Báo cáo Y học: Purification and characterization of the thyrotropin-releasing hormone (TRH)-degrading serum enzyme and its identification as a product of liver origin doc

Purification and characterization of the thyrotropin-releasing
hormone (TRH)-degrading serum enzyme and its identification
as a product of liver origin
Stephanie Schmitmeier
1,
*, Hubert Thole
1,
†, Augustinus Bader
2
and Karl Bauer
1
1
Max-Planck-Institut fu
¨
r experimentelle Endokrinologie, Hannover, Germany;
2
Gesellschaft fu
¨
r Biotechnologische Forschung,
Abt. Organ und Gewebekulturen, Braunschweig, Germany
Previous biochemical studies have indicated that t he mem-
brane-bound thyrotropin-releasing hormo ne (TRH)-degra-
ding enzyme (TRH-DE) from brain and liver and t he serum
TRH-DE are derived from the s ame gene. These studies also
suggested that the serum enzyme is of liver origin. The
present study was undertaken to verify these hypotheses. In
different species, a close relationship between the activities of
the serum enzyme and the particulate liver enzyme was
noticed. The activity of the serum enzyme decreased when
rats were treated with thioacetamide, a known hepatotoxin.
With hepatocytes cultured i n a sandwich configuration,

release of the TRH-DE into t he culture medium cou ld also
be demonstrate d. The trypsin-solubilized p articulate liver
TRH-DE and the serum TRH-DE were purified to elec-
trophoretic homogeneity. Both enzymes and the brain
TRH-DE were recognized by a monoclonal antibody gen -
erated with the purified brain enzyme as antigen. Lectin blot
analysis indicated that the serum enzyme a nd the liver
enzyme are glycopro teins containing a sugar structure of the
complex type, whereas the brain enzyme exhibits an oligo-
mannose/hybrid glycostructure. A molecular mass of
97 000 Da could be estimated for all three enzymes after
deglycosylation and SDS/PAGE followed by Western
blotting. Fragment analysis of the serum T RH-DE revealed
that the peptide sequences correspond to the cDNA deduced
amino-acid sequences of the membrane-bound brain
TRH-DE, whereby two peptides were identified that are
encoded by exon 1. These data strongly support the hypo-
thesis that the TRH-DEs are all derived f rom t he same gene,
whereby the serum enzyme is generated by proteolytic
cleavage of the particulate liver enzyme.
Keywords: TRH-degrading enzyme (TRH-DE); serum;
liver; b rain; c haracterization.
The signal substance thyrotropin-releasing hormone
(TRH), a h ypothalamic hypophysiotropic neuropeptide
hormone (reviewed in [1,2]) and a peptidergic neurotrans-
mitter/neuromodulator (reviewed in [3,4]), is known to be
rapidly inactivated by the brain TRH-degrading enzyme
(TRH-DE), a n e ctoenzyme located on the surface of
neuronal cells, as well as by the soluble serum TRH-DE.
The highest activity of the membrane-bound TRH-DE is

found in brain and significant activities are also detected in
retina, lung and liver but not in other tissues such as heart,
kidney and muscle [5–7]. Because the membrane-bound
brain T RH-DE a nd the serum TRH-DE exhibit the same
extraordinary high degree of substrate specificity and
identical enzyme-chemical characteristics [8–14] it has b een
suggested t hat both enzymes are derived from the same
gene.
Based o n the observation that the developmental pattern
of the particulate liver TRH-DE and the serum TRH-DE
are almost identical it has been proposed that the serum
TRH-DE, like most serum enzymes and p roteins, might be
of liver origin [9,15]. This i nterpretation w as supported by
the findings t hat the activities of the particulate liver
enzyme, like the serum enzyme [16–18], is a lso regulated
by thyro id h ormones [19]. Moreover, similar enzyme-
chemical properties between the particulate liver enzyme
and the serum enzyme were noticed [9,15].
To verify the hypothesis that the serum TRH-DE is of
liver origin we analyzed the TRH-DE i n serum and
tissue ho mogenates of different species and studied the
effect of thioacetamide, a hepatotoxin, on the expression
of the serum enzyme and t he particulate liver enzyme.
Furthermore, with hepatocytes in culture we analyzed the
release of the TRH-DE into the medium. Finally, we
purified the TRH-DE from pig serum and liver to
electrophoretic homogeneity and studied th e r elationship
between these e nzymes. By s equence analysis we also
verified the hypothesis that the membrane-bound brain
TRH-DE and the serum TRH-DE a re derived from the

