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.     

Two human trypsinogens. Purification, molecular properties, and N-terminal sequences

The two human trypsinogens have been isolated from human pancreatic juice in a sufficient amount to study molecular and structural properties. The purification procedure included filtration on Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The two trypsinogens represent 19% of total proteins of pancreatic juice. Trypsinogen 1, the major form, is present in a quantity twice that of trypsinogen 2, which is the most anionic protein in human pancreatic juice. The two proteins have partial immunological identity, close molecular weights (23 438 and 25 006 for trypsinogens 1 and 2, respectively) and similar amino acid compositions. The N-terminal sequences are the same for the first 9 residues: Ala-Pro-Phe-Asp4-Lys-Ile. The two proteins differ in the activation peptides released during the transformation to trypsins. Trypsinogen 2 liberates one octapeptide Ala-Pro-Phe-Asp4-Lys while trypsinogen 1 liberates two peptides, the same octapeptide and the pentapeptide (Asp)4-Lys.     

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.     

Identification of two major proteins of bovine pancreatic stones as immunoreactive forms of trypsinogens.

Two major proteins have been identified in sodium citrate extracts of bovine pancreatic stones from 15 glands with lithiasis. They were found to have a molecular weight of about 24 000 and were further characterized by a variety of methods, including polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, isoelectric focusing, two-dimensional electrophoresis, immunodiffusion, immunoelectrophoresis and determination of N-terminal residues. These two immunologically and electrophoretically different proteins were definitely shown to be immunoreactive forms of anionic and cationic trypsinogens, which are normal components of pancreatic juice. However, in contrast with both secretory trypsinogens, the stone proteins displayed an important charge heterogeneity under isoelectric-focusing conditions. A possible role for both secretory trypsinogens in pancreatic lithogenesis is suggested by the reproducibility of the data. Finally, two minor proteins with a lower molecular weight (about 11 000--13 000) have also been found to be present in all extracts, but have not yet been identified.     

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.     

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.     

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.     

Comprehensive proteomic analysis of human pancreatic juice

Proteomic technologies provide an excellent means for analysis of body fluids for cataloging protein constituents and identifying biomarkers for early detection of cancers. The biomarkers currently available for pancreatic cancer, such as CA19-9, lack adequate sensitivity and specificity contributing to late diagnosis of this deadly disease. In this study, we carried out a comprehensive characterization of the "pancreatic juice proteome" in patients with pancreatic adenocarcinoma. Pancreatic juice was first fractionated by 1-dimensional gel electrophoresis and subsequently analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 170 unique proteins were identified including known pancreatic cancer tumor markers (e.g., CEA, MUC1) and proteins overexpressed in pancreatic cancers (e.g., hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) and lipocalin 2). In addition, we identified a number of proteins that have not been previously described in pancreatic juice (e.g., tumor rejection antigen (pg96) and azurocidin). Interestingly, a novel protein that is 85% identical to HIP/PAP was identified, which we have designated as PAP-2. The proteins identified in this study could be directly assessed for their potential as biomarkers for pancreatic cancer by quantitative proteomics methods or immunoassays.     

Lipolytic enzymes of the human pancreas. II. Purification and properties of cholesterol ester hydrolase

Cholesterol ester hydrolase (sterol-ester acylhydrolase, EC 3.1.1.13) was purified from human pancreatic tissue by column chromatography and acetone precipitation, leading to a 400-fold enrichment. Isoelectric focusing of this product reveals a double-band at pH 4.5 and 4.6. The molecular weight was estimated at 320 kDa by means of Sephadex filtration on calibrated columns. Obviously these large molecules represent a tetrameric form of the monomeric subunit (molecular mass 76-80 kDa), which is also enzymatically active. It was found together with the dimeric form in pancreatic juice, where the tetrameric enzyme is responsible for the major part of the hydrolytic activity, splitting cholesterol ester as well as synthetic substrates, such as fluorescein or p-nitrophenyl esters. Attempts to split the tetrameric cholesterol ester hydrolase, isolated from pancreatic tissue, into active subunits found additionally in pancreatic juice by the influence of bile acids and proteolytic enzymes failed. The spectral shift method using Rhodamine fluorescence was employed in order to prove that fluorescein dilaurate forms micellar solutions and mixed micelles when bile salts are present.     

