- Email: [email protected]
Characterization of turkey pancreatic lipase
Biochimie 82 (2000) 153−159 © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400003795/FLA
Characterization of turkey pancreatic lipase Adel Sayaria, Hafedh Mejdoubb, Youssef Gargouria* a
Laboratoire de lipolyse enzymatique, ENIS, BPW 3038 Sfax, Tunisia b FSS, Route de Soukra, 3038 Sfax, Tunisia (Received 14 June 1999; accepted 25 November 1999)
Abstract — Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAESepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37 °C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37 °C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65 °C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (β-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N-terminal amino acid residues were sequenced. This sequence was similar to those of mammal’s pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS pancreatic lipase / colipase / amphiphiles / inhibition / reactivation
1. Introduction The hydrolysis of dietary triacylglycerol in mammals is catalyzed by the major lipases of the digestive tract: preduodenal and pancreatic lipases. In man, the first step of hydrolysis of dietary lipids begins in the stomach and is catalyzed by the acid-stable lipase present in gastric juice [1]. In 1981, the first amino acid sequence of porcine pancreatic lipase (PPL) was established by De Caro et al. [2]. Over the past few years, the amino acid sequences of some pancreatic lipases have been deduced from the corresponding cDNA in the case of several species, including man [3, 4], dog [5], guinea pig [6, 7], rat [8, 9], coypu [10, 11], and rabbit [12]. On the basis of the primary structure comparison, the pancreatic lipase family can be subdivided into three subgroups: i) classical pancreatic lipases; ii) pancreatic lipase-related protein 1
(PLRP1); and iii) pancreatic lipase-related protein 2 (PLRP2) [4, 10]. It is well known that in the guinea pig [6], the coypu [10], and the rat [9], PLRP2 displays different kinetic properties from those of the classical lipases. Other kinetic studies performed with emulsion of triacylglycerols as lipase substrate have shown that other amphiphiles such as synthetic detergents [13, 14] or proteins [15, 16] are also inhibitors of pancreatic lipase. The aim of this study is to compare some biochemical properties of a bird pancreatic lipase with mammal’s pancreatic lipases. To allow this comparison, we proposed to purify to homogeneity TPL in order to study the effect of amphiphiles and the kinetics properties of the pure enzyme. The N-terminal sequence of TPL was determined and compared to that of mammals. 2. Materials and methods
* Correspondence and reprints Abbreviations: DrPL, dromedary pancreatic lipase; HPL, human pancreatic lipase; PPL, porcine pancreatic lipase; TPL, turkey pancreatic lipase; BSA, bovine serum albumin; CMC, critical micellar concentration; GA, gum arabic; NaDC, sodium deoxycholate; NaTDC, sodium taurodeoxycholate; OO, olive oil; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TC2, triacetin; TC3, tripropionin; TC4, tributyrin; TX-100, Triton X-100.
2.1. Materials Tributyrin (99%; puriss), benzamidine and 4-methyl morpholine were from Fluka (Buchs, Switzerland); tripropionin (99%, GC) was from Janssen Chimica (Geel, Belgium), sodium taurodeoxycholate (NaTDC) and triacetin (99%; puriss) were from Sigma Chemical (St. Louis, USA); gum arabic was from Mayaud Baker LTD (Dagen-
154
Sayari et al.
ham, UK); acrylamide and electrophoresis grade, were from BDH (Poole, UK); marker proteins and supports of chromatography used for TPL purification: Sephacryl S-200 and DEAE-Sepharose gel were from Pharmacia (Uppsala, Sweden); PVDF membrane was purchased from Applied Biosystems (Roissy, France); trans blot cell apparatus was from Bio-Rad (Paris, France); HPLC column protein Pak 300 SW was from Waters (St. Quentin, France). All chemicals used on HPLC column were of analytical grade from Merck (Darmstadt, Germany); pH-stat was from Metrohm (Switzerland).
Lipase activity was also measured using TC2 or TC3 as substrate according to Ferrato et al. [20]. One lipase unit corresponds to 1 µmol of fatty acid liberated per min. The colipase activity was measured at pH 8.5 and 37 °C using a pH-stat. Assays were performed with olive oil emulsion as substrate in the presence of 6 mM NaTDC. The level of colipase of all pancreases was measured using an homogenate prepared in the same conditions described by Rathelot et al. [18]. One colipase unit is the amount of cofactor that increases bile salt inhibited pancreatic lipase activity by one enzyme unit [18].
2.2. Pancreas collections
2.6. Determination of protein concentration
Pancreases from different species were collected immediately after death from the local slaughterhouse of Sfax or Sidi Salem (Tunisia). A stock of turkey pancreases was kept at –20 °C before delipidation.
Protein concentration was determined as described by Bradford et al. [21].
2.3. Delipidation of turkey pancreases
Analytical polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate (SDSPAGE) was performed by the method of Laemmli [22]. Samples for sequencing were electroblotted according to Bergman and Jörnvall [23]. Protein transfer was performed during 1 h at 1 mA/cm2 at room temperature.
After decongelation, the turkey pancreases were cut into small pieces (1–2 cm3) and delipidated according to the method described previously [17]. After delipidation, 20 g of delipidated powder of turkey pancreases were obtained from 100 g of fresh tissue.
2.7. Analytical methods
2.8. Sugar content 2.4. Enzymes and proteins Bovine serum albumin fraction IV (BSA) and β-lactoglobulin A were from Sigma (St. Louis, USA). Crude porcine colipase was obtained after homogenization at pH 3.0 of delipidated powder of porcine pancreases (1 g/10 mL) (Laboratoire Industriel de Biologie, France) followed by a centrifugation at 10 000 rpm for 10 min. The supernatant, which contains all the colipase but no lipase activity, was adjusted to pH 6.0. After centrifugation at 10 000 rpm for 10 min, colipase sample was stored at –20 °C. The specific activity measured under standard conditions [18] is 350 U/mg. In some cases, pure porcine pancreatic colipase from Boehringer (Mannheim, France) was used (specific activity under standard conditions is 10 000 U/mg). No significant difference was observed when pure TPL activity was measured in the presence of crude or pure porcine colipase (data not shown).
Total sugar of the purified TPL was measured by anthrone-sulfuric acid method using glucose as a standard [24]. 2.9. Amino acid sequencing The N-terminal of TPL was sequenced by automated Edman’s degradation, using an Applied Biosystems 470A protein sequencer equipped with PTH 120A Analyser [25]. The sequence has kindly been determined by Dr. Reinbolt (IBMC, UPR 9002 du CNRS, Strasbourg, France). 3. Results and discussion 3.1. The level of pancreatic lipase and colipase in some animals
2.5. Lipase and colipase activity determination The lipase activity was measured titrimetrically at pH 8.5 and 37 °C with a pH-stat, under the standard assay conditions described previously, using tributyrin (0.25 mL) or liprocil (0.25 mL) in 30 mL of 2.5 mM Tris-HCl, pH 8.5, 100 mM NaCl, 5 mM CaCl2 [19] or olive oil emulsion [18] as substrate. Some lipase assays were performed in the presence of NaTDC, colipase, TX-100 or β-lactoglobulin A.
