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: karl.bauer@mpihan.mpg.de 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. 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(1997) Membrane protein secretases. Bio chem. J. 32 1, 265–279. 1286 S. Schmitmeier et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . 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,. 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. 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. )