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.     

Purification and characterization of human pancreatic colipase.

Two colipases, named colipase I and colipase II, have been isolated from extracts of human pancreatic gland. The two proteins can be separated by ion-exchange chromatography, isoelectric focusing and slab technique gel electrophoresis. The result of this study indicates that the two colipases, both of which are glycoproteins, have identical amino acid compositions. The pI values were found to be 6.1 for colipase I and 5.8 for colipase II. The different colipases have also been found in human pancreatic juice. The N-terminal amino acid was glycine for both colipase I (gland) and colipase II (juice). Only minor differences were found between the colipases isolated from gland and juice, and colipase I from gland alone was examined in detail.     

Purification and characterization of monkey salivary mucin.

Highly purified mucin was prepared from monkey (Macaca arctoides) extraparotid saliva by sequential chromatography on Sephadex G-200 (followed by reduction and alkylation of void volume materials), Sepharose CL-2B with 6 M urea, and CM52 cellulose with 6 M urea. Purity was critically ascertained by anion exchange chromatography, ultracentrifugal analysis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide electrophoresis, and crossed immunoelectrophoresis. Use of crossed immunoelectrophoresis to examine mucin preparations has not been previously reported. This technique was useful for assessing purity and displaying charge and size microheterogeneity in the purified S-carboxymethylated mucin. Threonine and serine comprised 37.8% of the total amino acids while the oligosaccharide moiety contained N-acetyl-glucosamine, N-acetylgalactosamine, fucose, galactose, N-acetylneuraminic acid, and sulfate. Following alkaline borohydride treatment, the carbohydrate chains were found to be linked O-glycosidically between N-acetylgalactosamine and threonine (serine).     

Purification and characterization of human pancreatic colipase

Two colipases, named colipase I and colipase II, have been isolated from extracts of human pancreatic gland. The two proteins can be separated by ionexchange chromatography, isoelectric focusing and slab technique gel electrophoresis. The result of this study indicates that the two colipases, both of which are glycoproteins, have identical amino acid compositions. The pI values were found to be 6.1 for colipase I and 5,8 for colipase II. The different colipases have also been found in human pancreatic juice. The N-terminal amino acid was glycine for both colipase I (gland) and colipase II (juice). Only minor differences were found between the colipases isolated from gland and juice, and colipase I from gland alone was examined in detail.     

Purification and characterization of fish surface mucin

Fish surface mucin from Pampus argenteus was extracted with different organic solvents and the residue passed through Sephadex G-200. The major peak was purified by DEAE-Cellulose chromatography and five fractions were obtained. Carbohydrate and protein contents showed that major peak is a glycoprotein. Rechromatography of this component on the Sephadex G-200 column gave a single peak, with an estimated minimal molecular weight of 6.9 X 10(5). Analysis of individual sugar components revealed the presence of galactose, glucose, mannose, arabinose, N-acetyl glucosamine, N-acetyl galactosamine and sialic acid. The most represented amino acids are threonine, serine, proline, glutamic acid and glycine. The N-terminal amino acid end was blocked. Nearly 47% of sulphate was acid labile. Sialic acid and fucose were released rapidly by mild acid hydrolysis. The presence of blood group-A activity suggests that some kind of terminal alpha-Gal-NAC may be present.     

Purification and characterization of a plasminogen activator secreted by cultured human pancreatic carcinoma cells.

A plasminogen activator secreted by cultured human pancreatic carcinoma (Mia PaCa-2) cells has been purified to apparent homogeneity by procedures including Sepharose-L-arginine methyl ester affinity chromatography, Sephadex G-200 gel filtration, isoelectric focusing, and sodium dodecyl sulfate gel electrophoresis. The plasminogen activator shares many properties with urokinase including: molecular weight (55 000), isoelectric point (8.7), heat stability (60 degrees C, 30 min), PH stability (1.5-10), and its mode of activation of plasminogen. The intracellular enzyme is membrane bound and can be solubilized by detergent. Solubilized activator has a molecular weight similar to that of the secreted enzyme as determined by sodium dodecyl sulfate gel electrophoresis. The production of plasminogen activator by Mia PaCa-2 cells is totally inhibited by actinomycin D and cycloheximide.     

