Biochemical and structural comparative study between bird and mammal pancreatic colipases

Three colipases were purified from pancreas of two birds (ostrich and turkey) and one mammal (dromedary). After acidic and/or heat treatment and precipitation by sulfate ammonium and then ethanol, cofactors were purified by Sephadex G-50 gel filtration followed by ion-exchange chromatography first on Mono S and then on Mono Q. One molecular form was obtained from each species with a molecular mass of ∼10 kDa. Cofactors were not glycosylated. The N-terminal sequences of the three purified cofactors showed high sequence homology. A 90 amino acid sequence of the ostrich cofactor was established based on peptide sequences from four different digests of the denaturated protein using trypsin, chymotrypsin, thermolysin, or staphylococcal protease. This sequence exhibited a high degree of homology with chicken and mammal cofactors. Bile salt-inhibited pancreatic lipases from five species were activated to variable extents by colipases from bird and mammal origins. The bird pancreatic lipase-colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. Our comparative study showed that mammal colipase presents a lower activation level toward bird lipases than the bird counterpart. Three-dimensional modeling of ostrich colipase suggested a structural explanation of this fact. Three colipases were purified from pancreas of two birds (ostrich and turkey) and one mammal (dromedary). After acidic and/or heat treatment and precipitation by sulfate ammonium and then ethanol, cofactors were purified by Sephadex G-50 gel filtration followed by ion-exchange chromatography first on Mono S and then on Mono Q. One molecular form was obtained from each species with a molecular mass of ∼10 kDa. Cofactors were not glycosylated. The N-terminal sequences of the three purified cofactors showed high sequence homology. A 90 amino acid sequence of the ostrich cofactor was established based on peptide sequences from four different digests of the denaturated protein using trypsin, chymotrypsin, thermolysin, or staphylococcal protease. This sequence exhibited a high degree of homology with chicken and mammal cofactors. Bile salt-inhibited pancreatic lipases from five species were activated to variable extents by colipases from bird and mammal origins. The bird pancreatic lipase-colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. Our comparative study showed that mammal colipase presents a lower activation level toward bird lipases than the bird counterpart. Three-dimensional modeling of ostrich colipase suggested a structural explanation of this fact. In mammals, the duodenal digestion of dietary triacylglycerols is accomplished essentially by the action of pancreatic lipase in the presence of colipase and bile salts. Colipase is a low molecular mass protein secreted by the exocrine pancreas that counteracts (in vitro) the inhibiting action of bile salts on lipase activity (1Borgstrom B. Erlanson-Albertsson C. Wieloch T. Pancreatic colipase: chemistry and physiology.J. Lipid Res. 1979; 20: 805-816Abstract Full Text PDF PubMed Google Scholar) via the formation of a specific lipase-colipase complex (2Donner J. Spink C.H. Borgstrom B. Sjoholm I. Interactions between pancreatic lipase, colipase and taurodeoxycholate in the absence of triglyceride substrate.Biochemistry. 1976; 15: 5413-5417Crossref PubMed Scopus (69) Google Scholar). Colipase has been purified from the pancreatic glands of several species (1Borgstrom B. Erlanson-Albertsson C. Wieloch T. Pancreatic colipase: chemistry and physiology.J. Lipid Res. 1979; 20: 805-816Abstract Full Text PDF PubMed Google Scholar). The first indication of the existence of a cofactor for pancreatic lipases was reported in 1910 by Rosenheim (3Rosenheim O. On pancreatic lipase. III. The separation of lipase from its co-enzyme.J. Physiol. 1910; 15: 14-16Google Scholar). Colipase, which has been isolated from the pancreas of many mammals (human, dog, horse, ox, pig, rat, and sheep) (4Sternby B. Borgstrom B. Purification and characterisation of human pancreatic colipase.Biochim. Biophys. Acta. 1979; 617: 371-382Google Scholar, 5Lee P.C. Comparative studies of canine colipase and lipases from bovine, porcine, canine, human and rat pancreas.Comp. Biochem. Physiol. 