Isolation and partial characterization of the pancreatic secretory trypsin inhibitor in the rat

Isolation in a 55% yield of the low molecular weight pancreatic secretory trypsin inhibitor was achieved by gel filtration of an acid extract of whole inactive rat pancreas juice on Sephadex G-50 at pH 2.5 followed by desalting and ion-exchange chromatography on SP Sephadex C-50 at pH 4.5. Two distinct chromatographic fractions were obtained, labeled fraction 1 and 2. Fractions 1 and 2 showed three, respectively two, distinct closely migrating cationic bands on gel electrophoresis in barbital buffer, pH 8.6. Each fraction demonstrated one band on polyacrylamide disc electrophoresis at pH 4.6. The inhibitor is homogenous on gel filtration and on the basis of its stoichiometry with active site titrated rat anionic trypsin. Its molecular weight is approx. 6024. The amino acid composition is included. Rat pancreatic secretory trypsin inhibitor is trypsin-specific and interacts on a 1:1 molar basis with rat trypsin. It is good inhibitor of bovine trypsin but a poor inhibitor of human cationic trypsin and its binding to trypsin is reversible by acidification. Like other inhibitors of this sort, it is present in about 0.1–0.2% of the total protein content of the juice, and normally exists in its free form. A simple procedure for the production of antiserum to the inhibitor which is a poor antigen is also described.     

Quantitative proteomic profiling of pancreatic cancer juice

Pancreatic juice is an exceptionally rich source of cancer-specific proteins shed from cancerous ductal cells into the pancreatic juice. Quantitative proteomic analysis of the proteins specific to pancreatic cancer juice has not previously been reported. We used isotope-code affinity tag (ICAT) technology and MS/MS to perform quantitative protein profiling of pancreatic juice from pancreatic cancer patients and normal controls. ICAT technology coupled with MS/MS allows the systematic study of the proteome and measures the protein abundance in pancreatic juice with the potential for development of biomarkers. A total of 105 proteins were identified and quantified in the pancreatic juice from a pancreatic cancer patient, of which 30 proteins showed abundance changes of at least twofold in pancreatic cancer juice compared to normal controls. Many of these proteins have been externally validated. This is the first comprehensive study of the pancreatic juice proteome by quantitative global protein profiling, and the study reveals numerous proteins that are shown for the first time to be associated with pancreatic cancer, providing candidates for diagnostic biomarkers. One of the identified proteins, insulin-like growth factor binding protein-2 was further validated by Western blotting to be elevated in pancreatic cancer juice and overexpressed in pancreatic cancer tissue.     

Separation of the proteins present in pancreatic juice using hydrophobic interaction chromatography

We have previously described a method for separating pancreatic juice proteins by reversed-phase HPLC. However, the solvents used in such a system denature the proteins, whose characterization therefore depends solely on molecular weight determinations. We have therefore tested the suitability of hydrophobic interaction chromatography as an alternative method of separation. Using a TSK phenyl 5PW column with a decreasing, four-stage ammonium sulfate concentration gradient, it was possible to separate the major proteins in rat pancreatic juice. The identity of each protein was confirmed by measuring its molecular weight and by assaying its enzyme activity. Hydrophobic interaction chromatography represents an improved system for separating pancreatic secretory enzymes in active form.     

Proteolysis of human trypsinogen 1. Pathogenic implication in chronic pancreatitis

SDS electrophoresis on polyacrylamide gels of purified trypsinogen 1 has shown the occurence of a proteolysis in some molecules during long storage at ?20°C. This proteolyzed trypsinogen gives a positive reaction with an antiserum directed against the precipitate protein, major protein of about 14 000 molecular weight extracted from precipitates present in the pancreatic juice of patients with chronic pancreatitis. The autoactivation of proteolyzed trypsinogen 1 liberates a polypeptide of 14 000 molecular weight which is immunologically identical to the precipitate protein. These results show that the major protein present in pancreatic precipitates (and pancreatic stones) of patients with chronic pancreatitis is a degradation product of trypsinogen 1 liberated by a proteolysis which necessarily requires a premature zymogen activation in the disease.     