Cloning and sequencing of a human pancreatic tumor mucin cDNA

A monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin was used to select human pancreatic mucin cDNA clones from a lambda gt11 cDNA expression library developed from a human pancreatic tumor cell line. The full-length 4.4-kilobase mucin cDNA sequence included a 72-base pair 5'-untranslated region and a 307-base pair 3'-untranslated region. The predicted amino acid sequence for this cDNA revealed a protein of 122,071 daltons containing 1,255 amino acid residues of which greater than 60% were serine, threonine, proline, alanine, and glycine. Approximately two-thirds of the protein sequence consisted of identical 20-amino acid tandem repeats which were flanked by degenerate tandem repeats and nontandem repeat sequences on both the amino-terminal and carboxyl-terminal ends. The amino acid sequence also contained five putative N-linked glycosylation sites, a putative signal sequence and transmembrane domain, and numerous serine and threonine residues (potential O-linked glycosylation sites) outside and within the tandem repeat position. The cDNA and deduced amino acid sequence of the pancreatic mucin sequence was over 99% homologous with a mucin cDNA sequence derived from breast tumor mucin, even though the native forms of these molecules are quite distinct in size and degree of glycosylation.     

Purification and characterization of a human NO synthase.

A NO synthase (NOS, EC 1.14.23) was isolated from human cerebellum by two sequential chromatography steps, that is affinity chromatography on 2'5'ADP sepharose and size exclusion chromatography on Superose 6. Human NOS migrated as a single band of 160 kDa on SDS/PAGE. The enzyme was Ca2+/calmodulin-regulated and NADPH/tetrahydrobiopterin (BH4)-dependent, which are characteristics of a type I NOS previously isolated from rat cerebellum. Antisera raised against purified rat cerebellar NOS crossreacted specifically with a 160 kDa protein in crude supernatant fraction of human cerebellum and purified human NOS but not in crude supernatant fraction of the temporal lobe. These findings provide evidence that nitrinergic signal transduction through conversion of L-arginine to L-citrulline and NO does also occur in humans and NO may function as a neurotransmitter in the human central nervous system.     

Proteogenomic characterization of pancreatic ductal adenocarcinoma

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Purification and characterization of human transcortin.

Human transcortin was purified to apparent homogeneity from plasma by a two-step procedure involving affinity and hydroxyapatite chromatography. The affinity gel incorporated denatured bovine serum albumin as the spacer and cortisol hemisuccinate as the ligand. Although isolated transcortin showed a propensity for spontaneous polymerization according to a geometric progression (1, 3, 9) only one band was observed on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Cortisol-binding activity of the isolated protein gave an apparent association constant of 2.5 X 10(8) M-1 at 4 degree C in equilibrium dialysis. Isoelectric focusing of purified native transcortin showed six discrete bands, five between pH 3.75 and 4.15 and another, possibly desialylated, at pH 6.15. Desialylated transcortin also gave six bands on isoelectric focusing, with pI values ranging from 4.90 to 6.30.     

Purification and characterization of ovine pancreatic alpha - amylase.

Ovine pancreatic amylase has been purified from pancreas homogenate by ammonium sulfate, acetone precipitation, DEAE-cellulose chromatography and finally by specific adsorption on polydextran gel. The enzyme is homogeneous and found as a single form as shown by disc electrophoresis, SDS gel electrophoresis, electrofocusing and ultracentrifugation. Its specific activity is similar to that of porcine amylase. The amino acid composition indicates a high content in aromatic and acidic amino acids as for the porcine enzyme; however the methionine and half cystine content differ widely. The N-terminal end is blocked. Also ovine amylase is glycosylated. The molecular weight (56,000-58,000) is slightly higher than for the porcine enzyme. The isoelectric point is acidic (pI = 3.2).     