1978; 60B: 373-378Google Scholar, 6Julien R. Rathelot J. Canioni P. Sarda L. Gregoire J. Rochat H. Horse pancreatic colipase: isolation by a detergent method and amino terminal sequence of the polypeptide chain.Biochimie. 1978; 60: 103-107Crossref PubMed Scopus (32) Google Scholar, 7Julien R. Canioni P. Rathelot J. Sarda L. Plummer Jr., T.H. Studies on bovine pancreatic lipase and colipase.Biochim. Biophys. Acta. 1972; 280: 215-224Crossref PubMed Scopus (45) Google Scholar, 8Erlanson C. Borgstrom B. Purification and further characterization of colipase from porcine pancreas.Biochim. Biophys. Acta. 1972; 271: 400-412Crossref PubMed Scopus (57) Google Scholar, 9Erlanson-Albertsson C. The existence of pro-colipase in pancreatic juice.Biochim. Biophys. Acta. 1981; 666: 299-300Crossref PubMed Scopus (37) Google Scholar, 10Canioni P. Julien R. Rathelot J. Sarda L. Inhibition of sheep pancreatic lipase activity against emulsified tributyrin by non-ionic detergents.Biochimie. 1976; 58: 751-753Crossref PubMed Scopus (23) Google Scholar) and from chicken (11Bosc-Bierne I. Rathelot J. Canioni P. Julien R. Bechis G. Gregoire J. Rochat H. Sarda L. Isolation and partial structural characterization of chicken pancreatic colipase.Biochim. Biophys. Acta. 1981; 667: 225-232Crossref PubMed Scopus (32) Google Scholar), was found in the pancreatic juice of higher mammals in a proform (procolipase). Procolipase is converted to its physiologically more active form by a tryptic cleavage of a single bond, arginine 5-glycine 6 (12Borgstrom B. Wieloch T. Erlanson-Albertsson C. Evidence for a pancreatic procolipase and its activation by trypsin.FEBS Lett. 1979; 108: 407-410Crossref PubMed Scopus (84) Google Scholar). The removal of the N-terminal pentapeptide of native colipase, conserved in all mammal colipases studied to date, was found to highly increase the capacity of colipase to anchor the lipase to a lipid-water interface (13Wieloch T. Borgstrom B. Pieroni G. Pattus P. Verger R. Porcine pancreatic procolipase and its trypsin-activated form: lipid binding and lipase activation on monomolecular films.FEBS Lett. 1981; 128: 217-220Crossref PubMed Scopus (25) Google Scholar, 14Rathelot J. Canioni P. Bosc-Bierne I. Sarda L. Kamoun A. Kaptein R. Cozzone P.J. Limited trypsinolysis of porcine and equine colipases. Spectroscopic and kinetic studies.Biochim. Biophys. Acta. 1981; 671: 155-163Crossref PubMed Scopus (36) Google Scholar, 15Erlanson-Albertsson C. Larsson A. Importance of the N-terminal sequence in porcine pancreatic colipase.Biochim. Biophys. Acta. 1981; 665: 250-255Crossref PubMed Scopus (22) Google Scholar). The three-dimensional structure of the colipase was determined in complex with the pancreatic lipase by X-ray crystallography (16Van Tilbeurgh H. Sarda L. Verger R. Cambillau C. Structure of the lipase-colipase complex.Nature. 1992; 359: 159-162Crossref PubMed Scopus (322) Google Scholar) and alone in solution by NMR (17Wieloch T. Borgström B. Falk K.E. Forsén S. High-resolution proton magnetic resonance study of porcine colipase and its interactions with taurodeoxycholate.Biochemistry. 1979; 18: 1622-1628Crossref PubMed Scopus (22) Google Scholar). Colipase belongs to a family of small cysteine-rich proteins. It is a fairly flat molecule of dimensions 25 × 30 × 35 Å with a three-finger topology comparable to that of snake toxins (16Van Tilbeurgh H. Sarda L. Verger R. Cambillau C. Structure of the lipase-colipase complex.Nature. 