Stimulatory effect of an endogenous peptide in rat pancreatic juice on pancreatic enzyme secretion in the presence of atropine: evidence for different mode of action of stimulation from exogenous trypsin inhibitors

A new factor which activated the secretion of pancreatic enzymes was discovered and purified from rat bile-pancreatic juice. A fraction below M.W.10,000 of rat bile-pancreatic juice enhanced trypsinogen secretion by injection into anesthetized rat duodenum. The factor was purified from this fraction using its biological activity as an index by Sephadex G-50, SP Sephadex C-50 and HPLC. This factor was a peptide of which molecular weight was about 6,000 and had trypsin inhibitory activity. From these and some other findings, it was suggested that the peptide was identical with the "Kazal type" inhibitor. In the anesthetized and atropine-treated rat, of which intestinal trypsin was removed by thoroughly washing with saline containing 5 microM soybean trypsin inhibitor (SBTI), pancreatic secretion became basal state, and was not stimulated by injection of SBTI into its duodenum any longer. Under this condition, however, injection of this purified peptide brought about markedly stimulation of pancreatic enzyme secretion. These results suggest that this peptide has a certain function which enhances pancreatic enzyme secretion by the different manner from exogenous trypsin inhibitors such as SBTI.     

Monoclonal antibody analysis of human pancreatic juice components related to the pancreatic calculi protein

A CaCO3-crystal growth inhibitor has recently been isolated from the calculi of patients affected by pancreatic lithiasis. It is a phosphoglycoprotein, with a molecular weight of 14,000, whose probable physiological role is the stabilization of exocrine pancreatic secretion which is supersaturated with respect to CaCO3. In order to isolate this inhibitor from human pancreatic juice, monoclonal antibodies to the protein were prepared and an immunoadsorbent column was developed. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of proteins fixed by the immunoadsorbent reveals the form having a molecular weight of 14,000, in addition to other protein bands which have higher molecular weights (16,000, 16,800, 18,000, and 18,800). All of these different proteins are also recognized by a monospecific polyclonal antibody to the 14,000 molecular weight form. Using the same monospecific polyclonal antibody only one messenger RNA coding for this inhibitor has been demonstrated. Thus, this heterogeneity might be explained by post-translational modifications.     

Purification of pancreatic phospholipase A2 from human duodenal juice

Phospholipase A2 (EC 3.1.1.4) was purified from delipidated human duodenal juice by hydrophobic and cation exchange chromatography, followed by molecular sieving on an HPLC column. The resulting enzyme preparation of phospholipase A2 had a molecular weight of 14 kDa, a specific activity of 2000 U/mg protein, and an N-terminal amino acid sequence which was characteristic for human pancreatic phospholipase A2.     

Effect of diversion of bile-pancreatic juice to the ileum on pancreatic secretion and adaptation in the rat

Cannulas were implanted to collect bile and pancreatic juice, and the collected secretions were pumped back into the intestine at the level of the duodenum or the proximal ileum. The effect of 6 days of such treatment on pancreatic secretion and on pancreatic growth was determined. The effect on pancreatic secretion was studied by measuring the pancreatic secretory response to a stimulus, provided by acute diversion of bile-pancreatic juice from the proximal intestine. Trophic effects were studied in a separate group of rats by measuring pancreatic weight, protein content, and chymotrypsin activity after an overnight fast. Stimulated pancreatic secretion was 2.1 times greater for protein output and 3.4 times greater for fluid output in rats with chronic diversion of bile-pancreatic juice to the ileum. Pancreatic weight, protein content, and chymotrypsin activity were increased 2.6, 2.9, and 4.8 times, respectively, by chronic diversion of bile-pancreatic juice to the ileum. These results indicate that pancreatic hypertrophy and hyperplasia reported in rats with bile-pancreatic duct transposition to the ileum are the result of loss of feed-back inhibition from bile-pancreatic juice in the proximal intestine.     