Pancreatic cholesterol esterases. 2. Purification and characterization of human pancreatic fatty acid ethyl ester synthase

Human pancreatic fatty acid ethyl ester synthase has been isolated and purified 1200-fold to homogeneity, and its activities, binding properties, and N-terminal amino acid sequence indicate that it is a member of the lipase family. This 52-kDa monomeric protein is present at 0.6-1.2 mg/g of pancreas, and it catalyzes the synthesis and hydrolysis of ethyl oleate at rates of 2400 nmol mg-1 h-1 and 30 nmol mg-1 h-1, respectively. Kinetic analyses reveal a pronounced substrate specificity for unsaturated octadecanoic fatty acids, with ethyl ester synthetic rates of 2400 nmol mg-1 h-1 (linoleic), 2400 nmol mg-1 h-1 (oleic), 400 nmol mg-1 h-1 (arachidonic), 300 nmol mg-1 h-1 (palmitic), and 100 nmol mg-1 h-1 (stearic). Like cholesterol esterase, the enzyme binds to immobilized heparin, and this property was critical for its purification to homogeneity. Its N-terminal amino acid sequence is virtually identical with that reported for human triglyceride lipase, NH2-X-Glu-Val-Cys-5Tyr-Glu-Arg-Leu-Gly-10Cys-Phe-Ser-Asp- Asp-15Ser-Pro-Trp-Ser-Gly-20Ile, and it differs by only four residues from that reported for porcine pancreatic lipase. The synthase purified here also cleaves triglycerides, hydrolyzing triolein at a rate of 30 nmol mg-1 h-1, and this activity is stimulated by colipase and inhibited by sodium chloride. Conversely, commercially available porcine triglyceride lipase exhibits fatty acid ethyl ester synthase activity (1530 nmol mg-1 h-1) and hydrolyzes triolein at a rate of 23 nmol mg-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of alpha-L-fucosidase from Bacillus circulans grown on porcine gastric mucin.

Bacillus circulans isolated from soil was found to produce two types of alpha-L-fucosidase differing in substrate specificity. One was able to liberate L-fucose from porcine gastric mucin (PGM), but not from artificial substrates, including p-nitrophenyl and methyl alpha-L-fucosides, while the other acted not on PGM but on p-nitrophenyl alpha-L-fucoside. The production of the former enzyme was enhanced about 150 times as much by PGM added to the medium as by glucose. The alpha-L-fucosidase acting on PGM was purified from the culture fluid obtained with PGM medium by ammonium sulfate fractionation and subsequent column chromatography. The purified enzyme was found to be homogeneous by PAGE and its molecular weight was estimated to be approximately 285,000. The optimum pH was found to be 5.5 to 6.5 and the stable pH range was 4.5 to 9.0. The enzyme decomposed various blood group O(H) active substances such as PGM, human milk and human saliva, and moreover acted on A-, B-, and O-erythrocytes. The enzyme was shown to cleave alpha-(1----2)-, (1----3)-, and (1----4)-L-fucosidic linkages in various glycoproteins and oligosaccharides, but failed to hydrolyze alpha-(1----6)-L-fucosic linkages in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine and intact bovine thyroglobulin.     

Purification and properties of human pancreatic deoxyribonuclease I.

Human pancreatic DNase I was purified extensively from duodenal juice of healthy subjects by a procedure including ammonium sulfate fractionation, ethanol fractionation, phosphocellulose fractionation, isoelectric focusing, and gel filtration. The final preparation was free of DNase II, pancreatic RNase, alkaline phosphatase, and protease. The enzyme had a molecular weight of approximately 30,000, as determined by gel filtration on Sephadex G-100, and showed maximum activity at pH 7.2-7.6. It required divalent cations for activity, and caused single-strand breaks by endonucleolytic attack on double- as well as single-stranded DNA molecules. The enzyme was inhibited by actin and bovine pancreatic DNase I antibody.     

Purification and characterization of human platelet proteoglycan.

Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.     

Purification and characterization of a calpain activator from human platelets.