same gene.
Correspondence to K. Bauer, Max-Planck-Institut fu
¨
r experimentelle
Endokrinologie, PO Box 610309, D-30603 Hannover, Germany.
Fax: + 49 5115359 203, Tel.: + 49 5115359 200,
E-mail:
Abbreviations: TRH, thyrotropin-releasing hormone; TRH-DE,
TRH-degrading enzyme; DFP, diisopropyl fluorophosphate; E C L,
enhanced chemiluminescence; SNA, S a mbucus nigra agglutinin;
GNA, Galanthus nivalis agglutinin; MPSP, membrane protein-
solubilizing protease; TACE, TNFa protease.
*Present address: Department of Biochemistry and Molecular B iology,
University of So uthern California, Keck S chool of Medicine and
Norris Comprehensive Cancer Center, Cancer Research Laboratory
#106, 1303 N. Mission Road, Los Angeles, CA 90033, USA.
Present address: Solvay Pharmaceuticals GmbH, PO Box 220,
D-30002 Hannover.
(Received 18 July 2001, revised 12 December 2001, a ccepted 8 January
2002)
Eur. J. Biochem. 269, 1278–1286 (2002) Ó FEBS 2002
MATERIALS AND METHODS
Chemicals
Diisopropyl fluorophosphate (DFP), thioacetamide,
2-iodoacetamide, dithioerythritol and calf thymus DNA
were ob tained from Sigma Aldrich Chemie GmbH (Tauf-
kirchen, Germany). Hoechst dye 33258 (bisbenzimidazol)
was from Calbiochem-Novabiochem GmbH (Bad Soden,
Germany). G lutamine and penicillin/streptomycin were
purchased from Life Technologies Gm bH (Karlsruhe,

Germany). Insulin was from Hoechst AG (Frankfurt,
Germany), prednisone from MSD Sharp & Dohme GmbH
(Haar, Germany), and glucagon from Novo Nordisk
Pharma GmbH (Mainz, Germany). Poly(ethylene glycol)
6000 was obtained from Serva (Heidelberg, Germany).
Digoxigenin-labeled lectins, antidigoxigenin antibodies con-
jugated either to alkaline phosphatase or to horseradish
peroxidase as well as endoglycosidase F/N-glycosidase
F enzyme preparation and endoproteinase Lys-C were
purchased from Roche Diagnostics GmbH (Mannheim,
Germany). Goat anti-(mouse IgG) Ig con jugated to alkaline
phosphatase was obtained from Bio-Rad Laboratories
GmbH (Munich, Germany). The enhanced chemilumines-
cence (ECL)-Western blotting detection kit was from
Amersham Pharmacia Biotech (Freiburg, Germany).
Nitrocellulose BA-S83 was f rom S chleich er & Schuell
(Dassel, Germany). 5-Bromo-4-chloro-indolylphosphate
and Nitro blue tetrazolium were purchased from Biomol
Feinchemikalien GmbH (Hamburg, Ge rmany).
Animals
Cows (Schwarz-bunte Rasse) and pigs (Deutsche Land-
rasse) w ere raised a nd maintained at the Institut fu
¨
r
Tierzucht und-verhalten, Mariensee, Germany. Sprague–
Dawley rats were maintained at our institute according to
the animal welfare committee of the Medizinische Hochsch-
ule Hannover, Germany. All animals had access t o s tandard
chow and water ad libitum .
Preparation of tissue homogenates and serum

After the animals were killed, blood and tissues were
immediately collected. S erum was obtained after clotting
overnight at 4 °C and centrifugation. Livers were thor-
oughly perfused w ith cold N aCl/P
i
(2.8 m
M
KH
2
PO
4
,
9.4 m
M
K
2
HPO
4
, 150 m
M
NaCl, pH 7.3). Brains and
perfused livers were minced and then homogenized in
3vol. of 10m
M
sodium phosphate buffer, pH 7.3
containing 0.04% NaN
3
(buffer A) by use of an Ultra
Turrax homogenizer (Jahnke and Kunkel, Staufen, Ger-
many).