1992; 359: 159-162Crossref PubMed Scopus (322) Google Scholar, 18Boisbouvier J. Albrand J.P. Blackledge M. Jaquinod M. Schweitz H. Lazdunzki M. Marion D. A structural homologue of colipase in black namba venom revealed by NMR floating disulphide bridge analysis.J. Mol. Biol. 1998; 283: 205-219Crossref PubMed Scopus (64) Google Scholar). The tips of the fingers contain the binding site to a lipolytic interface. Colipase binds to the noncatalytic C-terminal domain of pancreatic lipase and exposes the hydrophobic tips of its fingers at the opposite side of its hydrophilic lipase binding site. These hydrophobic tips probably help to bring the catalytic N-terminal domain of pancreatic lipase into close contact with the interface, where a drastic change occurs in the conformation of the lid domain, a surface loop controlling the access of the substrate to the active site, which pops open. As a result of this structural reorganization, the N-terminal part of colipase binds to the lid domain, forming a second lipase-colipase interaction site. The open lid and the extremities of the colipase fingers, as well as the β9 loop (19Withers-Martinez C. Carrière F. Verger R. Bourgeois D. Cambillau C. A pancreatic lipase with a phospholipase A1 activity: crystal structure of a chimeric pancreatic lipase-related protein 2 from guinea pig.Structure. 1996; 4: 1363-1374Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), form an impressive continuous hydrophobic plateau extending over >50 Å that might be able to interact strongly with a lipid-water interface. The interaction with the open lid suggested a second role of colipase: stabilization of an active (open) lipase conformation (20Lowe M.E. Colipase stabilizes the lid domain of pancreatic triglyceride lipase.J. Biol. Chem. 1997; 272: 9-12Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Other studies have investigated the interaction between colipase and the C-terminal domain of lipase and the relative importance of the putative lipase binding residues in the hydrophilic surface of colipase. Jennens and Lowe (21Jennens M.L. Lowe M.E. The C-terminal domain of human pancreatic lipase is required for stability and full activity but not colipase reactivation.J. Lipid Res. 1995; 36: 1029-1036Abstract Full Text PDF PubMed Google Scholar) demonstrated that deletion of the C-terminal domain greatly decreased lipase activity, but the truncated lipase still required colipase for activity in the presence of bile acids. These results raised questions about the importance of the interactions between colipase and the C-terminal domain of lipase. Later, Ayvazian et al. (22Ayvazian L. Crenon I. Hermoso J. Pignol D. Chapus C. Kerfelec B. Ion pairing between lipase and colipase plays a critical role in catalysis.J. Biol. Chem. 1998; 273: 33604-33609Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) demonstrated that disruption of the interaction between glutamate 45 of colipase and lysine 400 of the lipase C-terminal domain impaired lipolytic activity secondary to an inability of the colipase mutant to bind lipase. Furthermore, Crandall and Lowe (23Crandall W.V. Lowe M.E. Colipase residues Glu64 and Arg65 are essential for normal lipase mediated fat digestion in the presence of bile salt micelles.J. Biol. Chem. 2001; 275: 12505-12512Abstract Full Text Full Text PDF Scopus (23) Google Scholar) have suggested that the hydrophilic surface of colipase interacts with the lipase in solution to form an active lipase-colipase complex. This complex formation was shown to be influenced by bile salt micelles and to require the glutamate 64-arginine 65 colipase binding region. Several studies have provided evidence that no difference can be observed among mammals in the activation of a pancreatic lipase from one species by colipase from another species when emulsified triolein or tributyrin (TC4) is used as a substrate (24Rathelot J. Julien R. Bosc-Bierne I. Gargouri Y. Canioni P. Sarda L. Horse pancreatic lipase. Interaction with colipase from various species.Biochimie. 1981; 63: 227-234Crossref PubMed Scopus (59) Google Scholar, 25Sternby B. Borgstrom B. Comparative studies on the ability of pancreatic colipases to restore activity of lipases from different species.Comp. Biochem. Physiol. 1981; 68B: 15-18Google Scholar). Pancreatic lipase and colipase were purified from chicken (11Bosc-Bierne I. Rathelot J. Canioni P. Julien R. Bechis G. Gregoire J. Rochat H. Sarda L. Isolation and partial structural characterization of chicken pancreatic colipase.Biochim. Biophys. Acta. 1981; 667: 225-232Crossref PubMed Scopus (32) Google Scholar, 26Bosc-Bierne I. Rathelot J. Bechis G. Delori P. Sarda L. Evidence for the existence of procolipase in chicken pancreas and pancreatic juice.Biochimie. 