Molecular distribution of zinc in biliary and pancreatic secretions

Rat bile and pancreatic fluid were examined for the presence of low molecular weight zinc complexes. Fluids were collected separately by cannulation, and zinc distribution in collected samples was analyzed by gel filtration on Sephadex G-50. Most of the zinc in bile was associated with low molecular weight zinc complexes; only a small amount of zinc was present in the high molecular weight fraction. In contrast, pancreatic secretions did not contain low molecular weight zinc complexes, but there were considerable amounts of zinc bound to high molecular weight compounds. The addition of zinc to bile resulted in an increased amount of zinc in the low molecular weight fraction, while the addition of zinc to pancreatic fluid resulted primarily in an increase in zinc bound to the high molecular weight components. Like pancreatic fluid, homogenates of pancreatic tissue had no low molecular weight zinc complex. In rats whose bile and pancreatic fluid were removed and not returned into the intestine, the amount of zinc bound to low molecular weight complexes in intestinal homogenates was reduced. This alteration of the molecular distribution of zinc in intestinal homogenates by removal of bile and pancreatic fluid suggests the potential importance of low molecular weight zinc complexes for zinc homeostasis.     

Physiological and chemical characteristics of antibacterial activity of pancreatic juice.

Attempts were made to find and characterize an antibacterial activity (ABA) factor in porcine pancreatic juice (PJ). Its isolation requires several steps. Since ABA factor was found to be heat resistant, the first step was heating for 30 min at 65 degrees C. Afterwards column chromatography, ethanol precipitation and polyacrylamide gel electrophoresis were involved. Finally, we obtained a pancreatic juice fraction with antibacterial activity against Escherichia coli strain AB1157. In the presence of this fraction the number of living bacterial cells in overnight culture decreased about 10,000 fold and a spot-test gave clearly positive results. The results of analysis suggest that the antibacterial factor is a polypeptide active in a pH range 8.0-8.5, that migrates in polyacrylamide gel electrophoresis as a band under 14,000 Da. Mass spectroscopy analysis of active fraction showed high concentration of porcine pancreatic spasmolytic polypeptide (PSP). In conclusion, a polypeptide controlling bacterial homeostasis has been found in the porcine pancreatic juice.     

Exocrine pancreatic secretion of phospholipid, menaquinone-4, and caveolin-1 in vivo

The exocrine pancreas releases secretory products essential for nutrient assimilation. In addition to digestive enzymes, the release of lipoprotein-like particles containing the membrane trafficking protein caveolin-1 from isolated pancreatic explants has been reported. The present study examined: (1) if gastrointestinal hormones induce the apical secretion of phospholipid in vivo and (2) a potential association of caveolin-1 and the lipid-soluble vitamin K analog menaquinone-4 (MK-4) with these structures. Analysis of isolated acinar cells, purified zymogen granules, and pancreatic juice collected in vivo indicated the presence a caveolin-1 immunoreactive protein that was acutely released in response hormone stimulation. Chloroform-extracted fractions of pancreatic juice also contained high concentrations of MK-4 that was secreted in parallel to protein and phospholipid. The presence of caveolin-1 and MK-4 in the phospholipid fraction of pancreatic juice places these molecules in the secretory pathway of exocrine cells and suggests a physiological role in digestive enzyme synthesis and/or processing.     

Purification and properties of myosin from the "hatching muscle" (m. complexus) of geese

The present work is concerned with the study of myosin fractions prepared from the hatching muscle (m. complexus) and a control muscle (m. pectoralis) of the developing goose embryo. The m. complexus attained its maximum mass at hatching and in the 4-day-old bird the mass of this muscle was only one fourth of that recorded at hatching. The m. complexus was hypertrophied already on the 21st day. At days 21, 27 and 28 of incubation and at posthatching days myosin preparations were made from both muscles. Partial purification of myosins from both sources yielded a high molecular weight fraction characteristic of the adult bird and one other protein fraction with molecular mass half of myosin. Both preparations exhibited the characteristic properties of myosin. The lower molecular weight fraction was also shown to develop filamentous aggregates as did the higher molecular-weight, gel filtrated myosin. The phosphate content of the half molecular mass myosin fraction prepared from the embryonic m. complexus at days prior to hatching was considerably higher than that of the high molecular weight fraction and the predominant component was P-Arg. Since the embryonic myosin was still not available in the m. complexus of the 4-day-old birds and the hypertrophied muscle underwent regression after hatching it appears that this myosin fraction is actively involved in breaking through the shell during the hatching period in geese.     

