coumarin; ZPP, N-carbobenzoxy-prolyl-prolinal.
FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4415
is present in any locations inside or outside the cells
other than the cytoplasm, and no variants have been
predicted or reported. This has been considered para-

doxical, due to the extracellular location of the puta-
tive POP substrates [3]. Nevertheless, PE activity has
been detected in all biological fluids and in membranes
from most of the tissues studied, especially the brain.
Different extracellular proteins with PE activity have
been described. In serum, a PE activity, insensitive to
specific POP inhibitors, has been identified as fibro-
blast activation protein or seprase, but important levels
of PE activity itself, sensitive to POP-specific inhibi-
tors, have also been detected [4,5], although confirma-
tion of this enzyme’s identity, by direct sequencing or
by antibody binding, has not been provided.
Membrane-bound PE activity has been detected and
measured, and has been considered by several authors
to be POP activity [6–8]. However, isolated prepara-
tions have been analyzed and regarded as a different
peptidase, as these preparations have shown some
physical and enzymatic features that are different from
those of its classical cytoplasmic counterpart [9,10].
Recently, we have detected binding of antibody against
POP in internal membranes in immunohistochemistry
studies in rat brain [11]. However, no clear identifica-
tion of the protein responsible for this activity has been
provided. Here, we report the purification and identifi-
cation of the membrane-bound PE (herein referred to
as membrane-bound prolyl oligopeptidase, mPOP)
from pig brain and characterization of the enzyme’s
properties in comparison to POP. The nature of mPOP
association with membranes was also studied.
Results

Membrane-associated PE activity is tightly bound
preferentially to synaptosomes and endoplasmic
reticulum (ER)
Initial whole membrane fractionation of pig brain
homogenate resulted in the partitioning of total PE
activity between membrane-bound and soluble frac-
tions with a 40 : 60 ratio. As reported previously [10],
high-salt washes and a hypotonic treatment were
required to detach loosely bound PE activity from the
membranes. Accordingly, we found that a considerable
amount of PE activity bound to the membranes was
released upon a 0.5 m NaCl wash of total membrane
preparation (Table 1). A hypotonic wash and two fur-
ther salt washes were necessary to ensure that all the
loosely bound POP was released. Further washes
released no detectable activity from the membranes,
but detectable levels were tightly attached to them
(Table 1), and those were sensitive to specific POP
inhibitors (see below).
Following this series of washes, the membranes were
further fractionated by centrifugation on a sucrose gra-
dient to determine which types of membrane contained
PE activity. After a three-cushion gradient (0.8, 1.0
and 1.2 m sucrose), we were able to separate three dif-
ferent membrane fractions, low density (on top of the
0.8 m layer), medium density (0.8 and 1.0 m interface)
and high density (1.0 and 1.2 m interface). With the
use of specific enzyme marker assays (Fig. 1), we iden-
tified the heavy membranes as ER, whereas the mem-
branes of intermediate density were mainly composed

of synaptosomal and mitochondrial membranes. The
light fraction contained myelin membranes, as
described previously [10]. Although we detected the
presence of PE activity in all membrane fractions, this
activity was maximal in the synaptosomal fraction
(Table 2), similar to the observations reported by
O’Leary & O’Connor [9]. As the activity detected in
the various membrane fractions could be attributable
to other peptidases, we applied the purification proto-
col (detailed in Experimental procedures) to both ER
and synaptosomal membranes. All elution profiles
resulting from this purification scheme were identical,
regardless of the membrane fraction origin. Moreover,
analysis of these preparations revealed that the kinetic
properties of both fractions were also identical (data
not shown). Thus, we decided to employ the purifica-
tion protocol using whole membrane preparation as
starting material, as the yield was considerably higher.
A multistep protocol enriches membrane PE
activity > 2000-fold
PE activity solubilization from membranes was only
achieved by extraction with detergents, such as
Triton X-100 at 0.4%. This detergent treatment was
sufficient to solubilize all activity associated with
Table 1. POP activity partitioning during membrane preparation
from pig brain crude extract and membrane wash effects on recov-
ered activity.
Sample
Volume
(mL)

Protein
(mg)
Specific activity
(nmolÆmin
)1
Æmg
)1
)
Total activity
(nmolÆmin
)1
)
Crude extract 1080 8704 1.0 8645
Unwashed
membranes
500 2354 1.4 3384
0.5
M NaCl wash 450 305 9.0 2760
Water wash 400 107 13.8 1480
4
M NaCl wash 300 92 4.8 440
Washed
membranes
90 1005 0.2 161
Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.
4416 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS
membranes, and also produced a three-fold activation
of enzymatic activity (Table 3). This increase was not
due to assay conditions, such as the presence of reduc-
tants. Solubilized membrane PE activity was sub-

sequently purified by several chromatography steps.
The use of a DEAE column as a first step eliminated
all cationic and most weak anionic protein contami-
nants, which altogether constituted more than half of
the total protein (Table 3). Substantial further purifica-
tion was achieved with phenyl–Sepharose and hydroxyl-
apatite (HA) columns (see Fig. S1). Although this step
yielded more than 300-fold PE activity purification
(Table 3) with respect to total membrane, SDS ⁄ PAGE
revealed the presence of several protein bands (data
not shown). Thus, to further purify PE activity, we
dialyzed the HA pool to decrease the salt concentra-
tion and reapplied it to a DEAE column (see Fig. S1
for column chromatogram). This procedure enriched
activity 1700-fold, but SDS ⁄ PAGE analysis still
revealed several contaminating proteins. Consequently,
native gel electrophoresis was utilized to improve puri-
fication. Under these conditions, a single band was
revealed by silver staining that coincided with the PE
activity profile of the gel lane (Fig. 2). It is important
to note that once PE activity was solubilized, neither
stability nor activity was modified by detergent concen-
tration. Extensive removal of Triton X-100, by series
of dilutions and ultrafiltrations where the detergent
was undetectable (< 0.0001%), did not produce pro-
tein precipitation or loss of activity.
The gel filtration profile of solubilized membrane
PE activity varies with ionic strength, similarly to
that of cytoplasmic POP (cPOP)
It has been suggested previously that the enzyme

responsible for membrane PE activity is distinct from
POP, on the basis of the difference between their
molecular masses (87 kDa versus 65 kDa) obtained
in gel filtration experiments [9,10]. The theoretical
molecular mass of POP is 80 kDa, which agrees with
estimates from SDS ⁄ PAGE [1]. Initially, we also
thought that membrane PE was heavier than POP,
as under our conditions (20 mm potassium phos-
phate) it eluted with a molecular mass of 95 kDa by
gel filtration. However, when higher salt concentra-
tions were used, membrane PE activity also eluted
with a molecular mass of 65 kDa (Fig. 3). This
behavior did not depend on the Triton X-100 con-
centration, and was very similar to that of POP
when run in the same conditions.
Identification of the protein responsible for
membrane PE activity
Peptides produced by trypsin digestion of purified
membrane PE were analyzed by liquid chromatogra-
phy–MS ⁄ MS on Qstar (HPLC ⁄ Q ⁄ TOF) or MALDI-
TOF ⁄ TOF MS, which revealed that these fragments
correspond to the sequence of mammalian POP
0.075
0.050
0.025
0.000
TM
HM
Suc. dehydrog. (units·mg
–1