A calpain (Ca(2+)-activated neutral protease) activator was purified from human platelets by ammonium sulfate fractionation, gel-filtration, ion-exchange chromatography, followed by heat-treatment. The purified calpain activator with a Mr of 47.5 kDa was a heat-stable protein as demonstrated in other cells. The calpain activator did not change the Ca2+ sensitivity of calpain but activated calpain activity about 2-fold. This calpain activator may play an important role in the activation of the protease system leading to the Ca(2+)-mediated physiological process of platelets.     

Purification and characterization of human truncated prorenin.

Posttranslational processing of enzymatically inactive prorenin to an active form participates in the control of the activity of a key system involved in blood pressure regulation, growth, and other important functions. The issue is complicated because renin can be produced by a number of tissues throughout the body, in addition to the kidney, but the mechanism by which they process prorenin to renin is unknown and difficult to determine because of the small amounts of renin present. In the juxtaglomerular cell of the kidney, a 43 amino acid prosegment is cleaved from the amino terminus of prorenin to generate renin of molecular weight 44,000 [Do, Y. S., Shinagawa, T., Tam, H., Inagami, T., & Hsueh, W. A. (1987) J. Biol. Chem. 262, 1037-1043]. Using human uterine lining or a recombinant human prorenin system, we employed the same approach as that used in kidney, ammonium sulfate precipitation at pH 3.1 followed by pepstatin and H-77 affinity chromatography or gel filtration, to purify to homogeneity a 45,500-MW totally active renin. The specific activity of the active truncated prorenin was 850 Goldblatt units (GU)/mg of protein for chorion-decidua renin and 946 GU/mg of protein for recombinant renin, both similar to that reported for pure human renal renin. Both forms of renin cross-reacted with an antibody generated against 44,00-MW pure human renal renin and with an antibody generated against a peptide identical to the carboxy-terminal one-third of the prosegment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of alpha-amylase from rat pancreatic acinar carcinoma. Comparison with pancreatic alpha-amylase.

alpha-Amylase was purified to apparent homogeneity from normal pancreas and a transplantable pancreatic acinar carcinoma of the rat by affinity chromatography on alpha-glucohydrolase inhibitor (alpha-GHI) bound to aminohexyl-Sepharose 4B. Recovery was 95-100% for both pancreas and tumour alpha-amylases. They were monomeric proteins, with Mr approx. 54000 on SDS/polyacrylamide-gel electrophoresis. Isoelectric focusing of both normal and tumour alpha-amylases resolved each into two major isoenzymes, with pI 8.3 and 8.7. Tumour-derived alpha-amylase contained two additional minor isoenzymes, with pI 7.6 and 6.95 respectively. All four tumour isoenzymes demonstrated amylolytic activity when isoelectric-focused gels were treated with starch and stained with iodine. Two-dimensional electrophoresis, on SDS/10-20%-polyacrylamide-gradient gels after isoelectric focusing, separated each major isoenzyme into doublets of similar Mr values. Pancreatic and tumour-derived alpha-amylases had similar Km and Ki (alpha-GHI) values, but the specific activity of the tumour alpha-amylase was approximately two-thirds that of the normal alpha-amylase. Although amino acid analysis and peptide mapping with the use of CNBr, N-chlorosuccinimide or Staphylococcus aureus V8 proteinase gave comparable profiles for the two alpha-amylases, tryptic-digest fingerprint patterns were different. Antibodies raised against the purified pancreatic alpha-amylase and tumour alpha-amylase respectively showed only one positive band on immunoblotting after gel electrophoresis of crude extracts of rat pancreas and carcinoma, at the same position as that of the purified enzyme. More than 95% of the alpha-amylase activity in the pancreas and in the tumour was absorbed by an excess amount of either antibody, indicating that normal and tumour alpha-amylases are immunologically identical. The presence of additional isoenzymes in the carcinoma, and dissimilarity of tryptic-digest patterns, may reflect an alteration in gene expression or in the post-translational modification of this protein in this heterogeneously differentiated transplantable pancreatic acinar carcinoma.