In order to compare the level of TPL and colipase activities with other species, we measured, under the same conditions, the rate of hydrolysis of olive oil emulsion or tributyrin by bovine, chicken, dromedary, and sheep pancreatic lipases. Results reported in table I show a large variation in pancreatic lipases and colipases levels between species. For all species, except turkey, the TC4 was hydrolyzed more efficiently than the olive oil. The ratio short/long chains is about 2. In the case of turkey, the
Characterization of turkey pancreatic lipase
155
Table I. Pancreatic lipase and colipase levels in some animals. Species
Turkey Chicken Bovine Sheep Dromedary
Lipase activity (U/g of fresh pancreases) Olive oil
TC4
14 600 ± 2 400 3 350 ± 1 313 2 180 ± 790 2 620 ± 1 602 581 ± 73
5 700 ± 274 7 375 ± 2358 3 940 ± 1 392 5 300 ± 908 1 051 ± 135
Colipase activity (U/g of fresh pancreases) 3 000 ± 316 2 375 ± 530 1 080 ± 421 1 600 ± 300 517 ± 85
The determination of the lipase and colipase content of pancreases from all species was performed in an homogenate prepared in a Waring Blendor (2 × 30 s) with 10 mL of 10 mM Tris-HCl, pH 8, 2 mM benzamidine per gram of fresh tissue. After centrifugation at 10 000 rpm, for 15 min, the amount of enzyme was estimated on an aliquot of the supernatant using olive oil emulsion or tributyrin (0.25 mL of tributyrin in 30 mL of 2.5 mM Tris-HCl, pH 8.5, 100 mM NaCl, 5 mM CaCl2) as substrate in the presence of a molar excess of colipase (colipase to lipase molar ratio = 5) and 4 mM NaTDC. The lipase and colipase activities were measured trimetrically at pH 8.5 and 37 °C using a pH-stat. One lipase unit corresponds to 1 µmol of fatty acid liberated per min. One colipase unit corresponds to the amount of cofactor that increases bile salt inhibited pancreatic lipase activity by one enzyme unit. For each species, the activities represent the average ± standard error mean of five assays obtained from five different pancreases.
ratio short/long chains is about 0.5. When olive oil emulsion was used as substrate, TPL presented the highest level and about 16 000 U/g of fresh turkey pancreases were detected. The maximal ratio in lipase units per gram of different pancreases was observed between turkey and dromedary, more than 27-fold. Table I shows that the ratio lipase/colipase is about 1.5 for chicken, bovine, sheep and dromedary. This ratio reaches 5 in the case of turkey. 3.2. Purification of TPL 5 g of delipidated powder of turkey pancreases were suspended in 55 mL of buffer A: 25 mM Tris-HCl, pH 8.2, 2 mM benzamidine, 25 mM NaCl, and mixed mechanically twice for 30 s at 4 °C using the Waring Blendor system. The mixture was then stirred with a magnetic bar for 60 min at 4 °C, and then centrifuged for 20 min at 10 000 rpm. 3.2.1. Ammonium sulfate precipitation
The supernatant (50 mL) was brought to 60% saturation with solid ammonium sulfate (19.5 g) under stirring conditions and maintained for 60 min at 4 °C. After centrifugation (30 min at 10 000 rpm), the precipitate was resuspended in 15 mL of buffer A. Insoluble material was removed by centrifugation for 10 min at 10 000 rpm. 3.2.2. Filtration on Sephacryl S-200
The supernatant (15 mL) was loaded on a column (3 × 100 cm) of gel filtration Sephacryl S-200 equilibrated with buffer A. Elution of lipase was performed with buffer A at 40 mL/h. The fractions containing the lipase activity (eluted at 1.4 void volumes) were pooled. 3.2.3. Anion exchange chromatography
The pooled fractions of Sephacryl S-200 column were added to the DEAE-Sepharose anion exchanger equili-
brated in buffer A. The column (1.6 × 20 cm) was rinsed with 400 mL of buffer A. No lipase activity was detected in the washing flow. Proteins were eluted by a linear NaCl gradient (500 mL of 25 to 250 mM in buffer A). The protein elution profile obtained is shown in figure 1A. TPL activity was eluted between 80 and 100 mM NaCl. The fractions containing the lipase activity were pooled and lyophilized. The results of SDS-PAGE analysis of the pooled fraction of this DEAE-Sepharose chromatography are given in figure 1B and showed that TPL was contaminated by two proteins with molecular masses of 25 and 30 kDa. To remove these contaminants, the lyophilized proteins were resuspended in 5 mL phosphate buffer, pH 7. 100 µg of this enzyme solution was loaded on size exclusion HPLC column Protein Pak 300 SW (7.8 mm × 30 cm) equilibrated in phosphate buffer. The lipase emerged 12 min after injection. Elution was performed with phosphate buffer at 0.5 mL/min. The fractions containing the TPL activity were pooled and analyzed on SDS-PAGE (figure 1B). This figure shows that only one band was revealed for TPL with a molecular mass of about 45 kDa. The molecular mass of TPL estimated by gel filtration on HPLC column Protein Pak 300 SW (7.8 mm × 30 cm) was 44 kDa (data not shown). These results (SDS-PAGE and gel filtration) suggested that TPL was a monomeric protein like all pancreatic lipases described so far [26]. The presence of sugar in TPL was tested. Our results indicate that the TPL, like bovine and ovine pancreatic lipases, is not a glycoprotein (data not shown). It differs from the two porcine lipases, which are glycoproteins [26]. The purification flow sheet is given in table II which shows that the specific activity of TPL reaches 4300 U/mg
156
Sayari et al. activities were measured at pH 7 and 37 °C [21]. This result shows that, in contrast to all mammal [26] and chicken pancreatic lipases [27, 28], TPL hydrolyses more efficiently the long- than the short-chain triacylglycerols. The ratio of TPL activity long/short chains is about 2.5. 3.3. Characterization of TPL 3.3.1. Activation of TPL by interface
As has been recently shown by Ferrato et al. [20], among the short chain triacylglycerols tested as substrates (TC2, TC3, TC4), TC3 is the best system to check the interfacial activation of pancreatic lipases. In this study we have selected the TC3 to evaluate the presence of interfacial activation phenomenon in TPL. The hydrolysis rate of TC3 emulsified in 0.33% GA and 0.15 M NaCl (figure 2A) or in 0.33% GA (figure 2B) as a function of substrate concentration shows that TPL hydrolyzed very slowly the TC3 when it is in monomeric state, however, up to the solubility limit of TC3 (12 mM) the TPL activity increased dramatically to reach 900 U/mg at 24 mM of TC3. This result indicates that TPL, as PPL [20, 29], presents the interfacial activation phenomenon. 3.3.2. Effects of pH and temperature on activity and stability of TPL
The maximal activity of TPL was measured at pH 8.5 and 37 °C. Like all the mammal’s pancreatic lipases, TPL was found to be stable between pH 7 and pH 9, and to lose its activity at pH > 9 or pH < 5. When the enzyme was incubated at a temperature higher than 50 °C, TPL was inactivated after few minutes like all mammal’s pancreatic lipases [26]. 3.3.3. Effects of bile salts on TPL activity
Figure 1. A. Chromatography of TPL on DEAE-Sepharose. The column (1.6 × 20 cm) was equilibrated with 25 mM Tris-HCl, pH 8.2, 2 mM benzamidine, 25 mM NaCl; a linear salt concentration gradient (25–250 mM NaCl) was applied to the column; gradient chamber 250 mL; fraction 3 mL; flow rate, 25 mL/h. The substrate was olive oil emulsion. B. SDS-PAGE (13%). Lane a, molecular mass markers (Pharmacia); lane b, characterization of the TPL obtained after DEAE-Sepharose chromatography (15 µg); lane c, 12 µg of purified TPL. The gel was stained with Coomassie blue.