)
TM
MM
HM
LM
PSD-95
Calnexin
ER-60
90 kDa
50 kDa
90 kDa
LM
MM
Fig. 1. Identification of the different membrane fractions where PE
activity is bound. Various membrane fractions were obtained fol-
lowing application of washed total pig brain membrane preparation
to sucrose gradients. The mitochondrial marker, succinate dehydro-
genase, was measured in every fraction (upper panel), as described
in Experimental procedures. PSD-95 (synaptosomal marker), calnexin
and ER-60 (ER markers) were assayed by western blotting (lower
panels). TM, total membranes; LM, low-density membranes; MM,
medium-density membranes; HM, high-density membranes. The
amounts of protein loaded onto gels were as follows: for the PSD-
95 blot, 70 lg of TM, 96 lg of LM, 89 lg of MM, and 93 lgof
HM; for the calnexin blot, 70 lg of TM, 96 lg of LM, 89 lg
of MM, and 93 lg of HM; and for the ER-60 blot, 8 lgofMM
and 5 lg of HM.
Table 2. Membrane-bound POP activity of different-density mem-
brane fractions obtained by sucrose gradient centrifugation.
Sample

Total
volume
(mL)
Specific activity
(nmolÆmin
)1
Æmg
)1
)
Total activity
(nmolÆmin
)1
)
Washed membranes 60 0.50 145
Low-density membranes 2.3 0.61 9
Medium-density
membranes
4.5 0.45 24
High-density membranes 10 0.17 21
J. Tenorio-Laranga et al. Membrane-bound prolyl oligopeptidase: mPOP
FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4417
(EC 3.4.21.26) (Fig. 4). To further confirm this, wes-
tern blots were performed using an antibody specific
against POP. All active fractions obtained during the
membrane PE purification process reacted with the
antibody against POP, thereby confirming the sequence
similarity between these two variants (Fig. 5A). Analy-
sis of the membrane PE tryptic peptides by MS did
not provide any insights into the membrane associa-
tion mechanism, such as sugar or lipid attachments

(not shown).
Within the membranes, mPOP shows anomalous
SDS
⁄
PAGE migration
Prior to Triton X-100 solubilization, different brain
membrane preparations were analyzed by western blot-
ting, using POP as a control. A significant fraction of
mPOP migrated faster than POP when a whole mem-
brane preparation was subjected to SDS ⁄ PAGE, and
anti-POP reactive bands were detected by western blot-
ting (Fig. 5B). The proportion of this ‘lighter’ form,
relative to that which corresponds to the cytoplasmic
purified POP control, varied with the type of mem-
brane fraction analyzed. As can be seen in Fig. 5B, the
myelin fraction showed a band in the western blot at
the same size as the pure pig soluble recombinant POP
(PPP), but the synaptosomal fraction showed two anti-
POP reactive bands in a ratio of approximately 50%.
The heavier ER membrane fraction contained almost
Fig. 2. Membrane PE purification by native electrophoresis from
the concentrated HA active fractions. Three adjacent lanes of a
native Triton–PAGE gel were loaded with 10 lg each of protein.
After electrophoresis (see Experimental procedures), the central
lane was excised in 5 mm pieces along the lane vertical axes. Hori-
zontal blade cuts were around 8 mm long such that small incisions
at the edge of adjacent lanes were produced to find the corre-
sponding pieces in the western blot, made with the first lane, and
the protein stain (silver), made with the third lane.
Fig. 3. Membrane PE gel filtration on Superdex-200 in 100 mM

phosphate buffer ( ) and in 20 mM potassium phosphate buffer
(
), performed as described in Experimental procedures. The
positions of elution of molecular mass standards are indicated:
ribonuclease (Rib), 13.7 kDa; chymotrypsinogen (Chym), 25 kDa;
ovoalbumin (Ovo), 43 kDa; BSA, 37 kDa; aldolase (Ald), 158 kDa;
catalase (Cat), 232 kDa; ferritin (Fer), 440 kDa; and thyroglobulin
(Thyr), 669 kDa.
Table 3. Purification of mPOP from a total membrane preparation. POP was assayed as described in Experimental procedures. Total activity
is specific activity multiplied by total protein in milligrams; after Triton X-100 extraction, there is a > 3-fold activation of activity, which is
reflected by an increase in total activity. Yield percentage refers to the activity in detergent extract, which is shown in parentheses.
Sample
Total
volume (mL)
Total
protein (mg)
Specific activity
(nmolÆmin
)1
Æmg
)1
)
Total activity
(nmolÆmin
)1
) Yield (%)
Fold
purification
Total membranes 150 855 0.5 453 100 1
Triton X-100 extraction 1000 533 3.3 1759 388 (100) 6.6

DEAE 680 232 7.4 1717 379 (98) 14.8
Phenyl–Sepharose 160 43 39 1679 371 (95) 78
HA 25 9.5 169 1595 352 (90) 338
Second DEAE 6.5 0.9 857 771 170 (43) 1714
Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.
4418 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS
exclusively the light band. This anomalous behavior
was reproducible, and was consistently eliminated by
membrane solubilization with Triton X-100 (Fig. 5C).
In these cases, the anti-POP reactive band always ran
at the same molecular mass as the band that corre-
sponded to cPOP, or to the pure POP control, regard-
less of the membrane fraction that the sample was
prepared from.
Kinetic properties of purified soluble
membrane PE
We next attempted to differentiate membrane PE from
POP on the basis of kinetic properties. This compara-
tive analysis included semipure pig brain POP, PPP,
and membrane PE purified by HA chromatography.
Accordingly, different inhibitors and substrates were
tested, and the kinetic behavior of membrane PE, in
comparison to that of POP, was evaluated.
It is known that POP is inhibited by some divalent
cations [12,13], including the heavy metals Mn
2+
,
Cu
2+
,Ni