Induction of liver cirrhosis in rats
Adult male Sprague–Dawley rats weighing 380–400 g
were used. Over the experimental period 10 rats were
given t ap water containing 0.03% thioacetamide and 10
rats were kept as control. At given time intervals,
approximately 1 mL of blood was collected by retrobul-
bar puncture and after clotting serum was obtained by
centrifugation.
Hepatocyte isolation and culture
Hepatocytes were isolated from young male pigs
(about 7 w eeks old) as described previously [20]. Isolated
hepatocytes were adjusted to a density of 2 · 10
6
viable
cells per mL in Williams’ medium E supplemented with
fetal bovine serum (10%), insulin (0.17 IUÆmL
)1
), predni-
sone (0.85 lgÆmL
)1
), glucagon (0.015 lgÆmL
)1
), penicillin
(100 UÆmL
)1
), streptomycin (100 lgÆmL
)1
) and glutamine
(4.3 m
M

). The cells were plated onto 60-mm tissue culture
dishes coated with collagen and then cultured as desc ribed
by Bader et al. [21,22]. The rate of albumin secretion
into the culture medium was measured by electroimmu-
nodiffusion [23] using a polyclonal antibody against
porcine albumin. Lactate dehydrogenase activity in the
culture medium was determined by a modified method of
Bergmeyer & Bernt [24].
Protein and DNA analysis
The DNA content of c ultured hepatocytes was d etermined
according to the method described by Downs & Wilfinger
[25] using the fluorescent dye bisbenzimidazol (Hoechst dye
33258) and calf thymus DNA as standard. Protein was
determined by a modification of the Lowry method as
described by Peterson using bovine serum albumin as
standard [26].
Determination of TRH-DE activity
The assay was carried o ut as described previously using
[pyroGlu-
3
H] TRH a s s ubstrate [ 27]. In brief, samples were
incubated at 30 °C in a final reaction mixture of 50 lL
containing 27 n
M
[pyroGlu-
3
H] TRH and the inhibitors of
the c ytosolic TRH-DEs (1 m
M
DFP and 1 m

M
2-iodoacet-
amide for p ost proline cleaving enzyme and pyroglutamate
aminopeptidase, respectively). As a measure for the enzy-
matic activity, the initial rate of TRH-degradation was
determined by a four-point kinetic t est.
Purification of the TRH-DE from porcine serum
Porcineserum(1L)wasdilutedwith1LofbufferA.
Under constant stirring, 2 L of a poly(ethylene glycol) 6000
solution (dissolved 50% w/v in buffer A) were added
through a dropping funnel o ver a period of 3 h. After an
additional hour without stirring, the precipitated protein
was pelleted by centrifugation at 17 0 00 g for 3 h. The
supernatant was discarded and the protein pellet was
dissolved by stirring overnight with 3 L of buffer A. The
clear supernatant obtained a fter centrifugation at 17 000 g
for 1 h was subjected to the purification procedure as
described for the trypsin-solubilized membrane-bound
TRH-DE from pig brain [28].
Purification of the membrane-bound TRH-DE
from porcine liver
After homogenization of thoroughly perfused pig livers a nd
washing of the membranes, the membrane-bound TRH-DE
was solubilized by trypsin treatment and purified to
homogeneity by following the protocol described for the
isolation of TRH-DE from pig brain [28].
Ó FEBS 2002 Characterization of the TRH-DE from serum and liver (Eur. J. Biochem. 269) 1279
SDS/PAGE analysis
SDS/PAGE analysis was carried out according to Laemmli
[29]. The proteins were denatured under reducing co nditions

by boiling for 3 min in sample buffer containing 200 m
M
dithioerythritol.
Western blot analysis
After electrophoresis proteins were blotted onto a nitrocel-
lulose membrane as described by Towbin et al. [30]. After
blocking with NaCl/Tris (50 m
M
Tris/HCl, 150 m
M
NaCl,
pH 7.5) containing 0.1% Tween-20 (NaCl/Tris/Tween), the
membrane was incubated overnight at 4 °Cwithamono-
clonal antibody (41H2; 4 lgÆmL
)1
) generated against the
particulate TRH-DE from pig brain. The membrane was
then washed with NaCl/Tris/Tween and subsequently
incubated for 1 h at room temperature with goat anti-
(mouse IgG) Ig conjugated to alkaline phosphatase
(1 : 3000 in NaCl/Tris). After washing, the membrane was
incubated with a 5-bromo-4-chloro-indolylphosphate/Nitro
blue tetrazolium solution (335 l
M
5-bromo-4-chloro-indo-
lylphosphate, 400 l
M
Nitro blue tetrazolium in 200 m
M
Tris/HCl, 100 m