using gum arabic emulsified olive oil as substrate in the presence of colipase and 4 mM NaTDC at pH 8.5 and 37 °C. Under the same conditions, specific activities of 109, 900, 1600 and 2600 U/mg were obtained when TC2, TC3, TC4 and liprocil were used as substrates respectively. When TC2 and TC3 were used as substrate, the TPL
The effect of varying concentrations of NaTDC on the rate of hydrolysis of olive oil emulsion (figure 3A) or tributyrin (figure 3B)by TPL is presented in figure 3. This figure shows that bile salt acts as in vitro inhibitor of TPL activity when TC4 or olive oil emulsion is used as substrate. In the two systems, inhibition is reversed by addition of colipase even at bile salt concentrations largely exceeding their CMC [30, 31]. The effect of varying concentrations of synthetic detergents like Tween-20 (non-ionic detergent) or amphiphilic proteins like β-lactoglobulin A on the rate of hydrolysis of olive oil emulsion by TPL shows that the activity of TPL decreases rapidly at a given amphiphilic concentration threshold. The cofactor failed to counteract the inhibition in a large range of inhibitor concentration in contrast to that observed in the case of bile salt (data not shown). Comparable results were obtained using ionic or zwitterionic detergents (data not shown). Thus the inhibition of TPL can not be related to the charge or the structure of the synthetic detergents.
Characterization of turkey pancreatic lipase
157
Table II. Flow sheet of the TPL purification. Purification step Extract of TPL (pH 8.2) (NH4)2SO4 precipitation S-200 chromatography DEAE-Sepharose chromatography Filtration on HPLC
Total activitya (units)
Proteinb (mg)
Specific activity (U/mg)
Activity recovery (%)
Purification factor
120 000 95 000 40 500 28 000
3 529 208 15.2 7.4
34 456.7 2 664 3 783
– 79 33.75 23.3
1 13.43 78.3 111.2
17 500
4
4 364
14.58
128.3
1 unit: 1 µmol of fatty acid released per min using olive oil emulsion as substrate in the presence of 4 mM NaTDC and in the presence of a molar excess of colipase (colipase to lipase molar ratio = 5). b Proteins were estimated by Bradford method [27]. The experiments were conducted three times. a
The addition of increasing concentrations of NaTDC to the reaction system containing TPL, colipase (colipase to lipase molar ratio 5), and inhibitory concentration of tween 20 (0.6 mM) or β-lactoglobulin A (12 µM) fully restores lipolysis (data not shown). Then bile salt shows the ability to activate lipolysis in the presence of various inhibitory amphiphiles. These experiments confirm those described by Gargouri et al. [13, 32] which have showed that inhibition of pancreatic lipase activity by amphiphiles such as proteins or detergents appears to be a general phenomenon related
to a desorption of lipase from its substrate, occurs after a change in interfacial quality.
3.3.4. N-terminal sequence of TPL
The NH2-terminal sequencing of the blotted TPL allowed the identification of 22 residues, S-E-V-X-Y-DR-V-G-X-F-T-D-D-I-P-W-S-G-T-A-E (where residue X was not identified). Table III shows the N-terminal sequence of TPL, together with those of DrPL [33],
Figure 2. Hydrolysis rate of TC3 by TPL as function of substrate concentration. The TC3 solutions were systematically prepared by mixing (3 × 30 s in a warring blender) a given amount of TC3 in 30 mL of 0.33% GA and 0.15 M NaCl (A), or in 30 mL of 0.33% GA (B). The release of propionic acid was recorded continuously at pH 7 and 37 °C using a pH-stat. The CMC of TC3 (12 mM) is indicated by vertical dotted lines.
158
Sayari et al.
Figure 3. Effect of increasing concentration of bile salt. NaTDC on the rate of hydrolysis of olive oil emulsion (A) or tributyrin (B) by TPL. Lipolytic activity was measured at pH 8.5 and 37 °C in the absence or in the presence of a molar excess of colipase (colipase to lipase molar ratio = 5).
HPL [3], and PPL [2]. TPL was found to exhibit a high degree of homology with the other mammalian pancreatic lipases. 4. Conclusion TPL was isolated to electrophoretic purity from delipidated pancreases. The pure enzyme is not a glycoprotein. It is a monomer with a molecular mass of 45 kDa. TPL hydrolyses the long chains more efficiently than the short chain triacylglycerols. It is inactivated at 60 °C and it is not stable at pH less than 5. TPL presents the interfacial activation phenomenon. Natural detergents (NaTDC, NaDC) act as strong inhibitors of TPL activity and colipase reverses this inhibition. Other amphiphiles (synthetic detergents or proteins) are inhibitors of TPL activity. In contrast to that observed in the case of bile salt, colipase failed to counteract the inhibition in a large range of inhibitor concentration. Reactivation of TPL by colipase in presence of inhibitory concentration of proteins or synthetic detergents requires the presence of bile salt.
The sequence of the N-terminal part shows that TPL is highly similar to other known mammalian pancreatic lipases. Thus no significant difference was observed when the biochemical properties of the TPL were compared to those of mammals.
Acknowledgments
Our thanks are due to Dr. J. Reinbolt (IBMC, UPR 9002 du CNRS, Strasbourg, France) for the sequencing of the NH2terminal of TPL. We acknowledge the help of S. Makhlouf (ENIS) with the pancreas collect and Mr. A. Hajji (ENIS) for his help with the English. The present results were presented during the 10th days of the ATSB held in Monastir, Tunisia, March 20–22, 1999. This work is part of a doctoral thesis by Adel Sayari. This work received financial support from the DGRST (BIND 3478), (E19/C09) and CMCU projects (96/F0920).