Characterization of two glycoproteins of human pancreatic juice: P35, a truncated protease E and P19, precursor of protein X

Four glycoproteins were separated by SDS-polyacrylamide gel electrophoresis of proteins of human pancreatic juice devoid of free proteolytic activity. The two low molecular weight glycoproteins were isolated and characterized. Protein P19, the precursor family of protein X, was analyzed by its carbohydrate content which seemed to play an important role in protein solubility at pH 8.0. Protein P35 was found to be a Con A-binding protein rich in mannose. Its N-terminal amino acid sequence covering 33 residues revealed a strong homology with human protease E without the dipeptide Val-Val. Is P35 a protein homologous to the subunit III of bovine procarboxypeptidase A?     

Identity of a cytosolic neutral cholesterol esterase in rat liver with the bile salt stimulated cholesterol esterase in pancreas

A neutral cholesterol esterase has been purified to homogeneity from the cytosolic fraction of rat liver. The 105,000 x g supernatant fraction of rat liver was applied to a DEAE-cellulose column to isolate a partially purified fraction of hepatic cholesterol esterase. Immunoblot analysis of the partially purified liver fraction with the anti-porcine pancreatic cholesterol esterase IgG demonstrated a single band with a molecular weight of 67,000. The hepatic protein was then isolated by immunoaffinity chromatography technique using a column constructed with antibodies prepared against the pancreatic cholesterol esterase. Characterization of the hepatic cholesterol esterase revealed that the hepatic enzyme shared antigenic epitopes with the pancreatic cholesterol esterase and was similarly activated by addition of bile salt such as taurocholate. Moreover, amino-terminal sequencing analysis of the hepatic cholesterol esterase showed an identical sequence with the pancreatic enzyme. Taken together, these results showed that the cholesterol esterases in the liver and the pancreas are very similar and possibly identical proteins.     

Soluble high molecular weight derivatives of pancreatic trypsin inhibitor. Effect of electrostatic interaction of protein with the carrier on covalent binding and some physico-chemical properties of modified preparations of pancreatic inhibitor

Modification of pancreatic trypsin inhibitor by carboxymethyl dextran with the aid of carboxyl groups of the carrier activated by water-soluble carbodiimide has been carried out. A special role of electrostatic interactions of protein with the carrier during inhibitor modification has been shown. This modification results in active preparations with high protein concentration on the carrier; their molecular weight is practically equal to that of previously used carrier. A comparison of the thermal stability of high molecular weight preparations of pancreatic inhibitor with respect to protein concentration on the carrier and the number of links between protein and carrier has been carried out.     

Purification of classical pancreatic lipase from dog pancreas

The purification of canine classical pancreatic lipase from canine pancreatic juice, but not from pancreatic tissue, has been reported previously. Given the logistic difficulties associated with collection of pancreatic juice in dogs and efforts to minimize experiments in live animals the objective of this project was to purify canine classical pancreatic lipase from dog pancreas. Dog pancreata were collected from research dogs that had been sacrificed for unrelated research projects. Pancreatic tissue was delipidated using organic solvents. The delipidated pancreatic extract was further purified by extracting the enzymes in a Tris-buffer containing two different protease inhibitors, benzamindine and phenylmethylsulfonyl fluoride (PMSF), followed by anion exchange chromatography, gel-filtration, and cation exchange chromatography. The purified protein showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of approximately 50.7. Isoelectric focusing showed isoelectric points ranging from 6.0 to 6.2. N-terminal amino acid sequencing of the first 25 amino acid residues showed the sequence Lys-Glu-Val-X-Phe-Pro-Arg-Leu-Gly-X-Phe-Ser-Asp-Asp-Ser-Pro-Trp-Ala-Gly-Ile-Val-Glu-Arg-Pro-Leu. This sequence showed close homology with classical pancreatic lipase in pigs, horses, and human beings. We conclude that canine classical pancreatic lipase can be successfully purified from canine pancreatic tissue.     