1984; 665: 413-416Crossref Scopus (12) Google Scholar, 27Bosc-Bierne I. Rathelot J. Perrot C. Sarda L. Studies on chicken pancreatic lipase and colipase.Biochim. Biophys. Acta. 1984; 794: 65-71Crossref PubMed Scopus (17) Google Scholar) and their biochemical properties studied. Recently, turkey and ostrich pancreatic lipases (TPL and OPL) were purified in our laboratory (28Sayari A. Mejdoub H. Gargouri Y. Characterization of turkey pancreatic lipase.Biochimie. 2000; 82: 153-159Crossref PubMed Scopus (37) Google Scholar, 29Ben Bacha A. Gargouri Y. Ben Ali Y. Miled N. Reinbolt J. Mejdoub H. Purification and biochemical characterization of ostrich pancreatic lipase.Enzyme Microb. Technol. 2005; 37: 309-317Crossref Scopus (20) Google Scholar). The biochemical properties of these enzymes were reported to be similar to those of mammals and chicken. However, the main difference observed was the capacity of TPL to hydrolyze the long-chain triacylglycerols more efficiently than the short-chain triacylglycerols. On the other hand, in contrast to TPL and to mammal pancreatic lipases, OPL hydrolyzes both short- and long-chain triacylglycerols at comparable rates. To gain more information about the bird pancreatic lipase-colipase function, we report here the purification and some biochemical properties of one mammal and two bird colipases. The complete primary structure of ostrich colipase allowed its comparison with the known sequences of mammal colipases. This work also addressed the question of the specificity of interaction of bird colipases with lipases of different origins. Chymotrypsin (treated with l-1-tosylamido 2-lysyl chloromethyketone), trypsin (treated with l-1-tosylamido 2-phenylethyl chloromethyketone), thermolysin, proteinase V8, TC4 (99% pure), and benzamidine were from Fluka (Buchs, Switzerland). BSA, sodium deoxycholate (NaDC), sodium taurodeoxycholate, and Triton X-100 were from Sigma Chemical (St. Louis, MO). Gum arabic was from Mayaud Baker, Ltd. (Dagenham, UK). Acrylamide and bis-acrylamide (electrophoresis grade) were from BDH (Poole, UK). Marker proteins and supports of chromatography used for colipase purification, Sephadex G-50, Mono S, and Mono Q, were from Pharmacia (Uppsala, Sweden). Polyvinylidene difluoride membranes and the Procise 492 protein sequencer equipped with the 140 C HPLC system were purchased from Applied Biosystems (Roissy, France). The C-8 reverse-phase Eurospher 100 column was from Knauer. pH-stat was from Metrohm (Herisau, Switzerland). Pancreases from different species were collected immediately after slaughter and kept at −20°C. All pancreases were collected from a local slaughterhouse (Sfax, Tunisia) except ostrich pancreases (Nabeul, Tunisia). After decongelation, pancreases were cut into small pieces (1–2 cm2) and delipidated according to the method described previously (30Verger R. De Haas G.H. Sarda L. Desnuelle P. Purification from porcine pancreas of two molecular species with lipase activity.Biochim. Biophys. Acta. 1969; 188: 272-282Crossref PubMed Scopus (204) Google Scholar). After delipidation, ∼15 g of delipidated powder of each pancreas was obtained from 100 g of fresh tissue. Lipase activity was measured titrimetrically at pH 8.5 and 37°C with a pH-stat, under the standard assay conditions described previously, using olive oil emulsion (31Gargouri Y. Cudrey C. Mejboub H. Verger R. Inactivation of human pancreatic lipase by 5-dodecyldithio-2-nitrobenzoic acid.Eur. J. Biochem. 1992; 204: 1063-1067Crossref PubMed Scopus (34) Google Scholar) or TC4 (0.25 ml) in 30 ml of 2.5 mM Tris-HCl and 1 mM CaCl2, pH 8.5 (32Rathelot 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. 1975; 57: 1117-1122Crossref PubMed Scopus (139) Google Scholar), as substrate. Some lipase assays were performed in the presence or absence of NaDC and colipase. One lipase unit corresponds to 1 μmol of fatty acid liberated per minute. Colipase activity was measured at pH 8.5 and 37°C as described by Rathelot et al. (32Rathelot 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. 