Table III. Sequence comparison of TPL with HPL, PPL and DrPL. TPL HPL PPL DrPL
1 S E V – Y D R V G 10– F T D D I P W S G 20T A E KEVCYERLGCFSDDSPWSGITE S EVC F PR LG C F S D DAPWAG IVQ TEVC F E R LG C F R D DAPWAG I
This study [3] [2] [33]
Characterization of turkey pancreatic lipase References [1] Gargouri Y., Piéroni G., Rivière C., Saunière J.F., Lowe P.A., Sarda L., Verger R., Importance of human gastric lipase for duodenal lipolysis: an in vitro study, Biochim. Biophys. Acta 879 (1986) 419–423. [2] De Caro J., Boudouard M., Bonicel J., Guidoni A., Desnuelle P., Rovery M., Porcine pancreatic lipase. Completion of the primary structure, Biochim. Biophys. Acta 671 (1981) 129–138. [3] Lowe M.E., Rosenblun J.L., Strauss A.W., Cloning and characterization of human pancreatic lipase cDNA, J. Biol. Chem. 264 (1989) 20042–20048. [4] Giller T., Buchwald P., Blum-Kaelin D., Hunziker W., Two novel human pancreatic lipase related proteins, hHPLRP1 and hHPLRP2, J. Biol. Chem. 267 (1992) 16509–16516. [5] Kerfelec B., La Forge K.S., Pingserver A., Scheele G., Primary structures of canine pancreatic lipase and phospholipase A2 messagers RNAs, Pancreas 1 (1986) 430–437. [6] Hjorth A., Carriére F., Cudrey C., Wöldike H., Boel E., Lawson D.M., Ferrato F., Cambillau C., Dodson G.C., Thim L., Verger R., A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho) lipase, Biochemistry 32 (1993) 4702–4707. [7] Carriére F., Thirstrup K., Hjorth S., Boel E., Cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2, FEBS Lett. 338 (1994) 63–68. [8] Wicker-Planquart C., Puigserver A., Primary structure of rat pancreatic lipase mRNA, FEBS Lett. 296 (1992) 61–66. [9] Payne M., Sims H., Jennens M., Lowe M., Rat pancreatic lipase and two related proteins: enzymatic properties and mRNA expression during development, Am. J. Physiol. 266 (1994) G914–G921 [10] Thirstrup K., Verger R., Carriére F., Evidence for a pancreatic lipase superfamily with new kinetic properties, Biochemistry 33 (1994) 2748–2756. [11] Thirstrup K., Carrière F., Hjorth S., Rasmussen P.B., Nielsen P.F., Ladefoged C., Thim L., Boel E., Cloning and expression in insect cells of two pancreatic lipases and a procolipase from Myocastor coypus, Eur. J. Biochem. 227 (1995) 186–193. [12] Aleman-Gomes J.A., Colwell N.S., Sasser T., Kumar V.B., Molecular cloning and characterization of rabbit pancreatic triglyceride lipase, Biochim. Biophys. Res. Commun. 188 (1992) 964–971. [13] Gargouri Y., Julien R., Bois A.G., Verger R., Sarda L., Studies on the detergent inhibition of pancreatic lipase activity, J. Lipid Res. 24 (1983) 1336–1342. [14] Rathelot J., Julien R., Bosc-Bierne I., Gargouri Y., Canioni P., Sarda L., Horse pancreatic lipase. Interaction with colipase from various species, Biochimie 63 (1981) 227–234. [15] Borgström B., Erlanson C., Interaction of serum albumin and other proteins with porcine pancreatic lipase, Gastroenterology 75 (1978) 382–386. [16] Gargouri Y., Julien R., Pieroni G., Verger R., Sarda L., Studies of the inhibition of pancreatic and microbial lipases by soybean proteins, J. Lipid Res. 25 (1984) 1214–1221.
159 [17] Verger R., De Haas G.H., Sarda L., Desnuelle P., Purification from porcine pancreas of two molecular species with lipase activity, Biochim. Biophys. Acta 188 (1969) 272–282. [18] Rathelot J., Julien R., Canioni P., Coeroli C., Sarda L., Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improved method for the determination of pancreatic lipase and colipase, Biochimie 57 (1975) 1117–1122. [19] Gargouri Y., Cudrey C., Mejboub H., Verger R., Inactivation of human pancreatic lipase by 5-dodecyldithio-2-nitrobenzoicacid, Eur. J. Biochem. 204 (1992) 1063–1067. [20] Ferrato F., Carrière F., Sarda L., Verger R., A critical reevaluation of the phenomenon of interfacial activation, Methods Enzymol. 286 (1997) 327–346. [21] Bradford M.M., A rapid and sensitive method for the quantitation of quantities of protein utilising the principle of protein-drye binding, Anal. Biochem. 72 (1976) 248–254. [22] Laemmli U.K., Cleavage of structural protein during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [23] Bergman T., Jörnvall H., Electroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis, Eur. J. Biochem. 169 (1987) 9–12. [24] Spiro R.G., Analysis of sugars found in glycoproteins, Methods Enzymol. 8 (1966) 3–26. [25] Hewick R.M., Hunkapiller M.W., Hood L.E., Dreyer W.J., A gaz-liquid solid phase peptide and protein sequenator, J. Biol. Chem. 256 (1981) 7990–7997. [26] Verger R., pancreatic lipases, in: Borgström B., Brockman H.L. (Eds.), Lipase, Elsevier, Amsterdam, 1984, pp. 121–150. [27] Bosc-Bierne I., Rathelot J., Canioni P., Julien R., Bechis G., Gregoire J., Rochat H., Sarda L., Isolation and partial structural characterization of chicken pancreatic lipase, Biochim. Biophys. Acta 667 (1981) 225–232. [28] Bosc-Bierne I., Rathelot J., Christine P., Sarda L., Studies on chicken pancreatic lipase and colipase, Biochim. Biophys. Acta 794 (1984) 65–71. [29] Sarda L., Desnuelle P., Action de la lipase pancréatique sur les esters en émulsion, Biochim. Biophys. Acta 30 (1958) 513–521 [30] Maylié M.F., Charles M., Gache C., Desnuelle P., Isolation and partial identification of a pancreatic colipase, Biochim. Biophys. Acta 229 (1971) 286–289. [31] Morgan R.G.H., Hoffman N.E., The interaction of lipase, lipase cofactor and bile salts in triglycerides hydrolysis, Biochim. Biophys. Acta 248 (1971) 143–148. [32] Gargouri Y., Piéroni G., Rivière C., Sarda L., Verger R., Inhibition of lipases by proteins: A binding study using dicaprin monolayers, Biochemistry 24 (1986) 1733–1738. [33] Mejdoub H., Reinbolt J., Gargouri Y., Dromedary pancreatic lipase: Purification and structural properties, Biochim. Biophys. Acta 1213 (1994) 119–126.