2+
, and Zn
2+
[14]. Our results demonstrate
that there were no significant differences between
membrane PE and POP regarding their sensitivity to
these metals (Table 4). In addition, we compared the
effects of some general serine, cysteine and metallo-
protease inhibitors, specific POP inhibitors, such as
N-carbobenzoxy-prolyl-prolinal (ZPP) and JTP-4819,
the proteasome inhibitor N-carbobenzoxy-leucyl-leucyl-
leucyl-COH, the specific dipeptidyl peptidase IV
inhibitor HIV-1 tat (1–9) fragment with sequence
H-Met-Asp-Pro-Val-Asp-Pro-Asn-Ile-Glu-OH, and the
POP inhibitor a
2
-gliadin 33-mer peptide [15] (see
A
B
C
Fig. 5. Western blots of different membrane PE preparations
obtained using an antibody against POP. (A) Cross-reactivity against
membrane PE and recombinant POP of antibody against POP.
Membrane PE, 8 lg of protein; PPP, 120 ng of protein. (B, C) Blot
on total membranes (TM), light-density membranes (LM), medium-
density membranes (MM) and high-density membranes (HM) from
pig brain before (B) or after (C) Triton X-100 solubilization and com-
pared with PPP. Protein amounts were as follows: (B) PPP
0.15 lg, TM 30 lg, LM and HM 60 lg, and HM 120 lg. (C) PPP
0.15 lg, TM 12 lg, LM 23 lg, MM 23 lg, and HM 30 lg.

Fig. 4. POP amino acid sequence from Sus scrofa (accession num-
ber NP_001004050 Ver NP_001004050.1 GI:51592147). Peptides
from tryptic digestion of purified membrane PE are highlighted (see
Doc. S1 in Supporting information also).
Table 4. Effects of divalent cations, ionic strength and chelators on
POP activity of membrane PE (A) and on cPOP (B).
0 l
M 1 lM 10 lM 100 lM 1mM 10 mM
(A) Membrane PE
Mg
2+
100 100 101 104 101 98
Ca
2+
100 97 99 103 101 97
Mn
2+
100 98 96 86 80 28
Cu
2+
100 102 98 90 11 8
Ni
2+
100 96 94 78 8 8
Zn
2+
100 93 94 13 8 8
0m
M 100 mM 200 mM 400 mM 600 mM 800 mM
NaCl 100 124 127 153 138 133

0m
M 2.5 mM 5mM 10 mM 25 mM 125 mM
EDTA 100 89 78 62 42 26
(B) cPOP
Mg
2+
100 101 99 96 99 98
Ca
2+
100 96 98 104 93 95
Zn
2+
100 96 86 4 3 3
0m
M 2.5 mM 5mM 10 mM 25 mM 125 mM
EDTA100 86 85 64 46 24
J. Tenorio-Laranga et al. Membrane-bound prolyl oligopeptidase: mPOP
FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4419
Table S1). As expected, both membrane PE and POP
were very sensitive to the specific POP inhibitors. Fur-
thermore, the kinetic constants produced by careful
titration of ZPP, a very specific and potent POP inhib-
itor, were equivalent for both preparations (Fig. 6).
Both membrane PE and POP were partially resistant
to phenylmethanesulfonyl fluoride, a generic serine
protease inhibitor that has a low efficiency in inhibit-
ing POP [1]. We also found that membrane PE was
inhibited by SH-reactive compounds such as malei-
mides, similarly to POP, and that both were similarly
resistant to dipeptidyl peptidase IV and proteasome

inhibitors. In addition, membrane PE showed the same
sensitivity to the 33-mer peptide (see Table S1) as has
been reported for POP [15].
Previous reports suggest that membrane PE and
POP display similar specificity for several proline-
containing neuropeptides, including bradykinin, angio-
tensin II, neurotensin, substance P, and gonadoliberin
[10]. In addition to these substrates, we assayed several
other peptides in an effort to define functional differ-
ences between membrane PE and POP. However, as
shown in Table 5, no differences were observed for
any of the substrates tested.
Membrane PE probably associates with the
membrane by nonprotein hydrophobic anchoring
The nature of the association of membrane PE with
membranes is not known. We tried to address this
question in several ways. Analysis of the hydrophobic-
ity profile of the POP protein sequence revealed that
the presence of membrane-spanning segments is highly
improbable (see Doc. S2 in Supporting information).
Our experiments with membrane PE extracted with
Triton X-114 demonstrated that this enzyme activity is
quantitatively partitioned in the hydrophilic phase,
arguing against any important hydrophobic protein
domain that would link the protein to the intermem-
brane milieu.
To further confirm this, we ran a temperature
dependence assay with membrane PE-containing mem-
branes (Fig. 7). Intrinsic membrane proteins display a
break in the Arrhenius plot due to membrane phase

transition, and delipidation of these particulate
enzymes eliminates the discontinuity in the plots [16].
We did not observe any break in the membrane PE
Arrhenius plot; however, there was a significant change
in the slope of the temperature dependency curve when
compared with that obtained for POP (Fig. 7). Fur-
thermore, the membrane PE Arrhenius plot resembled
that of POP when the experiment was performed after
detergent solubilization of membrane PE.
POP has been implicated in axonal transport [17].
Soluble protein elements in these processes are recruited
to membranes, through interaction with other proteins
and SH-bonding and ⁄ or divalent cation (Ca
2+
)-depen-
dent mechanisms. To test whether a similar process
would mediate the membrane PE association with
membranes, we evaluated the expression levels of PE
in membranes after homogenization or washes with
N-ethylmaleimide or EDTA. The presence of N-ethyl-
Fig. 6. Inhibitory effect of ZPP (Z-Pro-Prolinal) on POP (s) or mem-
brane PE (
) activity. The assay was performed as described in
Experimental procedures in the presence of the corresponding
inhibitor concentrations during preincubation. The estimated IC50
values for membrane PE (continuous line) and POP (broken line)
were 0.48 n
M (± 0.005 nM) and 0.52 nM (± 0.005 nM) respectively.
Table 5. Substrate specificity studies on membrane PE as compared with POP. +, ZPP-sensitive cleavage of the peptide occurred;
), cleavage of the peptide did not occur.