1975; 57: 1117-1122Crossref PubMed Scopus (139) Google Scholar). One colipase unit corresponds to the amount of cofactor that increases bile salt-inhibited pancreatic lipase activity by 1 enzyme unit. OPL (25Sternby B. Borgstrom B. Comparative studies on the ability of pancreatic colipases to restore activity of lipases from different species.Comp. Biochem. Physiol. 1981; 68B: 15-18Google Scholar), dromedary pancreatic lipase (33Mejdoub H. Reinbolt J. Gargouri Y. Dromedary pancreatic lipase. Purification and structural properties.Biochim. Biophys. Acta. 1994; 1213: 119-126Crossref PubMed Scopus (23) Google Scholar), and colipase from pig (8Erlanson C. Borgstrom B. Purification and further characterization of colipase from porcine pancreas.Biochim. Biophys. Acta. 1972; 271: 400-412Crossref PubMed Scopus (57) Google Scholar) were prepared in our laboratory as described previously. Chicken pancreatic lipase and TPL were purified according to previous works (28Sayari A. Mejdoub H. Gargouri Y. Characterization of turkey pancreatic lipase.Biochimie. 2000; 82: 153-159Crossref PubMed Scopus (37) Google Scholar, 34Fendri A. Frikha F. Mosbah H. Miled N. Zouari N. Ben Bacha A. Sayari A. Mejdoub H. Gargouri Y. Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase.Arch. Biochem. Biophys. 2006; 451: 149-159Crossref PubMed Scopus (20) Google Scholar). Recombinant human pancreatic lipase (HPL) was a generous gift from Dr. R. Verger (Centre National de la Recherche Scientifique, Marseille, France). The activated form of the pig cofactor was obtained by limited trypsinolysis under controlled conditions (14Rathelot J. Canioni P. Bosc-Bierne I. Sarda L. Kamoun A. Kaptein R. Cozzone P.J. Limited trypsinolysis of porcine and equine colipases. Spectroscopic and kinetic studies.Biochim. Biophys. Acta. 1981; 671: 155-163Crossref PubMed Scopus (36) Google Scholar). Protein concentration was determined as described by Bradford (35Bradford M.M. A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217399) Google Scholar) using BSA (E1cm1% = 6.7) as a reference. Ostrich, turkey, and dromedary pancreatic colipases were purified under the same conditions. Twenty grams of delipidated powder of ostrich, turkey, or dromedary pancreas was suspended in 300 ml of water containing 2 mM benzamidine, 150 mM NaCl, and 0.2% Triton X-100 (v/v) and ground mechanically twice for 30 s at 4°C using the Waring Blendor system. The mixture was stirred with a magnetic bar for 30 min at 4°C and then centrifuged for 30 min at 12,000 rpm. To inactivate the lipase, the supernatant was incubated for 5 min at 70°C in the case of ostrich and dromedary colipases. After rapid cooling, insoluble material was removed by centrifugation for 30 min at 12,000 rpm. Afterward, the pH of the previous supernatant was brought to 3.0 by adding 6 N HCl under gentle stirring at 0°C. After centrifugation (30 min at 12,000 rpm), the clear supernatant was adjusted to pH 7 with 6 N NaOH. In the case of turkey colipase, the pH of the homogenate was brought to 1.5 by adding 6 N HCl under gentle stirring at 0°C and incubated for 5 min. We obtained a clear supernatant after centrifugation for 20 min at 12,000 rpm, which contained 20,000 colipase units per gram of delipidated pancreatic tissue. The results show that the bird pancreases presented higher colipase content than the mammal pancreases. The highest level was observed with turkey (20,000 colipase units per gram of delipidated pancreas). Ostrich presented only 10,000 colipase units per gram of delipidated powder, and dromedary presented the lowest level: 1,100 colipase units. Pancreatic extracts from ostrich, turkey, and dromedary pancreases containing 200,000, 400,000, and 22,000 colipase units, respectively, were brought to 60% saturation with solid ammonium sulfate under stirring conditions and maintained for 30 min at 4°C. After centrifugation (30 min, 12,000 rpm), precipitates were resuspended in a minimum of extraction solution (water containing 2 mM benzamidine, 150 mM NaCl, and 0.2% Triton X-100). Insoluble proteins were discarded by centrifugation (15 min, 12,000 rpm). Preparations of colipases contained between 70% and 80% of the starting amount of colipase. Supernatants