Isolation and convulsivant effect of the "insulin-like" protein obtained from the exocrine pancreas of Bradypus tridactylus L

1. A pancreatic insulin-like protein fraction with low electrophoretic mobility showing high molecular weight is present. 2. Isoelectric focussing studies showed that the high molecular weight protein has a pI about 7.4 when used at a pH range between 3.5 and 8.0. 3. The partially purified aldehyde-fuchsin positive high molecular weight protein fraction gave a positive effect for the convulsivant test in mice. 4. A high molecular weight insulin-like protein in pancreatic juice was found. 5. Insulin was found by radioimmunoassay in the basal and post stimulation pancreatic juice.     

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.     

库克Noni果汁多糖含量及分子量测定

建立测定库克Noni果汁中多糖含量及分子量的方法。通过醇沉法分离Noni果汁多糖,进一步分离纯化得到均一多糖组分;用精制Noni果汁多糖测得该多糖对葡萄糖的换算因子,对多糖含量进行定量测定;并通过SE-HPLC(Size Exclusion-High Performance Liquid Chromatography)法测定了该多糖的相对分子量。结果表明,多糖含量测定方法简便可行,供试液在4 h内显色稳定,重现性较好,平均回收率为99.3%,RSD为1.93%(n=3);测得该多糖的相对分子量为1140 KDa。     

Purification of human lysozyme from milk and pancreatic juice

Human milk lysozyme was purified by heparin-Sepharose affinity chromatography and Sepharose 4B gel-permeation chromatography. This procedure was also found applicable to the purification of human pancreatic juice lysozyme. Double-diffusion analyses indicated that human milk lysozyme was immunochemically identical to human saliva and human pancreatic juice lysozyme. Based on the identity of the N-terminal 10-amino-acid-residue sequence analyzed, it was suggested that human milk lysozyme and human pancreatic juice lysozyme are identical molecular entities.     

Comparison of the proteins in pancreatic juice in normal and diseased humans. Determination of serum proteins and demonstration of a particular protein in chronic calcifying pancreatitis

For a better understanding of the molecular mechanism leading to intraductal precipitation of proteins in primary chronic calcifying pancreatitis in man, we studied the composition of normal and pathological human pancreatic juice by immunotechniques. We found an increased level of serum proteins in pathological juices: 12.47% of total proteins compared to 1.8% in normal ones; albumin is 8.16% of the total proteins, IgG 2.84%, IgA 0.83% and IgM 0.91%. Transferrin and alpha 2-macroglobulin are present, but were not estimated. The albumin/IgA and albumin/IgG ratios favour the hypothesis of a local synthesis of these immunoglobulins as was shown in normal juice. The cross adsorption of antisera against pancreatic juice showed the presence in the pathological juice of a normal molecule in much higher concentration. The role of these proteins in precipitation is discussed.     

Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates

The ubiquitin-proteasome pathway is a major route of degradation of cell proteins. It also plays an essential role in maintaining cell homeostasis by degrading many rate-limiting enzymes and critical regulatory proteins. Alterations in proteasome activity have been implicated in a number of pathologies including Parkinson's disease, Alzheimer's disease and diabetes. The eukaryotic proteasome is a multicatalytic protease characterized by three activities with distinct specificities against peptide substrates. Although substrates were identified which could selectively measure the individual activities in the purified proteasome little data is available on how specific those substrates are for proteasomal activity when used with biological samples which may contain many other active peptidases. Here we examine the three major peptidase activities in lysates of two cell types and in a liver cytosol fraction in the presence of specific proteasome inhibitors and after fractionation by gel permeation chromatography. We demonstrate that other proteinases present in these preparations can degrade the commonly used proteasome substrates under the standard assay conditions. We develop a simple method for separating the proteasome from the lower molecular weight proteases using a 500kDa molecular weight cut-off membrane. This allows proteasome activity to be accurately measured in crude biological samples and may have quite broad applicability. We also identify low molecular weight tryptic activity in both the cell and tissue preparations which could not be inhibited by the proteasome inhibitor epoxomycin but was inhibitable by two cysteine proteinase inhibitors and by lactacystin suggesting that lactacystin may not be completely proteasome specific.     