Characterization of turkey pancreatic lipase Adel Sayaria, Hafedh Mejdoubb, Youssef Gargouria* a
Laboratoire de lipolyse enzymatique, ENIS, BPW 3038 Sfax, Tunisia b FSS, Route de Soukra, 3038 Sfax, Tunisia (Received 14 June 1999; accepted 25 November 1999)
Abstract — Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAESepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37 °C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37 °C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65 °C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (β-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N-terminal amino acid residues were sequenced. This sequence was similar to those of mammal’s pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS pancreatic lipase / colipase / amphiphiles / inhibition / reactivation
1. Introduction The hydrolysis of dietary triacylglycerol in mammals is catalyzed by the major lipases of the digestive tract: preduodenal and pancreatic lipases. In man, the first step of hydrolysis of dietary lipids begins in the stomach and is catalyzed by the acid-stable lipase present in gastric juice [1]. In 1981, the first amino acid sequence of porcine pancreatic lipase (PPL) was established by De Caro et al. [2]. Over the past few years, the amino acid sequences of some pancreatic lipases have been deduced from the corresponding cDNA in the case of several species, including man [3, 4], dog [5], guinea pig [6, 7], rat [8, 9], coypu [10, 11], and rabbit [12]. On the basis of the primary structure comparison, the pancreatic lipase family can be subdivided into three subgroups: i) classical pancreatic lipases; ii) pancreatic lipase-related protein 1
(PLRP1); and iii) pancreatic lipase-related protein 2 (PLRP2) [4, 10]. It is well known that in the guinea pig [6], the coypu [10], and the rat [9], PLRP2 displays different kinetic properties from those of the classical lipases. Other kinetic studies performed with emulsion of triacylglycerols as lipase substrate have shown that other amphiphiles such as synthetic detergents [13, 14] or proteins [15, 16] are also inhibitors of pancreatic lipase. The aim of this study is to compare some biochemical properties of a bird pancreatic lipase with mammal’s pancreatic lipases. To allow this comparison, we proposed to purify to homogeneity TPL in order to study the effect of amphiphiles and the kinetics properties of the pure enzyme. The N-terminal sequence of TPL was determined and compared to that of mammals. 2. Materials and methods
* Correspondence and reprints Abbreviations: DrPL, dromedary pancreatic lipase; HPL, human pancreatic lipase; PPL, porcine pancreatic lipase; TPL, turkey pancreatic lipase; BSA, bovine serum albumin; CMC, critical micellar concentration; GA, gum arabic; NaDC, sodium deoxycholate; NaTDC, sodium taurodeoxycholate; OO, olive oil; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TC2, triacetin; TC3, tripropionin; TC4, tributyrin; TX-100, Triton X-100.
2.1. Materials Tributyrin (99%; puriss), benzamidine and 4-methyl morpholine were from Fluka (Buchs, Switzerland); tripropionin (99%, GC) was from Janssen Chimica (Geel, Belgium), sodium taurodeoxycholate (NaTDC) and triacetin (99%; puriss) were from Sigma Chemical (St. Louis, USA); gum arabic was from Mayaud Baker LTD (Dagen-
154
Sayari et al.
ham, UK); acrylamide and electrophoresis grade, were from BDH (Poole, UK); marker proteins and supports of chromatography used for TPL purification: Sephacryl S-200 and DEAE-Sepharose gel were from Pharmacia (Uppsala, Sweden); PVDF membrane was purchased from Applied Biosystems (Roissy, France); trans blot cell apparatus was from Bio-Rad (Paris, France); HPLC column protein Pak 300 SW was from Waters (St. Quentin, France). All chemicals used on HPLC column were of analytical grade from Merck (Darmstadt, Germany); pH-stat was from Metrohm (Switzerland).
Lipase activity was also measured using TC2 or TC3 as substrate according to Ferrato et al. [20]. One lipase unit corresponds to 1 µmol of fatty acid liberated per min. The colipase activity was measured at pH 8.5 and 37 °C using a pH-stat. Assays were performed with olive oil emulsion as substrate in the presence of 6 mM NaTDC. The level of colipase of all pancreases was measured using an homogenate prepared in the same conditions described by Rathelot et al. [18]. One colipase unit is the amount of cofactor that increases bile salt inhibited pancreatic lipase activity by one enzyme unit [18].
2.2. Pancreas collections
2.6. Determination of protein concentration
Pancreases from different species were collected immediately after death from the local slaughterhouse of Sfax or Sidi Salem (Tunisia). A stock of turkey pancreases was kept at –20 °C before delipidation.
Protein concentration was determined as described by Bradford et al. [21].
2.3. Delipidation of turkey pancreases
Analytical polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate (SDSPAGE) was performed by the method of Laemmli [22]. Samples for sequencing were electroblotted according to Bergman and Jörnvall [23]. Protein transfer was performed during 1 h at 1 mA/cm2 at room temperature.
After decongelation, the turkey pancreases were cut into small pieces (1–2 cm3) and delipidated according to the method described previously [17]. After delipidation, 20 g of delipidated powder of turkey pancreases were obtained from 100 g of fresh tissue.
2.7. Analytical methods
2.8. Sugar content 2.4. Enzymes and proteins Bovine serum albumin fraction IV (BSA) and β-lactoglobulin A were from Sigma (St. Louis, USA). Crude porcine colipase was obtained after homogenization at pH 3.0 of delipidated powder of porcine pancreases (1 g/10 mL) (Laboratoire Industriel de Biologie, France) followed by a centrifugation at 10 000 rpm for 10 min. The supernatant, which contains all the colipase but no lipase activity, was adjusted to pH 6.0. After centrifugation at 10 000 rpm for 10 min, colipase sample was stored at –20 °C. The specific activity measured under standard conditions [18] is 350 U/mg. In some cases, pure porcine pancreatic colipase from Boehringer (Mannheim, France) was used (specific activity under standard conditions is 10 000 U/mg). No significant difference was observed when pure TPL activity was measured in the presence of crude or pure porcine colipase (data not shown).
Total sugar of the purified TPL was measured by anthrone-sulfuric acid method using glucose as a standard [24]. 2.9. Amino acid sequencing The N-terminal of TPL was sequenced by automated Edman’s degradation, using an Applied Biosystems 470A protein sequencer equipped with PTH 120A Analyser [25]. The sequence has kindly been determined by Dr. Reinbolt (IBMC, UPR 9002 du CNRS, Strasbourg, France). 3. Results and discussion 3.1. The level of pancreatic lipase and colipase in some animals
2.5. Lipase and colipase activity determination The lipase activity was measured titrimetrically at pH 8.5 and 37 °C with a pH-stat, under the standard assay conditions described previously, using tributyrin (0.25 mL) or liprocil (0.25 mL) in 30 mL of 2.5 mM Tris-HCl, pH 8.5, 100 mM NaCl, 5 mM CaCl2 [19] or olive oil emulsion [18] as substrate. Some lipase assays were performed in the presence of NaTDC, colipase, TX-100 or β-lactoglobulin A.