Peptide Sequence Membrane PE POP
12-mer H
2
N-QLQPFPQPQLPY-OH ++
33-mer LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF ))
HIV H-MDPVDPNIE-OH ++
PEP-3 YGRKKRRQRRRG-NH
2
))
PEP-26 RGTICKKTMLDGLNNYCTGVGR-NH
2
))
PEP-48_2 Ac-LINEEEFFDAVEAALDRQ-NH
2
))
PEP-50 Ac-PYSRSSSMSSIDLVSASDDVHRFSSQ-NH
2
))
PEP-52 Ac-CDPGYIGSR-NH
2
++
Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.
4420 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS
maleimide or EDTA, or both, during homogenization
or membrane washes did not alter the amount of PE
detected in the membrane fraction (data not shown).
An alternative explanation is that membrane PE is
attached to membranes through a hydrophobic anchor
that has been added to the protein post-translationally.
A web-based (expasy) analysis of the POP sequence

for post-translational modifications related to glycosyl-
ation, myristoylation, prenylation and glycosylphos-
phatidyl inositol anchoring returned very low scores
(see Doc. S2 in Supporting information). Furthermore,
POP lacks a signal sequence required for some of these
modifications. The only possibility that was found is
palmitoylation of the Cys563 within the sequence
GGLLVATCANQRPDL(556–570), which, according
to the css-palm server (-ust-
c.org/css_palm/), has a relatively good score for modifi-
cation (see Doc. S2 in Supporting information and
Fig. 8).
Using [
3
H]palmitate, or [
3
H]palmitoyl-CoA, we have
tried to measure in vitro or metabolic palmitoylation,
but our attempts have been unsuccessful, in part
because of the relatively low expression levels of
endogenous membrane PE. These results, however, do
not rule out this possibility.
Discussion
This article reports the identification of the protein
responsible for PE activity in membranes isolated
from pig brain, which we now call mPOP. For more
than 20 years, it has been known that some PE
activity it associated with membranes from almost
all tissues and especially from brain [18–20]. Further-
more, mPOP activity has been found to change with

age [6,21]. Purification of mPOP from bovine brain
has been attempted previously [9,10], and on the
basis of those studies, it was concluded that this
peptidase is expressed mainly in the synaptosomal
fraction and has a heavier mass (87 kDa) than POP
(65 kDa). Furthermore, on the basis of sensitivity to
thiol-reactive inhibitors, mPOP was thought to be a
thiol-dependent metallopeptidase [10], but the basic
problem was that this enzyme has never been readily
identified before.
In an attempt to clarify the identity of mPOP, we
undertook the task of purifying and characterizing
particulate POP from pig brain. We have confirmed
that a significant amount of PE activity can be mea-
sured in the particulate fraction from crude pig brain
homogenates. In our preparations, this activity
accounted for around 40% of the total homogenate
activity, similar to the 50% reported previously for
the corresponding fraction from bovine brain [9].
However, after osmotic shock and high-salt treat-
ment, our total membrane preparation contained less
than 5% of total activity, as compared with 20%
recovery reported earlier for washed synaptosomal
membranes from bovine brain [9,10]. This discrep-
ancy may be species-related, as the preparation con-
ditions were essentially the same. The PE activity
present in our washed membrane preparation was
very tightly bound, and was solubilized only after
detergent treatment. Upon sucrose gradient fraction-
ation, some PE activity was present in the heavier

membrane fraction containing ER, but the majority
was detected within the synaptosomal membrane
fraction, consistent with a role for mPOP in synapse
function. This is consistent with the finding of bind-
ing of antibody against POP to internal neuronal
membranes in rat brain slices [11].
Kinetic experiments demonstrated that the substrate
preference and the inhibitor sensitivity of purified
mPOP are identical to those of POP (see Table 4 and
Table S1). Furthermore, POP-specific antibodies
cross-reacted with mPOP on western blots. Analysis
of peptide fragments generated by trypsin digestion
identified mPOP protein as POP. Thus, we found
only two features that distinguish the two forms, and
those are attributed to the membrane milieu where
mPOP resides. One was the tight membrane associa-
tion, which could only be disrupted by detergent solu-
bilization. The second feature was the different
Fig. 7. Arrhenius plots of membrane PE ( ), POP ( ) and solubi-
lized membrane PE (d). Activity assays were performed in dupli-
cate as described in Experimental procedures, but reaction tubes
were preincubated at every temperature (every 2 °C between 10
and 40 °C) and the reaction was incubated at the corresponding
temperature for 40 min. Solid lines represent the linear regres-
sions.
J. Tenorio-Laranga et al. Membrane-bound prolyl oligopeptidase: mPOP
FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4421
temperature dependence between mPOP activity,
when is bound to membranes, and POP activity. For
transmembrane enzymes, Arrhenius plots generally

show a break that corresponds to the membrane
melting point. At temperatures above this point, the
more fluid environment produces a decrease of activa-
tion energy, producing a convex Arrhenius plot [22].
However, we found that the Arrhenius plot of mPOP
did not show any break but did reflect an increase of
activation energy in relation to POP or Triton-solubi-
lized mPOP. Interestingly, these two latter forms were
very similar to each other (Fig. 6). We interpret this
as an inhibitory effect of the membranous milieu, or
of the putative anchor, or both, on mPOP activity, as
the Arrhenius profile switched to a POP profile of
lower activation energy upon detergent solubilization.
It is remarkable that Triton X-100 activated mPOP
activity at least by a factor of 3 (Table 3). It was also
observed that the SDS ⁄ PAGE migration of mPOP,
when still attached to the native membranes, was
atypical. Consistently, different membrane fractions
showed a reactive band for antibody against POP
that migrated at a slightly, but perceptibly, lower
molecular mass, present at different proportions for
the different fractions (Fig. 5B). In total membranes,
this lighter band was a major component, becoming
almost exclusive in the heavy membrane fraction.
Medium-density membranes presented two bands: the
light band and the one that matched the soluble POP
control. On the other hand, in the light membrane
fraction, the latter band was the only one appearing.
It was remarkable that this lower molecular mass
band completely disappeared from all membrane frac-

tions when the very same samples were solubilized
with Triton X-100. After this treatment, only one
band around 80 kDa, as with the cPOP control, was
detected in all cases. It can be argued that the mem-
brane-associated state induces a more compact and
tighter conformation in which the anchor site is less
accessible to reduction, thereby resulting in faster
migration. After solubilization and membrane disrup-
tion, the protein anchor might be more accessible to
reducing agents, and thus could be readily dissociated
to yield the soluble form with normal migration in
SDS ⁄ PAGE (Fig. 5B,C). The fact that both normally
migrating and abnormally migrating bands, from the
different membrane fractions, appear in different
proportions could be explained by bringing into play
Fig. 8. Localization of Cys563 on the 3D
model of pig POP. Peptidase_S9 domain
residues are shown in magenta; propeller
domain chains are represented in navy,
brown, green, gray and orange; Cys563 is
shown in yellow. Modeled from file mmd-
bId:21074, for POP from porcine brain, from
the NCBI’s Entrez Structure database, and
handled by
CN3D v. 4.1 (NCBI) software.
Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.
4422 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS
the different physical and chemical properties of the
corresponding membranes, which would obviously
affect the interactions with mPOP.