M
NaCl, 10 m
M
MgCl
2
,pH9.5)for
visualization.
Lectin blot analysis
Lectin blot an alysis was performed according to the method
described by Haselbeck et al. [31]. After Western blotting
and blocking, the membrane was cut and individual strips
were incubated for 1 h with d igoxigenin-conjugated lectins
(1 : 1000 in NaCl/Tris containing 1 m
M
MgCl
2
,1m
M
MnCl
2
,1m
M
CaCl
2
and 1 m
M
ZnCl
2
, pH 7 .5). The strips
were then washed with NaCl/Tris/Tween and s ubseq uently

incubated for 1 h with sheep anti-digoxigenin Ig conjugated
either to alkaline phosphatase (0.75 UÆmL
)1
) or to horse-
radish peroxidase (0.1 UÆmL
)1
). After washing of the s trips
with NaCl/Tris/Tween, the reaction products of alkaline
phosphatase or peroxidase were visualized by incubation
with the 5-bromo-4-chloro-indolylphosphate/Nitro blue
tetrazolium solution or b y u sing the E CL-Western blotting
detection kit, respectively.
Deglycosylation
Deglycosylation o f t he purified TRH-DE from liver, s erum
and b rain was performed as described previously [28] using
the endoglycosidase F/n-glycosidase F enzyme preparation.
Briefly, the enzymes (30 lL containing 0.4–0.5 lgprotein)
were denatured by boiling for 3 min in the presence of 0 .1%
SDS. After adding n-octylglycoside in a threefold e xc ess to
SDS, the glycosidase mixture (0.2 U in 50 lL) was added
and the reaction mixture was incubated for 24 h at 25 °C.
Following SDS/PAGE and Western blotting, the enzymes
were visualized by using the monoclonal antibody 41H2 as
described above.
Enzyme fragmentation and peptide sequencing
After isolation, the serum TRH-DE (100 lg, approximately
860 pmol) was either e xposed to cyanogen bromide in 70%
formic acid or digested by endoproteinase Lys-C as
described for the particulate TRH-DE from rat and pig
brain [32]. Enzyme fragments were isolated by reverse-phase

HPLC on C
4
or C
8
Vydac columns using acetonitrile in
0.1% trifluoroacetic acid as eluant. Isolated f ragments were
analyzed by gas-phase sequencing using the Applied
Biosystem 477A sequenator.
RESULTS
Degradation of TRH by serum and tissue homogenates
from different species
For comparative studies the specific activities of t he TRH-
DEs we re dete rmined i n se rum as well as in brain and liver
homogenates from cow, pig and rat (Table 1). For all t hree
species, high e nzymatic activities were found in brain. In rat
and pig, high enzymatic activities were also detected in
serum and significant activities in liver. In c ontrast, very low
activities were measured in liver homogenates and serum
from cows.
Table 1. Specific activities of the TRH-DEs in serum, liver and brain
from rat, pig and cow. Serum and tissue homogenates were prepared
and analyzed as described in Materials and methods (n ¼ 3 for pig and
cow, n ¼ 8 for rats, values are me ans ± SD).
Specific activity of the TRH-degrading enzyme
(% TRH-degradedÆmin
)1
Æmg protein
)1
)
Species Serum Liver Brain

Rat 3.92 ± 0.45 0.95 ± 0.06 8.56 ± 1.05
Pig 11.92 ± 1.67 1.78 ± 0.26 10.27 ± 0.52
Cow 0.19 ± 0.03 0.04 ± 0.004 6.60 ± 0.51
Fig. 1. Effect of thioacetamide, a hepatotoxin, on the activity of the
serum TRH-DE . Thioacetamide (0.03%) was e ither added or not to
the drinking water of adult m ale r ats. At the indicated time points
1 mL of blood was collected from control (d) and thioacetamide-
treated (s) animals. Serum was prepared and tested for the TRH-
degrading a ctivity a s described in Materials a nd methods (values are
means±S D).
1280 S. Schmitmeier et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Influence of thioacetamide, a hepatotoxin,
on the activity of the serum TRH-DE
When r ats were t reated with thioacetamide, an agent which
is known to induce liver cirrhosis [33,34], we observed a
rapid d ecrease in the activity o f t he serum TRH-DE w ithin
18 days (Fig. 1 ). Histological examinations revealed dis-
tinctive evidence of damage in liver sections of animals
treated for 46 days. In contrast to control a nimals, in liver
of thioacetamide-treated rats loss of the liver cell structure
and intercellular granula, increase of connective tissue and
appearance of noduli and fibrotic septa were noticed (data
not shown).
Synthesis of the TRH-DE by hepatocytes in culture
In contrast to hepatocytes kept as monolayers in primary
cultures, hepatocytes cultured in a sandwich configuration
continue to synthesize and secrete serum enzymes and
proteins after an initial lag phase required for cell recovery
[35,36]. This lag phase is characterized by a decline of the
lactate dehydrogenase activity released into the culture