issued from ammonium sulfate precipitation were subjected to fractionation using ethanol. We added an equal volume of ethanol at 0°C. Insoluble proteins were removed by centrifugation, and the ethanol (4 v/v) was added slowly to the supernatant, bringing the solvent concentration to 90% (v/v) at 0°C. Precipitated proteins, which contained ∼60% of the starting amount of colipase, were collected and solubilized in minimum of 10 mM acetate buffer, pH 4.5, containing 0.05% Triton X-100 and 2 mM benzamidine (buffer A). In this study, we found this step critical to eliminate the last traces of lipids, facilitating the filtration chromatography step. The colipase sample was submitted to gel filtration through a Sephadex G-50 column (95 cm × 2.6 cm) equilibrated with buffer A. Elution of proteins was performed with the same buffer at 30 ml/h. The fractions containing the colipase activity eluted between 1.4 and 2 void volumes were pooled. Active fractions eluted from Sephadex G-50 were subjected to cation-exchange chromatography using a Mono S column (2.6 cm × 20 cm) equilibrated with 10 mM acetate buffer, pH 4.5, containing 0.05% Triton X-100 (buffer B). Nonbound proteins were washed out with 200 ml of buffer B. After a wash with 100 ml of 0.1 M NaCl in buffer B, elution was performed with a linear gradient of NaCl (0.1–0.4 M). Ostrich colipase activity emerged in a single peak at a NaCl concentration of ∼180 mM (Fig. 1A ). Similar results were obtained with turkey and dromedary cofactors (data not shown). The colipase-containing fractions were pooled. Benzamidine was added at a final concentration of 2 mM, and then the fractions were lyophilized for the purpose of concentration. The recovery of colipase activity after the Mono S step was ∼75% for all purified colipases. The purified colipases were dissolved in ∼10 ml of 10 mM Tris-HCl buffer, pH 8.2, containing 10 mM NaCl (buffer C). Each preparation was filtered through a column (2.6 cm × 100 cm) of Sephadex G-50 equilibrated in buffer C. The active fractions were pooled and then deposited on a Mono Q Sepharose column (1.5 cm × 10 cm) equilibrated in the same buffer. After washing with buffer C until the eluent was free of proteins, elution of fixed proteins was carried out using a linear gradient of NaCl from 10 to 250 mM in buffer C. Each colipase was eluted in a single peak corresponding to a NaCl concentration of ∼100 mM (Fig. 1B). Pure colipases were lyophilized and conserved at −20°C. The presence of glycan chains in the purified cofactors was checked by the anthrone-sulfuric acid method using glucose as a standard (36Spiro R. Analysis of sugar found in glycoproteins.Methods Enzymol. 1966; 256: 3-26Crossref Scopus (953) Google Scholar). The alkylation of Cys residues of colipase was performed using the technique of Okazaki, Yamada, and Imoto (37Okazaki K. Yamada H. Imoto T. A convenient S-2 aminoethylation of cysteinyl residues in reduced proteins.Anal. Biochem. 1985; 149: 516-520Crossref PubMed Scopus (37) Google Scholar). One milligram of cofactor in 1 ml of 10 mM Tris-HCl and 10 mM NaCl, pH 8.2, was denaturated in 375 μl of 8 M guanidine hydrochloride, 125 μl of 1 M Tris-HCl, 4 mM EDTA, pH 8.5, and 80 mM DTT during 30 min at 60°C. S-Pyridylethylation of cysteine residues of protein was performed by adding 4 μl of vinyl pyridine during 3 h at 25°C. The modified colipase was dialyzed against water for N-terminal sequencing. Purified ostrich pancreatic colipase was denaturated, reduced, and carboxymethylated as described previously and dialyzed against 50 mM Tris-HCl buffer, pH 8.5, without benzamidine. The cofactor solution (1 mg/ml) was digested at 37°C with the selected endopeptidase. The endopeptidase/cofactor varied from 1% to 10% (w/w). The samples (20 μl) were withdrawn from the incubation mixture at various times. The reaction was stopped by the addition of acetic acid (20% final concentration). For V8 digestion, some assays were performed with the denaturated cofactor solution (1 mg/ml) in 50 mM sodium acetate buffer, pH 5.2. The resulting peptides from enzymatic cleavage were then separated by chromatography on a C-8 reverse-phase column (250 mm × 4.6 mm). Elution was carried out with a gradient from 0 to 80% solvent B for 30 min at a flow rate of 0.6 ml/min (solvent A, 0.01% trifluoroacetic acid in water; solvent B, acetonitrile). Analytical SDS-PAGE was performed by the method of Laemmli (38Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207487) Google Scholar). The proteins were stained with either Coomassie Brilliant Blue or silver nitrate. Samples for sequencing were electroblotted according to Bergman and Jörnvall (39Bergman T. Jörnvall H. Electroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis.Eur. J. Biochem. 