Detection of two high molecular weight hydrophobic forms of the human estrogen receptor

The human estrogen receptor gene encodes a single protein of molecular weight 65,000 daltons. However, using a sensitive and rapid technique of high-performance hydrophobic interaction chromatography we have detected two distinct estrogen receptor species both of which are high molecular weight proteins (ca. 60A) as determined by high-performance size-exclusion chromatography. These are detected either in the presence or absence of sodium molybdate; rechromatography of individual isoform indicates that the two protein complexes have independent hydrophobic contact points. Consistent elution patterns of the two receptor species indicates they are formed selectively. We conclude that different post-translational modifications of the estrogen receptor protein could allow their specific interaction with non-receptor components resulting in the formation of two distinct high molecular weight complexes which would be rapidly resolved by high-performance hydrophobic interaction chromatography.     

High-performance gel-permeation chromatography of chitosan samples

The conformational properties of chitosan, a copolymer of 2-acetamido-2-deoxy- -glucose and 2-amino-2-deoxy- -glucose, have been examined both in solution and in the solid state. Little has been reported previously on the determination of molecular weight using high-performance gel-permeation chromatography (HPGPC) and no attempt has been devoted to an examination of molecular weight distribution. An HPGPC method for evaluating the above-mentioned parameters for chitosan samples having different molecular weights and different degrees of acetylation was therefore developed. Calibration using sodium polystyrene sulfonate commercial standards of narrow molecular weight distribution could not be carried out in the solvent system used for chitosan. Calibration was therefore performed by means of chitosan samples obtained by depolymerization.     

Chemical differences between seeds and elaiosomes indicate an adaptation to nutritional needs of ants

Ant-dispersed plants usually produce seeds with appendages (elaiosomes) as reward for ants. Plants that produce high-quality elaiosomes benefit because ants preferentially disperse their diaspores. We therefore hypothesized that seeds and elaiosomes differ in chemical composition in ways that make elaiosomes of high nutritional quality for ants, capable of providing essential dietary components that explain the increased fitness and higher gyne production documented for colonies with elaiosome consumption. To test the hypothesis we analysed the content and composition of lipids, amino acids, soluble carbohydrates, proteins and starch in seeds and elaiosomes of 15 central European ant-dispersed plants. After separating the different fractions, total lipids were determined gravimetrically, fatty acids and soluble carbohydrates were detected by gas chromatography (GC) and GC–mass spectrometry, free amino acids by an amino acid analyser while starch and protein were analysed photometrically. Seeds accumulated high molecular weight compounds such as proteins and starch, whereas elaiosomes accumulated more easily digestible low molecular weight compounds such as amino acids and monosaccharides. Analysis of similarities and similarity percentages analysis demonstrated that the composition of fatty acids, free amino acids and carbohydrates differed markedly between elaiosomes and seeds. The most important difference was in total amino acid content, which was on average 7.5 times higher in elaiosomes than in seeds. The difference was especially marked for the nitrogen-rich amino acid histidine. The availability of essential nutrients and, in some species, the higher nitrogen content in elaiosomes suggest that their nutritional value for larvae plays a key role in this interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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?     

Analysis of protein-protein interaction by simulation of small-zone size-exclusion chromatography: application to an antibody-antigen association

The association of two or more macromolecules results in the formation of a complex characterized by a larger Stokes radius than that of its components. Therefore, analytical procedures such as ultracentrifugation and size-exclusion gel chromatography that resolve molecules on the basis of size have been used to characterize the association. In this paper we describe an iterative computer simulation of small-zone size-exclusion gel filtration. The simulation describes univalent and bivalent interactions of proteins of equal and nonequal molecular weight and appears to have both qualitative and quantitative application to the evaluation of protein-protein interaction as revealed by alteration of chromatographic elution profiles. To test the validity of the simulation, the model was applied to an antibody-antigen interaction by determining the association constant (Ka) for the interaction between the binding fragment derived from a human immunoglobulin A rheumatoid factor and the antigenic fragment obtained from a human myeloma immunoglobulin G. The self-consistency of the estimated Ka values obtained with a valence value of 2 in contrast to the lack of self-consistency if an antigenic valence of 1 was assumed was taken to support the ability of the algorithm to reasonably emulate the chromatographic processes of interacting proteins. In conjunction with the computer simulation, a sensitive microcomputer-interfaced chromatography system was assembled, which is capable of analyzing 300 ng of protein in less than 1 h. This combination of rapid reagent-conservative chromatography and simulation analysis may contribute to the usefulness of small-zone gel filtration in studies of protein-protein interaction.