medium due to cell leakage (Fig. 2). After 2 days of
cultivation and restoration of the cell membrane integrity,
the c oncentration of a lbumin a nd the activity of t he
TRH-DE in the culture medium increased in a correlative
manner (Fig. 2).
Purification of the TRH-DEs from serum and liver
The membrane-bound live r TRH-DE was s olubilized and
purified by following exactly the procedure elaborated for
the purification of the particulate TRH-DE from rat and
pig brain [28]. For the purification of the serum TRH-
DE fractionation by poly(ethylene glycol) precipitation
was used as the first step not only to partially purify the
enzyme but also to reduce the ionic strength due to salt.
At a poly(ethylene glycol) c oncentration of 25% the
serum enzyme completely precipitated. The enzyme was
recovered almost completely (97%) from the protein
pellet and could be applied directly to the Q-Sepharose
column. Elution from this column and further purifica-
tion followed again the procedure described previously
[28].
Characterization of the TRH-DEs from serum and liver
Molecular mass estimation. An approximate molecular
mass of 260 000 Da has been determined before for the
serum TRH-DE by gel filtration of porcine serum [9]. By
the same method, a molecular mass of % 250 000 Da
could be e stimated for t he trypsin-solubilized and purified
membrane-bound liver TRH-DE (Fig. 3). When the
purified enzymes were s ubjected to SDS/PAGE under
reducing conditions a molecular mass of % 125 000 Da
could be estimated for both enzymes, the serum TRH-

DE and the trypsin-solubilized membrane-bound liver
TRH-DE (Fig. 4 A), indicating that both enzymes consist
of two identical subunits. In contrast, but in agreement
with our previous data [28] a molecular mass of
116 000 Da could be determined for the trypsin-solubi-
lized membrane-bound TRH-DE from pig brain
(Fig. 4 A).
Fig. 2. Activity of the TRH-DE in the medium of cultured hepatocytes.
As de scribed in Materials and methods pig hepatocytes were isolated
and s eeded onto a collagen layer. A ft er cultivation for 24 h, the c ells
were covered by a second layer of collagen (arrow). After gelatinization
of th e s econd layer for 4 h, c ulture m edium was ad ded. Th e medium
was changed every 24 h and used to determine the concentration of
albumin ( r), the activity o f lactate dehydrogenase (d) and the activity
of th e TRH-DE (s)asdescribedinMaterialsandmethods(n¼10;
values are mean±SD).
Fig. 3. Estimation of the trypsin-solubilized TRH-DE f rom pig liver by
gel fi ltration on a TSK-G 3000 SW-column. After partial purification,
the trypsin-solubilized membrane-bound liver TRH-DE was subjected
to gel filtration on a calibrated TSK-G 3000 SW-column. The protein
elution profile was monitored b y following the absorbance at 280 n m
(ÆÆÆ). The enzyme activity (s) was determined as described in Materials
and meth ods. For calibration, a m ixture of standard proteins ( d)of
known molecular mass, namely: thyroglo bulin (1; M
r
669 000), ferritin
(2; M
r
450 0 00), catalase (3; M
r

245 0 00) a nd o valbumin ( 4; M
r
45 000)
was applied to t he same column.
Ó FEBS 2002 Characterization of the TRH-DE from serum and liver (Eur. J. Biochem. 269) 1281
Western blot analysis. To verify the hypothesis that the
brain T RH-DE, the serum TRH-DE and the liver TRH-DE
arederivedfromthesamegene,theenzymepreparations
were subjected to Western blotting. All three enzymes w ere
recognized by the monoclonal antibody 41H2 which was
generated by using purified TRH-DE from pig brain as
antigen (Fig. 4B). This finding indicates t hat t hese enzymes
are immunologically very similar. At this point, it is worth
noting that this antibody is specific to the enzymes of
porcine origin and does not react with t he enzymes from rat
or mouse.
Identification as glycoproteins. Asinthecaseofthebrain
TRH-DE [28], the TRH-DEs from liver and serum also
bind strongly to the Lentil-lectin Se pharose columns
which were used for the purification of these enzymes.
Thus, both enzymes could be identified as glycoproteins.
To gain more information as to the carbohydrate
structure, the three enzymes were subjected to lectin blot
analysis. As shown in T able 2, the serum enzyme and the
liver enzyme exhibit identic al properties, distinctly differ-
ent from th e brain enzyme. For example, the liver enzyme
and t he serum e nzyme are re adily recognized by the
lectin SNA (Sambucus nigra agglutinin) but not by
the lectin GNA (Galanthus nivalis agglutinin), whereas
the opposite is true for the brain enzyme. The collected