1987; 169: 9-12Crossref PubMed Scopus (73) Google Scholar). Protein transfer was performed during 1 h at 1 mA/cm2 at room temperature. The molecular masses of purified colipases were determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The N-terminal sequences of colipases and peptides were determined by automated Edman degradation, using an Applied Biosystems Procise 492 protein sequencer equipped with the 140 C HPLC system (40Hewick R.M. Hunkapiller M.W. Hood L.E. Dreyer W.J. A gas-liquid solid phase peptide and protein sequenator.J. Biol. Chem. 1981; 256: 7990-7997Abstract Full Text PDF PubMed Google Scholar). The structure of the ostrich colipase was modeled using the three-dimensional coordinates of the open form of the HPL/colipase complex (Protein Data Base code 1lpa) as template using the structure-modeling program Deep View/Swiss-PDB Viewer version 3.7 (SP5) (http://www.expasy.org/spdbv/). The model was then subjected to energy minimization using the GROMOS96 software implementation of Swiss-Pdb Viewer http://iqc.ethz.ch/gromos). Five cycles of minimization were performed (5,000 steps of steepest descent, 5,000 steps of conjugate gradients, 5,000 steps of steepest descent, cutoff at 30 Å) using a harmonic constraint and a cutoff of 30 Å. The geometry of the final models was stereochemically analyzed using the PROCHECK program (41Laskowski R.A. Mac Arthur M.W. Moss D.S. Thornton J.M. PROCHECK: a program to check the stereochemical quality of protein structures.J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The accessible surfaces of the two models were calculated with Surface Racer, a computer program for fast calculation of accessible and molecular surface areas (42Tsodicov Jr., O.V. Record M.T. Sergeev Y.V. Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature.J. Comput. Chem. 2002; 23: 600-609Crossref PubMed Scopus (351) Google Scholar). Three colipases were purified from the pancreases of two birds (ostrich and turkey) and one mammal (dromedary) according to the procedure described in Materials and Methods. The results of SDS-PAGE analysis of the colipases eluted from the Mono Q column (Fig. 1C) showed that each of the three purified cofactors exhibited one band corresponding to a molecular mass of ∼10 kDa. This was in agreement with molecular mass estimation using a gel filtration Superose 12 column by fast-protein liquid chromatography. On the other hand, molecular masses of 9,593.03, 9,138.12, and 9,245.23 Da were determined by mass spectrometry for ostrich, turkey, and dromedary pancreatic colipases, respectively (data not shown). Altogether, these results suggest that bird colipases are monomeric proteins, as was found for mammal colipases. The presence of glycan chains in pure colipase molecules was checked. Our results showed that the three purified proteins are not glycosylated (data not shown). The purification flow given in Table 1 shows that the specific activity of pure proteins reached 13,000, 11,000, and 10,000 U/mg for turkey, ostrich, and dromedary colipases, respectively, when olive oil emulsion was used as a substrate at pH 8.5 and 37°C and in the presence of 6 mM NaDC.TABLE 1Flow sheet of ostrich, turkey, and dromedary pancreatic colipase purificationPurification StepSpeciesTotal Activity (units)Protein (mg)Specific Activity (U/mg)Activity Recovery (%)Purification FactorHeat and/or acidic treatmentOstrich200,0001,091183.331001Turkey400,0002,046.04195.5Dromedary22,0002,00011(NH4)2SO4 precipitation (60%)Ostrich165,00060027582.51.5Turkey336,0001,518.3221.3841.13Dromedary16,5001