Several studies indicate that POP interacts with cyto-
skeleton proteins and that it is probably involved
in axonal transport [17]. Components of neuronal
membrane trafficking are tightly associated with
membrane-bound organelles in a chaperone-mediated
or chaperone-sensitive way, and are therefore resistant
to in vitro treatments, including high salt, which release
most peripheral membrane proteins [23]. Homogeni-
zation with buffers containing millimolar levels of
SH-modifying agents, such as N-ethylmaleimide, or
chelators of divalent cations, such as EDTA, is known
to significantly release these proteins from membranes.
Proteins such as annexins associate with membranes in
aCa
2+
-dependent way, and EDTA disrupts this asso-
ciation and solubilizes the proteins [24]. We did not
note any effects of N-ethylmaleimide or EDTA during
homogenization or membrane washing on the mem-
brane PE levels (not shown). These data indicate that
the mechanism of mPOP membrane association is
most probably not mediated by SH interaction or by
divalent cations.
On the whole, our results may suggest that POP
undergoes a post-translational modification in which a
membrane anchor is added to the protein, attaching it
to membranes. The nature of this putative anchor
remains to be determined, but it is most likely a hydro-
phobic chain, as POP does not contain any substantial
hydrophobic domain that could be used to embed it in

cellular membranes (see Doc. S2 in Supporting infor-
mation). Our analysis of the POP amino acid sequence
suggests palmitoylation as a possible post-translational
modification; the well-conserved sequence of pig POP,
GGLLVATCANQRPDL(556–570), yields a very good
score for this kind of modification according to css-
palm software [25]. Examination of the 3D structure
of pig POP revealed that a critical cysteine is situated
very near the surface of the enzyme, on the bottom
side of the catalytic domain, making it a good candi-
date for the putative palmitoylation (Fig. 8). In fact,
this cysteine residue is totally conserved among all
eukaryotic POP genes sequenced [26]. Although
S-palmitoylation is theoretically sensitive to SH group-
reducing agents, this site could be buried when mPOP
is anchored to the membrane. Although our attempts
to measure in vitro or metabolic palmitoylation have
not succeeded, the possibility of this post-translational
modification is still open.
It is also important to note that we did not use any
inhibitors for lytic enzymes during mPOP preparation,
and it is possible that the putative membrane anchor
was removed during the process of isolation and purifi-
cation, or during trypsinization and peptide extraction,
preventing its identification by MS. Additionally,
membrane-bound proteins, which interact directly with
the hydrophobic inner membrane phase, require the
presence of an amphiphilic compound, such as a deter-
gent or a phospholipid, for stability and activity. We
found that once mPOP is solubilized from membranes,

the concentration of Triton X-100 can be considerably
decreased without any protein precipitation or loss of
activity. This strongly points to the intrinsically soluble
nature of mPOP, as strongly hydrophobic proteins
tend to aggregate and lose activity during lipid or
detergent removal.
The cytoplasmic location of POP is paradoxical
when considering its major role in the metabolism of
extracellular neuropeptides [3]. Recently, other roles
have been suggested for POP, such as axonal transport
and ⁄ or modulation of intracrine peptide regulation [1].
The existence of an alternative particulate form with
PE activity, a different enzyme but with the same func-
tional properties as POP, that is responsible for the
degradation of neuropeptides in the synaptosomal
cleft, would solve the localization problem. However,
attempts to identify this enzyme have been unsuccess-
ful [9,10]. Contrary to expectations, we found several
pieces of evidence suggesting that the particulate form
is, in fact, a variant of POP (EC 3.4.21.26); mPOP has
the same gel filtration, immunological, activity and
inhibitory properties as soluble POP, and even MS
data provided high confidence in the identification.
Our results additionally suggest that a post-transla-
tional modification is necessary for POP to be associ-
ated with membranes. Furthermore, the data reported
here also suggest that this modification is sensitive to
the reducing state of the environment, and this may
indicate the existence of specific cell machinery that
controls the association–dissociation event. One funda-

mental question is the orientation of mPOP in the
synaptosomes. Fast and effective neuropeptide degra-
dation in the synaptosomal cleft would only be possi-
ble if mPOP was facing that side. Another interesting
aspect invoked is the possibility that the membrane
association of POP is connected to its transport out of
the cell, the membrane-bound form being only a tran-
sitory stage.
Experimental procedures
Total membrane preparation
Eighty grams of freshly isolated pig brain were homoge-
nized in 320 mL of ice-cold 0.32 m sucrose in 100 mm
J. Tenorio-Laranga et al. Membrane-bound prolyl oligopeptidase: mPOP
FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4423
potassium phosphate buffer (pH 7.4), sonicated, and centri-
fuged at 1000 g for 10 min. The resulting supernatant was
sonicated again, and centrifuged at 155 100 g for 30 min.
The pellet was washed first with 0.5 m NaCl, once with dis-
tilled water, twice with 4 m NaCl, and finally with distilled
water. The resulting membranes were resuspended in
100 mm potassium phosphate buffer (pH 7.4).
Membrane fractionation
An aliquot of the total membrane preparation was brought
to 0.32 m sucrose and 100 mm potassium phosphate buffer
(pH 7.4), and layered on a discontinuous sucrose gradient
(1.2, 1 and 0.8 m sucrose). The tubes were centrifuged at
80 000 g for 90 min, in a swing rotor. After centrifugation,
the membrane layers were carefully collected by pipetting.
Each fraction was diluted to 0.32 m sucrose, centrifuged
at 155 100 g, and resuspended in 100 mm potassium