In order to compare the level of TPL and colipase activities with other species, we measured, under the same conditions, the rate of hydrolysis of olive oil emulsion or tributyrin by bovine, chicken, dromedary, and sheep pancreatic lipases. Results reported in table I show a large variation in pancreatic lipases and colipases levels between species. For all species, except turkey, the TC4 was hydrolyzed more efficiently than the olive oil. The ratio short/long chains is about 2. In the case of turkey, the
Characterization of turkey pancreatic lipase
155
Table I. Pancreatic lipase and colipase levels in some animals. Species
Turkey Chicken Bovine Sheep Dromedary
Lipase activity (U/g of fresh pancreases) Olive oil
TC4
14 600 ± 2 400 3 350 ± 1 313 2 180 ± 790 2 620 ± 1 602 581 ± 73
5 700 ± 274 7 375 ± 2358 3 940 ± 1 392 5 300 ± 908 1 051 ± 135
Colipase activity (U/g of fresh pancreases) 3 000 ± 316 2 375 ± 530 1 080 ± 421 1 600 ± 300 517 ± 85
The determination of the lipase and colipase content of pancreases from all species was performed in an homogenate prepared in a Waring Blendor (2 × 30 s) with 10 mL of 10 mM Tris-HCl, pH 8, 2 mM benzamidine per gram of fresh tissue. After centrifugation at 10 000 rpm, for 15 min, the amount of enzyme was estimated on an aliquot of the supernatant using olive oil emulsion or tributyrin (0.25 mL of tributyrin in 30 mL of 2.5 mM Tris-HCl, pH 8.5, 100 mM NaCl, 5 mM CaCl2) as substrate in the presence of a molar excess of colipase (colipase to lipase molar ratio = 5) and 4 mM NaTDC. The lipase and colipase activities were measured trimetrically at pH 8.5 and 37 °C using a pH-stat. One lipase unit corresponds to 1 µmol of fatty acid liberated per min. One colipase unit corresponds to the amount of cofactor that increases bile salt inhibited pancreatic lipase activity by one enzyme unit. For each species, the activities represent the average ± standard error mean of five assays obtained from five different pancreases.
ratio short/long chains is about 0.5. When olive oil emulsion was used as substrate, TPL presented the highest level and about 16 000 U/g of fresh turkey pancreases were detected. The maximal ratio in lipase units per gram of different pancreases was observed between turkey and dromedary, more than 27-fold. Table I shows that the ratio lipase/colipase is about 1.5 for chicken, bovine, sheep and dromedary. This ratio reaches 5 in the case of turkey. 3.2. Purification of TPL 5 g of delipidated powder of turkey pancreases were suspended in 55 mL of buffer A: 25 mM Tris-HCl, pH 8.2, 2 mM benzamidine, 25 mM NaCl, and mixed mechanically twice for 30 s at 4 °C using the Waring Blendor system. The mixture was then stirred with a magnetic bar for 60 min at 4 °C, and then centrifuged for 20 min at 10 000 rpm. 3.2.1. Ammonium sulfate precipitation
The supernatant (50 mL) was brought to 60% saturation with solid ammonium sulfate (19.5 g) under stirring conditions and maintained for 60 min at 4 °C. After centrifugation (30 min at 10 000 rpm), the precipitate was resuspended in 15 mL of buffer A. Insoluble material was removed by centrifugation for 10 min at 10 000 rpm. 3.2.2. Filtration on Sephacryl S-200
The supernatant (15 mL) was loaded on a column (3 × 100 cm) of gel filtration Sephacryl S-200 equilibrated with buffer A. Elution of lipase was performed with buffer A at 40 mL/h. The fractions containing the lipase activity (eluted at 1.4 void volumes) were pooled. 3.2.3. Anion exchange chromatography
The pooled fractions of Sephacryl S-200 column were added to the DEAE-Sepharose anion exchanger equili-
brated in buffer A. The column (1.6 × 20 cm) was rinsed with 400 mL of buffer A. No lipase activity was detected in the washing flow. Proteins were eluted by a linear NaCl gradient (500 mL of 25 to 250 mM in buffer A). The protein elution profile obtained is shown in figure 1A. TPL activity was eluted between 80 and 100 mM NaCl. The fractions containing the lipase activity were pooled and lyophilized. The results of SDS-PAGE analysis of the pooled fraction of this DEAE-Sepharose chromatography are given in figure 1B and showed that TPL was contaminated by two proteins with molecular masses of 25 and 30 kDa. To remove these contaminants, the lyophilized proteins were resuspended in 5 mL phosphate buffer, pH 7. 100 µg of this enzyme solution was loaded on size exclusion HPLC column Protein Pak 300 SW (7.8 mm × 30 cm) equilibrated in phosphate buffer. The lipase emerged 12 min after injection. Elution was performed with phosphate buffer at 0.5 mL/min. The fractions containing the TPL activity were pooled and analyzed on SDS-PAGE (figure 1B). This figure shows that only one band was revealed for TPL with a molecular mass of about 45 kDa. The molecular mass of TPL estimated by gel filtration on HPLC column Protein Pak 300 SW (7.8 mm × 30 cm) was 44 kDa (data not shown). These results (SDS-PAGE and gel filtration) suggested that TPL was a monomeric protein like all pancreatic lipases described so far [26]. The presence of sugar in TPL was tested. Our results indicate that the TPL, like bovine and ovine pancreatic lipases, is not a glycoprotein (data not shown). It differs from the two porcine lipases, which are glycoproteins [26]. The purification flow sheet is given in table II which shows that the specific activity of TPL reaches 4300 U/mg
156
Sayari et al. activities were measured at pH 7 and 37 °C [21]. This result shows that, in contrast to all mammal [26] and chicken pancreatic lipases [27, 28], TPL hydrolyses more efficiently the long- than the short-chain triacylglycerols. The ratio of TPL activity long/short chains is about 2.5. 3.3. Characterization of TPL 3.3.1. Activation of TPL by interface
As has been recently shown by Ferrato et al. [20], among the short chain triacylglycerols tested as substrates (TC2, TC3, TC4), TC3 is the best system to check the interfacial activation of pancreatic lipases. In this study we have selected the TC3 to evaluate the presence of interfacial activation phenomenon in TPL. The hydrolysis rate of TC3 emulsified in 0.33% GA and 0.15 M NaCl (figure 2A) or in 0.33% GA (figure 2B) as a function of substrate concentration shows that TPL hydrolyzed very slowly the TC3 when it is in monomeric state, however, up to the solubility limit of TC3 (12 mM) the TPL activity increased dramatically to reach 900 U/mg at 24 mM of TC3. This result indicates that TPL, as PPL [20, 29], presents the interfacial activation phenomenon. 3.3.2. Effects of pH and temperature on activity and stability of TPL
The maximal activity of TPL was measured at pH 8.5 and 37 °C. Like all the mammal’s pancreatic lipases, TPL was found to be stable between pH 7 and pH 9, and to lose its activity at pH > 9 or pH < 5. When the enzyme was incubated at a temperature higher than 50 °C, TPL was inactivated after few minutes like all mammal’s pancreatic lipases [26]. 3.3.3. Effects of bile salts on TPL activity
Figure 1. A. Chromatography of TPL on DEAE-Sepharose. The column (1.6 × 20 cm) was equilibrated with 25 mM Tris-HCl, pH 8.2, 2 mM benzamidine, 25 mM NaCl; a linear salt concentration gradient (25–250 mM NaCl) was applied to the column; gradient chamber 250 mL; fraction 3 mL; flow rate, 25 mL/h. The substrate was olive oil emulsion. B. SDS-PAGE (13%). Lane a, molecular mass markers (Pharmacia); lane b, characterization of the TPL obtained after DEAE-Sepharose chromatography (15 µg); lane c, 12 µg of purified TPL. The gel was stained with Coomassie blue.