data shown in Table 2 indicate that the brain enzyme con-
tains an oligomannose/hybrid glycostructure, whereas the
serum enzyme and the liver enzyme belong to the groups
of glycoproteins with a glycostructure of the complex type.
To substantiate the notion that the TRH-DEs from liver,
serum and brain differ only i n the carbo hydrate moiety, the
three enzymes were incubated with the endoglycosidase
F/N-glycosidase F enzyme p reparation. After Western
blotting, a molecular mass of97 000 Da could b e determined
for all three enzymes (Fig. 4C) and thus a carbohydrate
content of 22% could be estimated for t he liver enzyme and
the serum enzyme vs. 16% for the brain e nzyme.
Peptide sequences of the serum TRH-DE
No sequence information could be obtained when the
purified serum enzyme was subjected directly to sequencing,
indicating that the aminoterminus is blocked. Therefore,
serum T RH-DE w as either s ubjected to cyanogen bromide
cleavage or to enzymatic digestion with endoproteinase
Lys-C. Overall 25 peptides could be isolated and sequenced.
Ten p eptides a re liste d in Table 3. Interestingly, four
peptides (3, 4, 8 and 10) were identical with the sequences
determined before when fragments of the membrane-bound
Table 2. Lectin blot analysis of the TRH-DEs from pig brain, serum and liver. The enzyme preparations were subjected to SDS/PAGE followed b y
Western b lotting. T he nitrocellulose membrane was then c ut and individual strips were incubated with digoxigenin-conjugated lectins. Anti-
digoxigenin a ntibodies conjugated either to alkaline phosphatase or to horseradish peroxidase were used for visualization as described in Materials
and methods.
Lectin
TRH-degrading
Brain enzyme Serum enzyme Liver enzyme
SNA (Sambucus nigra A.) – + +

GNA (Galanthus nivalis A.) + – –
MAA (Maackia amurensis A.) – – –
DSA (Datura stramonium A.) – – –
ConA (Concanavalin A) + + +
WGA (Wheat germ A.) + + +
PHA-L (Phytohaemagglutinin-L) + – –
PHA-E (Phytohaemagglutinin-E) + – –
RCA
120
(Ricinus communis A. I) + – –
Fig. 4. SDS/PAGE and Western blot analysis of the purified porcine
TRH-DE from brain, liver and serum. As described i n Materials and
methods the solubilized and purified membrane-bound TRH-DE from
brain (lane 1) and liver (lane 3) as well a s the purified serum TRH-DE
(lane 2) were subjected to SDS/PAGE and v isualized either by silver
staining (A) or immun ologically after Western blottin g ont o a nitro-
cellulose membrane by use of the monoclonal antibody 41H2 (B). For
the identification as glycoprotein (C), the purified enzymes were either
treated (T) or not (NT) with the endoglycosidase F /N-glyco sidase F
enzyme preparation as d escrib ed in Materials and m ethods and then
subjected t o S DS/P AGE f ollow ed b y Western blotting. The proteins
were again id entified by use o f the monoclonal antibody 41H2.
1282 S. Schmitmeier et al. (Eur. J. Biochem. 269) Ó FEBS 2002
TRH-DE from pig brain were analyzed [32]. This result
clearly demonstrates that both enzymes are derived from the
same gene. Comparison of the pep tide sequences of the
serum T RH-DE w ith the cDNA deduced amino-acid
sequences of the TRH-DE from rat [32] and human [37]
brain reveals that only eight amino acids (2.8%) were
different out of the 288 amino acids i dentified, whereby at

six positions the amino acids of the enzyme fro m pig and
human were identical and d ifferent from the rat enzyme and
only at two positions were the amino acids of the porcin e
peptide sequence different from that of rat and human,
which in turn were identical.
DISCUSSION
Even before TRH was finally isolated and structurally
elucidated, rapid inactivation of the biologically active
material by serum enzyme(s) had been demonstrated [38].
Subsequently, the serum enzyme catalyzing the hydrolysis
of TRH at the pyroGlu-His bond has been characterized
[8,9]. The findings that the activity of this enzyme is
regulated by thyroid hormones [16–18] strongly suggest a
physiological role of this peptidase for the inactivation of
TRH released into the peripheral circulation. This inter-
pretation is also supported by the high su bstrate specificity
of the enzyme [10] which therefore has also been named
ÔthyroliberinaseÕ. The physiological importance of this
peptidase was also supported by the observation that the
TRH-degrading enzyme (TRH-DE) is absent in the p lasma
of neonatal rats, whereas TRH is rapidly inactivated by
plasma of adult rats [39]. The endocrinological importance
of this enzyme was subsequently questioned by the findings
that the activity o f this peptidase varies considerably among
species and is almost absent in the plasma of beagle dogs [5].
In this study, the half-life of TRH after incubation with
various homogenates from different s pecies was also
examined but a correlation between the half-life of TRH
and TRH-degrading activities of the tissue homogenates
between s pecies was not observed. This result i s not