phosphate buffer (pH 7.4).
Enzymatic assays
PE activity was assayed by measuring the fluorescence
released from the substrate N-carbobenzoxy-glycyl-prolyl-7-
amido-4-methyl-coumarin (Z-Gly-Pro-AMC) (200 lm), as
previously reported [27], by incubating protein samples in
100 mm sodium phosphate buffer (pH 7.0). The assay was
stopped by the addition of 1 m sodium acetate buffer
(pH 4.2). A succinate dehydrogenase assay was performed in
0.3 mL containing 0.01 m sodium succinate, 0.05 m phos-
phate (pH 7.5), and 0.4 mg (in 50 lL) of each membrane
fraction. The mixtures were incubated at 37 °C for 15 min,
0.1 mL of 2.5 mgÆmL
)1
p-iodotetrazolium violet was added,
and incubation was continued for a further 10 min. The
reaction was stopped with 1 mL of ethyl acetate ⁄ ethanol ⁄
trichloroacetic acid (5 : 5: 1), and centrifuged at 16 000 g for
1 min; absorbance was measured at 490 nm.
Protein determination
Protein was determined by the Bradford method (Bio-Rad,
Hercules, CA, USA) using BSA (Sigma-Aldrich, St Louis,
MO, USA) as standard, and in the presence of 0.1%
Triton X-100 when required.
SDS ⁄ PAGE and western blotting
Samples were diluted 1 : 1 with loading buffer (100 mm
Tris ⁄ HCl, pH 6.8, 70% glycerol, 2% SDS, 0.005% bromo-
phenol blue, 10 mm b-mercaptoethanol) and separated on
8% or 10% polyacrylamide ⁄ bis-acrylamide Tris ⁄ HCl dis-
continuous gels. Gels were stained for protein or trans-

ferred to nitrocellulose for blotting. For protein staining,
gels were fixed with methanol ⁄ acetic acid ⁄ water (4 : 5 : 4)
for 30 min, washed with water for 30 min (two changes),
sensitized with 0.02% sodium thiosulfate for 1–2 min, and
incubated with 0.1% silver nitrate for 20 min at room tem-
perature. Gels were then rinsed twice with distilled water,
and bands were visualized by incubating for 1–2 min with
2% sodium carbonate and 0.04% formaldehyde. Western
blotting was performed under standard conditions using
primary antibody [28] diluted 1 : 5000 (with 0.5 m NaCl,
20 mm Tris-HCL pH 7.5 and 0.05% Tween 20), and the
anti-(chicken horseradish peroxidase) complex diluted
1 : 50 000 (Pierce, Rockford, IL, USA). Protein visualiza-
tion was performed using an ECL kit (Amersham-Biosci-
ence, Little Chalfont, UK), following the manufacturer’s
instructions.
Purification of mPOP
The total membrane preparation (see above) was
extracted with 0.4% Triton X-100 (at 1 mg of Triton
X-100 per mg of protein) in 20 mm buffer and 100 mm
NaCl on ice for 1 h. After the extraction, the samples
were centrifuged at 155 100 g for 30 min and the pellet
was discarded. Buffer was exchanged by dilution and
ultrafiltration (CentriPrep 50; Amicon, Millipore Corp.
Billerica, MA, USA), with DEAE equilibration buffer
[50 mm Tris, pH 7.4, 1 mm EDTA, 5 mm dithiothreitol,
0.05% Triton X-100], bound to an equilibrated DEAE–
Sepharose Fast Flow column (1.6 · 10 cm; Amersham,
Uppsala, Sweden), washed with DEAE equilibration buf-
fer, and step-eluted with 500 mm NaCl in the same buf-

fer. The eluted pool was concentrated and the buffer was
exchanged (as stated above) for phenyl–Sepharose equili-
bration buffer [900 mm (NH
4
)
2
SO
4
,50mm Tris ⁄ HCl,
pH 7.4, 5 mm dithiothreitol, 1 mm EDTA], and loaded
onto a phenyl–Sepharose High Performance column
(0.7 · 2.5 cm; Amersham). Activity eluted within the
flow-through. Most of the contaminating protein was
retained in the column and eluted by a wash without
(NH
4
)
2
SO
4
. Peak fractions were pooled, the buffer was
exchanged for HA equilibration buffer (10 mm potassium
phosphate, pH 7.4, 5 mm dithiothreitol, 1 mm EDTA,
0.05% Triton X-100), and the sample was loaded onto a
0.59 · 3.6 cm HA column (Bio-Rad). Activity was eluted
at around 250 mm potassium phosphate over a 10–
500 mm linear gradient (see Fig. S1). The buffer of the
HA pool was exchanged with DEAE buffer 2 (EDTA
1mm,5mm dithiothreitol, 50 mm Tris ⁄ HCl, pH 6.6),
loaded onto an equilibrated DEAE HiTrap Fast Flow

column (0.7 · 2.5 cm; Amersham), and eluted with a
0–500 mm NaCl gradient. Activity eluted at around
200 mm salt. All chromatographic steps were accom-
plished using an A
¨
KTA prime system and monitored
with primeview plus software (both from Amersham).
All chromatography profiles are shown in Fig. S1.
Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.
4424 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS
Partial purification of cPOP
The soluble protein obtained after the first centrifugation
from whole brain homogenate (see Total membrane pre-
paration) was DEAE buffer exchanged and loaded onto
an equilibrated DEAE–Sepharose Fast Flow column
(1.6 · 10 cm; Amersham). The elution was performed in a
gradient from 0 to 500 mm NaCl. The active fractions
(around 200 mm NaCl) were pooled and concentrated. This
fraction was used for further kinetic experiments and is
referred to as cPOP.
Purification of PPP
PPP was expressed in Escherichia coli and purified as
described previously [29].
Peptide digestion assay
The assay mixture (140 lL) was composed of 50 mm
Tris ⁄ HCl (pH 7.0) and cPOP, or mPOP, to 4 nmolÆmin
)1
of activity. Each peptide was added (prewarmed) at a final
concentration of 140 lm. The reaction was carried out at
30 °C for 60 min and stopped by the addition of trifluoro-

acetic acid to a final concentration of 0.1%. The resultant
mixture was microfuged for 30 min at maximum speed, and
the supernatant was filtered and analyzed by RP-HPLC
(C-18 5 lm Licrospher; Merck, Darmstadt, Germany) in a
25-min linear gradient of 10–80% acetonitrile in 0.1%
trifluoroacetic acid.
Gel filtration chromatography
HA activity fractions were pooled and buffer was
exchanged for 20 mm potassium phosphate buffer (pH 7.0),
1mm EDTA and 1 mm dithiothreitol, with or without
0.05% Triton X-100, or for 100 mm potassium phosphate
buffer (pH 7.0), 1 mm EDTA and 1 mm dithiothreitol, with
or without 0.05% Triton X-100. The samples were applied
to a calibrated Superdex-200 column (1 · 30 cm; Amer-
sham) equilibrated with the appropriate buffer. Apparent
molecular mass was calculated by regression of the calibra-
tion curve (ribonuclease, 13.7 kDa; chymotrypsinogen,
25 kDa; ovoalbumin, 43 kDa; BSA, 37 kDa; aldolase,
158 kDa; catalase, 232 kDa; ferritin, 440 kDa; thyroglobu-
lin, 669 kDa).
Triton X-114 partition experiments
Membranes were extracted under the same conditions
stated for Triton X-100, but using Triton X-114 instead,
according to Bordier [30]. The resulting supernatant was
layered on 6% (w ⁄ v) sucrose buffer, incubated for 15 min
at 30 °C, and centrifuged at 200 g at room temperature for
10 min. Both layers, hydrophobic and hydrophilic, were
assayed for POP activity.
Native PAGE
Native PAGE was performed basically as described by Lae-