using gum arabic emulsified olive oil as substrate in the presence of colipase and 4 mM NaTDC at pH 8.5 and 37 °C. Under the same conditions, specific activities of 109, 900, 1600 and 2600 U/mg were obtained when TC2, TC3, TC4 and liprocil were used as substrates respectively. When TC2 and TC3 were used as substrate, the TPL
The effect of varying concentrations of NaTDC on the rate of hydrolysis of olive oil emulsion (figure 3A) or tributyrin (figure 3B)by TPL is presented in figure 3. This figure shows that bile salt acts as in vitro inhibitor of TPL activity when TC4 or olive oil emulsion is used as substrate. In the two systems, inhibition is reversed by addition of colipase even at bile salt concentrations largely exceeding their CMC [30, 31]. The effect of varying concentrations of synthetic detergents like Tween-20 (non-ionic detergent) or amphiphilic proteins like β-lactoglobulin A on the rate of hydrolysis of olive oil emulsion by TPL shows that the activity of TPL decreases rapidly at a given amphiphilic concentration threshold. The cofactor failed to counteract the inhibition in a large range of inhibitor concentration in contrast to that observed in the case of bile salt (data not shown). Comparable results were obtained using ionic or zwitterionic detergents (data not shown). Thus the inhibition of TPL can not be related to the charge or the structure of the synthetic detergents.
Characterization of turkey pancreatic lipase
157
Table II. Flow sheet of the TPL purification. Purification step Extract of TPL (pH 8.2) (NH4)2SO4 precipitation S-200 chromatography DEAE-Sepharose chromatography Filtration on HPLC
Total activitya (units)
Proteinb (mg)
Specific activity (U/mg)
Activity recovery (%)
Purification factor
120 000 95 000 40 500 28 000
3 529 208 15.2 7.4
34 456.7 2 664 3 783
– 79 33.75 23.3
1 13.43 78.3 111.2
17 500
4
4 364
14.58
128.3
1 unit: 1 µmol of fatty acid released per min using olive oil emulsion as substrate in the presence of 4 mM NaTDC and in the presence of a molar excess of colipase (colipase to lipase molar ratio = 5). b Proteins were estimated by Bradford method [27]. The experiments were conducted three times. a
The addition of increasing concentrations of NaTDC to the reaction system containing TPL, colipase (colipase to lipase molar ratio 5), and inhibitory concentration of tween 20 (0.6 mM) or β-lactoglobulin A (12 µM) fully restores lipolysis (data not shown). Then bile salt shows the ability to activate lipolysis in the presence of various inhibitory amphiphiles. These experiments confirm those described by Gargouri et al. [13, 32] which have showed that inhibition of pancreatic lipase activity by amphiphiles such as proteins or detergents appears to be a general phenomenon related
to a desorption of lipase from its substrate, occurs after a change in interfacial quality.
3.3.4. N-terminal sequence of TPL
The NH2-terminal sequencing of the blotted TPL allowed the identification of 22 residues, S-E-V-X-Y-DR-V-G-X-F-T-D-D-I-P-W-S-G-T-A-E (where residue X was not identified). Table III shows the N-terminal sequence of TPL, together with those of DrPL [33],
Figure 2. Hydrolysis rate of TC3 by TPL as function of substrate concentration. The TC3 solutions were systematically prepared by mixing (3 × 30 s in a warring blender) a given amount of TC3 in 30 mL of 0.33% GA and 0.15 M NaCl (A), or in 30 mL of 0.33% GA (B). The release of propionic acid was recorded continuously at pH 7 and 37 °C using a pH-stat. The CMC of TC3 (12 mM) is indicated by vertical dotted lines.
158
Sayari et al.
Figure 3. Effect of increasing concentration of bile salt. NaTDC on the rate of hydrolysis of olive oil emulsion (A) or tributyrin (B) by TPL. Lipolytic activity was measured at pH 8.5 and 37 °C in the absence or in the presence of a molar excess of colipase (colipase to lipase molar ratio = 5).
HPL [3], and PPL [2]. TPL was found to exhibit a high degree of homology with the other mammalian pancreatic lipases. 4. Conclusion TPL was isolated to electrophoretic purity from delipidated pancreases. The pure enzyme is not a glycoprotein. It is a monomer with a molecular mass of 45 kDa. TPL hydrolyses the long chains more efficiently than the short chain triacylglycerols. It is inactivated at 60 °C and it is not stable at pH less than 5. TPL presents the interfacial activation phenomenon. Natural detergents (NaTDC, NaDC) act as strong inhibitors of TPL activity and colipase reverses this inhibition. Other amphiphiles (synthetic detergents or proteins) are inhibitors of TPL activity. In contrast to that observed in the case of bile salt, colipase failed to counteract the inhibition in a large range of inhibitor concentration. Reactivation of TPL by colipase in presence of inhibitory concentration of proteins or synthetic detergents requires the presence of bile salt.
The sequence of the N-terminal part shows that TPL is highly similar to other known mammalian pancreatic lipases. Thus no significant difference was observed when the biochemical properties of the TPL were compared to those of mammals.
Acknowledgments
Our thanks are due to Dr. J. Reinbolt (IBMC, UPR 9002 du CNRS, Strasbourg, France) for the sequencing of the NH2terminal of TPL. We acknowledge the help of S. Makhlouf (ENIS) with the pancreas collect and Mr. A. Hajji (ENIS) for his help with the English. The present results were presented during the 10th days of the ATSB held in Monastir, Tunisia, March 20–22, 1999. This work is part of a doctoral thesis by Adel Sayari. This work received financial support from the DGRST (BIND 3478), (E19/C09) and CMCU projects (96/F0920).