surprising as in tissue homogenates TRH is not only
inactivated by one enzyme as in serum or plasma but is
degraded by three peptidases (reviewed in [13,40]) namely
pyroglutamate aminopeptidase and post proline cleaving
enzyme (both are cytosolic enzymes), and the membrane-
bound TRH-DE, whereby the latter peptidase exhibits
identical enzyme-chemical characteristics as the serum
TRH-DE. Using enzyme-specific conditions to determine
the activity of the TRH-specific T RH-DEs indeed we found
high enzymatic a ctivities in b rain homogenates of all three
species examined. In contrast, considerable differences in
the TRH-degrading activities were noticed in the serum of
these animals, w hereby the enzyme activity is a lmost absent
in the serum from cow. Interestingly, we also observed a
correlation between the activity of the serum TRH-DE a nd
the activity of the TRH-DE in liver homogenates, suggest-
ing that the serum enzyme m ay be of liver origin.
At present we do not have an explanation for the late
development of the serum TRH-DE or for the extremely
low activity of this enzyme in some species. Nevertheless,
these results support the notion that the serum T RH-DE,
like most serum enzymes and proteins, is derived f rom
the liver. The decrease of the activity of the TRH-DE in
rats treated with t hioacetamide, a known hepatotoxin
which induces liver c irrhosis [33,34] seemed to be in line
with this interpretation. However, a rapid decrease in the
enzymatic activity w as already observed within a few
days after thioacetamide treatment. As liver cirrhosis is
generally a long-term process and is observed histolog-
ically only after treatment with thioacetamide for several

weeks, this decrease in the enzymatic activity seems to be
related to other effects of thioacetamide on hepatocytes
such as the reported inhibition of respiratory metabolism,
binding to metal-containing enzymes, blockade of
mRNA transport and loss of the cell’s ability t o store
glycogen [41].
Our experiments with hepatocytes in primary culture
provided more d irect evidence for the notion t hat the serum
TRH-DE is of liver origin. While hepatocytes in monolayer
cultures appear to dedifferentiate a nd rapidly stop secreting
liver-derived proteins, these cells maintain their function
(e.g. secretion of albumin, transferrin, a
1
-antitrypsin) when
cultured in a collageneous matrix [20–22,35,36]. After
seeding and establishing the cultures in a sandwich confi-
guration, we observed a decrease in the activity of lactate
dehydrogenase (a m arker for the r estoration of the i ntegrity
of the cell membrane) released into the culture medium.
Correspondingly, we found an increase in the amount of
albumin (a liver specific marker protein) a nd an increase in
Table 3. Sequence o f peptide fragm ents. The serum TRH-DE was either subjected to cyanogen bromide cleavage (+) or d igested with endopro-
teinase Lys-C (à). The peptides were isolated by reverse-p hase HPLC an d sequenced. The peptides which had been identified before from digests of
TRH-DE from pig brain [32] are marked with an asterisk. The numbers refer to the position of the amino acid as deduced from the cDNA of rat [32]
or human [37] brain TRH-DE. Differences are found at position 604 (F in pig; L in human and rat), 607 (T in pig and human; M in rat), 614 (I in pig
and human; L in rat), 680 (L in pig and human; I in rat), 760 (K in pig; R in rat and human), 991 (A in pig and human; S in rat), 1009 (M in pig and
human; R in r at) and 1023 (L in pig and hu man; M in rat).
Peptide 1+ 160-E XFTFSGEVNV EIA
Peptide 2à 192-VQLAEDRAF GAVPVAGFFL YPQTQVLVVV L
Peptide 3+ *485-EKQRFL TDVLHEV