mmli [31], but introducing modifications according to Orn-
stein [32], in which Triton X-100 substituted for SDS. Gels
were prerun for 30 min at 100 V, and resolved at 200 V
over 1 h. All of this process was carried out at 4 ° C. The
zymogram was performed by cutting the gel lanes with a
tandem razor blade set-up that gave pieces 1 mm long.
Every slice was then transferred to a different well in a
48-well titer plate, and minced with a blade in 500 lLof
prewarmed incubation buffer (see Enzymatic assays);
Z-Gly-Pro-AMC was then added to 100 lm. After 60 min
of incubation at 30 °C, generated amido-4-methylcoumarin
(AMC) fluorescence was measured.
Trypsin peptide identification
Samples (silver-stained bands from gel or activity pool from
chromatography fractions) were reduced, alkylated, trypsi-
nized and analyzed by liquid chromatography–MS ⁄ MS in
the Proteomics facility of the Centro de Investigacio
´
n Prı
´
n-
cipe Felipe, Valencia, Spain (a member of PROTEORED
SPAIN). The peptides resulting from digestion were applied
to an RP-HPLC PepMap C-18 75 lm · 150 mm (LC-Pack-
ing, Sunnyvale, CA, USA) at a flow rate of 200 nLÆmin
)1
,
and separated by a 14–47% acetonitrile gradient in 0.1%
formic acid. For the Qstar XL instrument (Applied Biosys-
tems, Framingham, MA, USA), the eluate was directly

applied to a nanospray source of the mass spectrometer,
and information-dependent acquisition analysis was carried
out with acquisition cycles in MS and MS ⁄ MS mode along
all the chromatogram. For the MALDI-TOF ⁄ TOF MS
instrument, the eluate was collected in a MALDI plate
(24 · 12 fractions) at 20 s per fraction with 2.5 mgÆmL
)1
a-cyano-4-hydroxycinnamic acid in 50% acetonitrile and
0.1% trifluoroacetate. Peptide mass spectra were analyzed
on a MALDI TOFTOF 4700 Proteomics analyzer (Applied
Biosystems), by 3000 shots in MS ⁄ MS mode, selecting the
six most intense precursors in each fraction (not analyzed
previously) for MS ⁄ MS analysis (3500 shots per precursor).
All resulting MS ⁄ MS spectra were sent to the mascot server
() using the mascot daemon
program (Matrix Science, London, UK).
Acknowledgements
We thank Ms Pirjo Ha
¨
nninen for excellent technical
assistance and Drs Sanchez del Pino, Luz Valero
Rustarazo, and Carmen Aguado Velasco, from the
CIPF Proteomics facility, for the critical discussion on
J. Tenorio-Laranga et al. Membrane-bound prolyl oligopeptidase: mPOP
FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4425
MS peptide analysis. We thank Dr Enrique Pe
´
rez-Paya
´
for the peptides used in this work. We would also like

to acknowledge Drs Zoltan Szeltner and Deborah Bur-
ks for critically reviewing the manuscript. This work
was supported by Fundacio
´
n Valenciana Centro de
Investigacio
´
n Prı
´
ncipe Felipe grant I-19 to J. Arturo
Garcı
´
a-Horsman, by Finnish Academy grants
No. 210758 ⁄ 2004 and No. 117881 ⁄ 2006, by Juselius
Foundation and Helsinki University Research Funds
grants to P. T. Ma
¨
nnisto
¨
, and by European Commis-
sion FP7-HEALTH-2007 grant 223077. J. Arturo
Garcı
´
a-Horsman was supported by the Ministerio de
Educacio
´
n y Ciencia (Ramo
´
n y Cajal Program).
References

1 Garcı
´
a-Horsman JA, Ma
¨
nnisto
¨
PT & Vena
¨
la
¨
inen JI
(2007) On the role of prolyl oligopeptidase in health
and disease. Neuropeptides 41, 1–24.
2Ma
¨
nnisto
¨
PT, Vena
¨
la
¨
inen J, Jalkanen A & Garcı
´
a-
Horsman JA (2007) Prolyl oligopeptidase: a potential
target for the treatment of cognitive disorders. Drug
News Perspect 20, 293–305.
3 Brandt I, Scharpe S & Lambeir AM (2007) Suggested
functions for prolyl oligopeptidase: a puzzling paradox.
Clin Chim Acta 377, 50–61.

4 McMahon G, Collins P & O’Connor B (2003) Charac-
terisation of the active site of a newly-discovered and
potentially significant post-proline cleaving endopepti-
dase called ZIP using LC-UV-MS. Analyst 128, 670–
675.
5 Cunningham DF & O’Connor B (1997) Identification
and initial characterisation of a N-benzyloxycarbonyl-
prolyl-prolinal (Z-Pro-prolinal)-insensitive 7-(N-benzyl-
oxycarbonyl-glycyl-prolyl-amido)-4-methylcoumarin
(Z-Gly-Pro-NH-Mec)-hydrolysing peptidase in bovine
serum. Eur J Biochem 244, 900–903.
6 Agirregoitia N, Irazusta A, Ruiz F, Irazusta J & Gil J
(2003) Ontogeny of soluble and particulate prolyl endo-
peptidase activity in several areas of the rat brain and
in the pituitary gland. Dev Neurosci 25 , 316–323.
7 Irazusta J, Larrinaga G, Gonzalez-Maeso J, Gil J,
Meana JJ & Casis L (2002) Distribution of prolyl
endopeptidase activities in rat and human brain.
Neurochem Int 40, 337–345.
8 Agirregoitia N, Casis L, Gil J, Ruiz F & Irazusta J
(2007) Ontogeny of prolyl endopeptidase and pyroglut-
amyl peptidase I in rat tissues. Regul Pept 139, 52–58.
9 O’Leary RM & O’Connor B (1995) Identification and
localisation of a synaptosomal membrane prolyl endo-
peptidase from bovine brain. Eur J Biochem 227, 277–
283.
10 O’Leary RM, Gallagher SP & O’Connor B (1996) Puri-
fication and characterization of a novel membrane-
bound form of prolyl endopeptidase from bovine brain.
Int J Biochem Cell Biol 28, 441–449.