Table III. Sequence comparison of TPL with HPL, PPL and DrPL. TPL HPL PPL DrPL
1 S E V – Y D R V G 10– F T D D I P W S G 20T A E KEVCYERLGCFSDDSPWSGITE S EVC F PR LG C F S D DAPWAG IVQ TEVC F E R LG C F R D DAPWAG I
This study [3] [2] [33]
Characterization of turkey pancreatic lipase References [1] Gargouri Y., Piéroni G., Rivière C., Saunière J.F., Lowe P.A., Sarda L., Verger R., Importance of human gastric lipase for duodenal lipolysis: an in vitro study, Biochim. Biophys. Acta 879 (1986) 419–423. [2] De Caro J., Boudouard M., Bonicel J., Guidoni A., Desnuelle P., Rovery M., Porcine pancreatic lipase. Completion of the primary structure, Biochim. Biophys. Acta 671 (1981) 129–138. [3] Lowe M.E., Rosenblun J.L., Strauss A.W., Cloning and characterization of human pancreatic lipase cDNA, J. Biol. Chem. 264 (1989) 20042–20048. [4] Giller T., Buchwald P., Blum-Kaelin D., Hunziker W., Two novel human pancreatic lipase related proteins, hHPLRP1 and hHPLRP2, J. Biol. Chem. 267 (1992) 16509–16516. [5] Kerfelec B., La Forge K.S., Pingserver A., Scheele G., Primary structures of canine pancreatic lipase and phospholipase A2 messagers RNAs, Pancreas 1 (1986) 430–437. [6] Hjorth A., Carriére F., Cudrey C., Wöldike H., Boel E., Lawson D.M., Ferrato F., Cambillau C., Dodson G.C., Thim L., Verger R., A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho) lipase, Biochemistry 32 (1993) 4702–4707. [7] Carriére F., Thirstrup K., Hjorth S., Boel E., Cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2, FEBS Lett. 338 (1994) 63–68. [8] Wicker-Planquart C., Puigserver A., Primary structure of rat pancreatic lipase mRNA, FEBS Lett. 296 (1992) 61–66. [9] Payne M., Sims H., Jennens M., Lowe M., Rat pancreatic lipase and two related proteins: enzymatic properties and mRNA expression during development, Am. J. Physiol. 266 (1994) G914–G921 [10] Thirstrup K., Verger R., Carriére F., Evidence for a pancreatic lipase superfamily with new kinetic properties, Biochemistry 33 (1994) 2748–2756. [11] Thirstrup K., Carrière F., Hjorth S., Rasmussen P.B., Nielsen P.F., Ladefoged C., Thim L., Boel E., Cloning and expression in insect cells of two pancreatic lipases and a procolipase from Myocastor coypus, Eur. J. Biochem. 227 (1995) 186–193. [12] Aleman-Gomes J.A., Colwell N.S., Sasser T., Kumar V.B., Molecular cloning and characterization of rabbit pancreatic triglyceride lipase, Biochim. Biophys. Res. Commun. 188 (1992) 964–971. [13] Gargouri Y., Julien R., Bois A.G., Verger R., Sarda L., Studies on the detergent inhibition of pancreatic lipase activity, J. Lipid Res. 24 (1983) 1336–1342. [14] Rathelot J., Julien R., Bosc-Bierne I., Gargouri Y., Canioni P., Sarda L., Horse pancreatic lipase. Interaction with colipase from various species, Biochimie 63 (1981) 227–234. [15] Borgström B., Erlanson C., Interaction of serum albumin and other proteins with porcine pancreatic lipase, Gastroenterology 75 (1978) 382–386. [16] Gargouri Y., Julien R., Pieroni G., Verger R., Sarda L., Studies of the inhibition of pancreatic and microbial lipases by soybean proteins, J. Lipid Res. 25 (1984) 1214–1221.
159 [17] Verger R., De Haas G.H., Sarda L., Desnuelle P., Purification from porcine pancreas of two molecular species with lipase activity, Biochim. Biophys. Acta 188 (1969) 272–282. [18] Rathelot J., Julien R., Canioni P., Coeroli C., Sarda L., Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improved method for the determination of pancreatic lipase and colipase, Biochimie 57 (1975) 1117–1122. [19] Gargouri Y., Cudrey C., Mejboub H., Verger R., Inactivation of human pancreatic lipase by 5-dodecyldithio-2-nitrobenzoicacid, Eur. J. Biochem. 204 (1992) 1063–1067. [20] Ferrato F., Carrière F., Sarda L., Verger R., A critical reevaluation of the phenomenon of interfacial activation, Methods Enzymol. 286 (1997) 327–346. [21] Bradford M.M., A rapid and sensitive method for the quantitation of quantities of protein utilising the principle of protein-drye binding, Anal. Biochem. 72 (1976) 248–254. [22] Laemmli U.K., Cleavage of structural protein during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [23] Bergman T., Jörnvall H., Electroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis, Eur. J. Biochem. 169 (1987) 9–12. [24] Spiro R.G., Analysis of sugars found in glycoproteins, Methods Enzymol. 8 (1966) 3–26. [25] Hewick R.M., Hunkapiller M.W., Hood L.E., Dreyer W.J., A gaz-liquid solid phase peptide and protein sequenator, J. Biol. Chem. 256 (1981) 7990–7997. [26] Verger R., pancreatic lipases, in: Borgström B., Brockman H.L. (Eds.), Lipase, Elsevier, Amsterdam, 1984, pp. 121–150. [27] Bosc-Bierne I., Rathelot J., Canioni P., Julien R., Bechis G., Gregoire J., Rochat H., Sarda L., Isolation and partial structural characterization of chicken pancreatic lipase, Biochim. Biophys. Acta 667 (1981) 225–232. [28] Bosc-Bierne I., Rathelot J., Christine P., Sarda L., Studies on chicken pancreatic lipase and colipase, Biochim. Biophys. Acta 794 (1984) 65–71. [29] Sarda L., Desnuelle P., Action de la lipase pancréatique sur les esters en émulsion, Biochim. Biophys. Acta 30 (1958) 513–521 [30] Maylié M.F., Charles M., Gache C., Desnuelle P., Isolation and partial identification of a pancreatic colipase, Biochim. Biophys. Acta 229 (1971) 286–289. [31] Morgan R.G.H., Hoffman N.E., The interaction of lipase, lipase cofactor and bile salts in triglycerides hydrolysis, Biochim. Biophys. Acta 248 (1971) 143–148. [32] Gargouri Y., Piéroni G., Rivière C., Sarda L., Verger R., Inhibition of lipases by proteins: A binding study using dicaprin monolayers, Biochemistry 24 (1986) 1733–1738. [33] Mejdoub H., Reinbolt J., Gargouri Y., Dromedary pancreatic lipase: Purification and structural properties, Biochim. Biophys. Acta 1213 (1994) 119–126.