Peptide 4+ *543-GHSVFQRQ LQDYLTIHKY GNAARNDLWNT LSEA
Peptide 5+ 598-GYP VITIFGNTTA ENRII
Peptide 6à 677-GSWL LGNI
Peptide 7à 751-DFLPWHAASK
Peptide 8+ *958-NSK LISGVTEFLN TEGELKELKN
Peptide 9à 985-SYDGVA AASFSRAVET VEANVRW
Peptide 10à *1009-M LYQDELFQWL GKALRH
Ó FEBS 2002 Characterization of the TRH-DE from serum and liver (Eur. J. Biochem. 269) 1283
the activity of the TRH-DE released into the culture
medium, suggesting that the increase in the enzymatic
activity is due to the increased synthetic a ctivity of
hepatocytes and not due to cell leakage.
For direct analysis we purified the membrane-bound liver
TRH-DE after solubilization by trypsin and the serum
TRH-DE to elec trophoretic homogeneity b y following the
procedure described for the isolation of the membrane-
bound TRH-DE from pig brain [28]. By gel filtration a
molecular mass of 250 000 Da could b e estimated for the
truncated liver enzyme, a value which corresponds well with
the molecular mass of the p apain-solubilized liver enzyme
[15] and the molecular mass of the serum enzyme [9]
reported before (260 000 Da) but differs from the molecular
mass of 230 000 Da determined for the trypsin-solubilized
brain enzyme [28]. After SDS/PAGE under reducing
conditions a molecular mass of 125 000 Da was estimated
for the liver enzyme and the serum enzyme and a molecular
mass of 116 000 Da for the brain enzyme, indicating that all
these enzymes exist as homodimers, like many surface
peptidases [42]. Interestingly, TRH-DEs from brain, liver
and s erum were all r ecognized by the monoclonal antibody

41H2 which was generated afte r immunizing m ice with the
TRH-DE from pig brain.
As the brain TRH-DE has b een identified as a glycopro-
tein [28], the immunological identity o f this enzyme, the
serum e nzyme and th e liver enzyme strongly suggeste d that
these proteins differ only in their carbohydrate moiety.
Analysis of the carbohydrate structures revealed that the
brain enzyme contains a glycostructure of the oligoman-
nose/hybrid type. The occurrence of terminal nonsu bstitu-
ted m annose and galactose residues is a general feature of
most brain glycoproteins [43] a nd thus the brain TRH-DE is
a ÔbrainÕ type glycoprotein [44]. In contrast, T RH-DE f rom
both liver and serum contained terminal nonsubstituted
a(2–6)-sialic acid units linked to galactose and were thus
characterized as glycoproteins of the ÔserumÕ type [45]. The
presence of sialic acid units in serum proteins is of biological
importance, as on hepatocytes desialyated proteins are
recognized by asialoglyc oprotein-specific receptors and are
thus removed from the circulation by the liver [46]. As
glycosylation is a species- and tissu e-specifi c p rocess [47–50]
the three enzyme preparations were enzymatically degly-
cosylated and subsequently subjected to SDS/PAGE. For
all t hree enzymes a band with a molecular mass of
97 000 Da could be visualized immunologically, indicating
the polypeptide chain of these enzymes is very similar or
identical. These results s trongly support the hypothesis that
the serum TRH-DE and the membrane-bound TRH-DE
from brain and liver are derived from the same gene,
whereby the soluble enzyme might be either generated by
alternative splicing of the mRNA (e.g. as reported for

immunoglobulin l [51,52]) or by proteolytic cleavage of the
membrane-bound liver enzym e as demonstrated for various
membrane-bound proteins with soluble c ounterparts
(reviewed in [53]). By fragmentation a nalysis of t he purified
serum TRH-DE, two peptide sequences (peptide 1 and 2)
(Table 1) could be identified which correspond to the
sequences 160–173 and 192–221 of the cDNA deduced
amino-acid sequence of the membrane-bound brain
TRH-DE. As both peptides are encoded b y e xon 1 which
ends at the position of amino-acid 260 [37,54], we can
conclude that the serum enzyme is not a product of
alternative mRNA s plicing but must b e generated by
proteolysis. Whe ther the s erum enzyme is released f rom the
plasma membrane of hepatocytes by proteases acting as
sheddases o r secretases (also designated as membrane
protein-solubilizing proteases, MPSPs) [53,55–57] remains
to be elucidated. Preliminary experiments indicate that the
release of the serum enzyme is not affected by inhibitors
directed against well characterized sheddases [name ly
b-secretase, c-secretase and TNFa protease (TACE)]. The
present results indicate furthermore that the serum e nyzme
might be generated intracellularly b ecause after homogeni-
zation of isolated hepatocytes and high speed centrifuga-
tion, 40% of the TRH-degrading activity could be found in
the cytosolic fraction and 60% of the e nzyme activity was
recovered from the particulate fraction ( Schmitmeier, S. &
Bauer, K., unpublished observation). This asp ect warrants
further investigation.
ACKNOWLEDGEMENTS
We would like to thank Prof Dr P. W. Jungblut for his interest and

encouragement and for providing the antibodie s against serum
albumin. We also than k H. O. Bader, S. Thiele for an imal care,
P. Affeldt for advice and help, and V. Ashe for t yping and for l inguistic
help in preparing the manuscript. Supported by the Deutsche
Forschungsgemeinschaft.
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