Comparative proteomic analysis of human pancreatic juice: methodological study

Pancreatic cancer is the most lethal of all the common malignancies. Markers for early detection of this disease are urgently needed. Here, we optimized and applied a proteome analysis of human pancreatic juice to identify biomarkers for pancreatic cancer. Pancreatic juice samples, devoid of blood or bile contamination, were collected from patients with pancreatic cancer (n = 5), benign pancreatic diseases (n = 6), or cholelithiasis (n = 3) during endoscopic retrograde cholangiopancreatography (ERCP). After ultramembrane centrifugation sample preparation, pancreatic juice proteins were separated by 2-DE and identified by MALDI-TOF-MS. A 2-DE dataset of pancreatic juice from patients with cholelithiasis was established, consisting of 76 protein spots representing 22 different proteins. Disease-associated obstruction of the pancreatic duct strongly effected the protein composition of pancreatic juice. Concurrently, pancreatic juice from patients with pancreatic cancer was compared to nonmalignant controls with comparable obstruction of pancreatic ducts. Seven protein spots were identified that consistently appeared at changed levels in pancreatic juice from patients with pancreatic cancer. In conclusion, comparative proteomic analysis of human pancreatic juice can be used to identify biomarkers of pancreatic cancer.     

Analysis of a 5S RNA-protein complex isolated from the ribosomes of rye embryos

Rye embryo ribosomes were dissociated into subunits and the large subunit fraction was treated with formamide. A low molecular weight complex of RNA and protein (RNP) was released. Electrophoresis of the RNP in polyacrylamide gels containing sodium dodecyl sulphate yielded an RNA band and a single protein band. The protein had a molecular weight of approximately 41 000 and the RNA of the complex was shown to be 5S ribosomal RNA. Embryos were germinated in the presence of [32P]orthophosphate and the labelled RNP was isolated from their ribosomes. The RNA component was partially digested with pancreatic A ribonuclease and the parts protected from degradation by the protein were determined by sequence analysis. Although the whole 5S RNA molecule was shielded to some extent, the portion most protected was between nucleotides 68 and 108. This is, therefore, probably the part of plant cytosol 5S RNA which is primarily involved in the interaction with protein in the complex and possibly in the ribosome as well.     

Isolation and partial characterization of a soluble mucin in human pancreatic juice.

Morphological and histochemical abnormalities in pancreatic mucin occur in many pancreatic disorders. However, the composition of pancreatic mucin is poorly understood. Purified mucin was isolated from pure pancreatic juice by sequential chromatography on Sepharose CL-2B and CL-4B followed by CsCl density gradient ultracentrifugation. The mucin preparation consists of 24% protein and 73% carbohydrate. Reduction of the macromolecule (greater than 2 x 10(6)) by mercaptoethanol resulted in the formation of subunits of molecular weight 500,000 and released several small molecular weight proteins, including a glycoprotein of an average molecular weight of 116,000. Cellulose acetate electrophoresis separated the mucin into three species of different staining properties for periodic acid-Schiff reagent and Alcian blue, suggesting the presence of microheterogeneity with respect to sulphation and sialation. Threonine, serine, and proline composed 48% of the total amino acids, while the oligosaccharide moiety contained N-acetylglucosamine, N-acetylgalactosamine, fucose, galactose, sialic acid, and sulphate. We also detected the presence of C16:0 and C18:0 fatty acids which were probably noncovalently bound to the pancreatic mucin.     

Evidence of a new serine protease in the rat pure pancreatic juice that degrades somatostatin

Somatostatin (SS) is found in the endocrine pancreas and has been reported in the pure pancreatic juice (PPJ) of different species. Characterization by gel filtration of immunoreactive SS (irSS) in the rat PPJ (rPPJ) results in a single peak corresponding to 23kDa molecular weight. Incubation of the 23kDa fraction with labeled or synthetic SS results in time dependent degradation of both peptides. This degradation is inhibitable by PMSF, calcium and by heat, whereas specific inhibitors of trypsin and chymotrypsin are without effect. These data suggest that irSS previously measured in rPPJ samples by RIA without confirmation of radioactive tracer stability may lead to false positive results. Indeed, our study indicates the presence of a 23kDa enzyme in the rPPJ degrading radiolabeled somatostatin during the RIA procedure. This putative new enzyme found into the rPPJ may thus be partially responsible for the apparent irSS presence.