11 Myo
¨
ha
¨
nen TT, Vena
¨
la
¨
inen JI, Garcı
´
a-Horsman JA,
Piltonen M & Ma
¨
nnisto
¨
PT (2008) Cellular and sub-
cellular distribution of rat brain prolyl oligopeptidase
and its association with specific neuronal neurotransmit-
ters. J Comp Neurol 507, 1694–1708.
12 Bausback HH & Ward PE (1986) Vascular, post proline
cleaving enzyme: metabolism of vasoactive peptides.
Adv Exp Med Biol A 198, 397–404.
13 Kato T, Nakano T, Kojima K, Nagatsu T & Sakaki-
bara S (1980) Changes in prolyl endopeptidase during
maturation of rat brain and hydrolysis of substance P
by the purified enzyme. J Neurochem 35, 527–535.
14 Kato T, Okada M & Nagatsu T (1980) Distribution of
post-proline cleaving enzyme in human brain and the
peripheral tissues. Mol Cell Biochem 32, 117–121.
15 Garcı

´
a-Horsman JA, Vena
¨
la
¨
inen JI, Lohi O, Auriola
IS, Korponay-Szabo IR, Kaukinen K, Ma
¨
ki M &
Ma
¨
nnisto
¨
PT (2007) Deficient activity of mammalian
prolyl oligopeptidase on the immunoactive peptide
digestion in coeliac disease. Scand J Gastroenterol 42,
562–571.
16 Brasitus TA, Schachter D & Mamouneas TG (1979)
Functional interactions of lipids and proteins in rat
intestinal microvillus membranes. Biochemistry 18,
4136–4144.
17 Schulz I, Zeitschel U, Rudolph T, Ruiz-Carrillo D,
Rahfeld JU, Gerhartz B, Bigl V, Demuth HU & Ross-
ner S (2005) Subcellular localization suggests novel
functions for prolyl endopeptidase in protein secretion.
J Neurochem 94, 970–979.
18 Checler F, Emson PC, Vincent JP & Kitabgi P (1984)
Inactivation of neurotensin by rat brain synaptic
membranes. Cleavage at the Pro10-Tyr11 bond by
endopeptidase 24.11 (enkephalinase) and a peptidase

different from proline-endopeptidase. J Neurochem 43,
1295–1301.
19 Dalmaz C, Netto CA, Volkmer N, Dias RD &
Izquierdo I (1986) Distribution of proline endopeptidase
activity in sub-synaptosomal fractions of rat hypothala-
mus. Braz J Med Biol Res 19, 685–690.
20 Del Bel EA, Gambarini AG & Martins AR (1986)
Neuropeptide-metabolizing peptidases in neuro-2a
neuroblastoma and C6 glioma cells. J Neurochem 47,
938–944.
21 Agirregoitia N, Gil J, Ruiz F, Irazusta J & Casis L
(2003) Effect of aging on rat tissue peptidase activities.
J Gerontol A Biol Sci Med Sci 58, B792–B797.
22 Truhlar D & Kohen A (2001) Convex Arrhenius plots
and their interpretation. Proc Natl Acad Sci USA 98,
848–851.
23 Tsai MY, Morfini G, Szebenyi G & Brady ST (2000)
Release of kinesin from vesicles by hsc70 and regula-
Membrane-bound prolyl oligopeptidase: mPOP J. Tenorio-Laranga et al.
4426 FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS
tion of fast axonal transport. Mol Biol Cell 11, 2161–
2173.
24 Willems GM, Janssen MP, Comfurius P, Galli M,
Zwaal RF & Bevers EM (2000) Competition of
annexin V and anticardiolipin antibodies for binding to
phosphatidylserine containing membranes. Biochemistry
39, 1982–1989.
25 Zhou F, Xue Y, Yao X & Xu Y (2006) CSS-Palm:
palmitoylation site prediction with a clustering and
scoring strategy (CSS). Bioinformatics 22, 894–896.

26 Vena
¨
la
¨
inen JI, Juvonen RO & Ma
¨
nnisto
¨
PT (2004)
Evolutionary relationships of the prolyl oligopeptidase
family enzymes. Eur J Biochem 271, 2705–2715.
27 Vena
¨
la
¨
inen JI, Garcı
´
a-Horsman JA, Forsberg MM,
Jalkanen A, Wallen EA, Jarho EM, Christiaans JA,
Gynther J & Ma
¨
nnisto
¨
PT (2006) Binding kinetics and
duration of in vivo action of novel prolyl oligopeptidase
inhibitors. Biochem Pharmacol 71, 683–692.
28 Myo
¨
ha
¨

nen TT, Vena
¨
la
¨
inen JI, Tupala E, Garcı
´
a-
Horsman JA, Miettinen R & Ma
¨
nnisto
¨
PT (2007)
Distribution of immunoreactive prolyl oligopeptidase
in human and rat brain. Neurochem Res 32, 1365–
1374.
29 Vena
¨
la
¨
inen JI, Juvonen RO, Forsberg MM, Garcı
´
a-
Horsman A, Poso A, Wallen EA, Gynther J & Ma
¨
n-
nisto
¨
PT (2002) Substrate-dependent, non-hyperbolic
kinetics of pig brain prolyl oligopeptidase and its tight
binding inhibition by JTP-4819. Biochem Pharmacol 64,

463–471.
30 Bordier C (1981) Phase separation of integral mem-
brane proteins in Triton X-114 solution. J Biol Chem
256, 1604–1607.
31 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
32 Ornstein L (1964) Disc electrophoresis. I. Background
and theory. Ann NY Acad Sci 121, 321–349.
Supporting information
The following supporting information is available:
Fig. S1. Chromatography profiles of membrane PE
purification.
Table S1. Effect of inhibitors on membrane PE.
Doc. S1. MS data.
Doc. S2. POP sequence in silico analysis.
This supporting information can be found in the
online version of this article.
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supporting
information supplied by the authors. Any queries
(other than missing material) should be directed to the
corresponding author for the article.
J. Tenorio-Laranga et al. Membrane-bound prolyl oligopeptidase: mPOP
FEBS Journal 275 (2008) 4415–4427 ª 2008 The Authors Journal compilation ª 2008 FEBS 4427