Immunohistochemical and immunoelectron microscopic analyses of alpha-amylase isozymes in human intrahepatic biliary epithelium and hepatocytes.

The expression and localization of the pancreatic and salivary isozymes of alpha-amylase in the intrahepatic biliary epithelium and hepatocytes were examined by the immunohistochemical method with polyclonal and monoclonal antibodies in 45 normal autopsied human livers. Immunoelectron microscopic studies with the protein A-gold method were performed with the monoclonal antibodies (MAb) on seven of the livers. The intrahepatic biliary system was divided into large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands. Immunohistochemically, pancreatic isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands in almost all livers. Interlobular ducts expressed pancreatic isozyme in only four (9%) livers. Bile ductules and hepatocytes were negative for pancreatic isozyme in all cases. Expression of salivary isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands in almost all livers, although the expression in interlobular ducts and bile ductules was weak. Hepatocytes were weakly positive for salivary isozyme. Immunoelectron microscopy revealed that both pancreatic and salivary isozymes were located in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands, and that hepatocytes had no pancreatic isozyme but contained salivary isozyme. These data suggest that pancreatic and salivary isozymes of alpha-amylase are produced by the intrahepatic biliary epithelium and secreted into intrahepatic biliary lumens, and that they may play an important role in the physiology of the intrahepatic biliary tree and hepatic bile. It is also suggested that hepatocytes produce a small amount of salivary alpha-amylase that may be secreted into the biliary tree.     

Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast

A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes.     

The molecular characteristics of a human pancreatic acidic phosphoprotein that inhibits calcium carbonate crystal growth.

A CaCO3-crystal-growth inhibitor was isolated from human pancreatic stones by using EDTA demineralization, followed by DEAE-Trisacryl chromatography. The isolated inhibitor was found to be a phosphoglycoprotein with Mr 14017 and having an unusual chemical composition. It is characterized by a high (42%) acidic amino acid content, but lacks methionine and gamma-carboxyglutamic acid. The protein contains 2.65 mol of P/mol of protein, as phosphoserine (2 mol) and phosphothreonine (0.5 mol). Isoelectric focusing of the protein yields one major band corresponding to an isoelectric point of 4.2. Immunochemical quantification of the crystal-growth inhibitor in pure pancreatic juice reveals that it constitutes 14% of the normal exocrine secretion. Our findings demonstrate that this is a novel secretory protein, which has no enzymic activity and which maintains pancreatic juice in a supersaturated state with respect to CaCO3.     

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.     

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.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

Genetic control of several alpha-amylase isozymes in winter hexaploid wheat

alpha-Amylase isozymes were detected via electrophoretic separation in a Tris-glycine polyacrylamide gel system (pH 8.4). Three chromosome 6B loci controlling the alpha-amylase isozyme composition were identified by studying the grain alpha-amylase patterns in an F --> infinity self-pollinating population of winter common wheat (Donskoi Mayak). The loci were found to take the following order in the long arm of chromosome 6B: cen.-alpha-Amy-B3-alpha-Amy-B6-alpha-Amy-B1-.     

Porcine pancreatic alpha-amylase shows binding activity toward N-linked oligosaccharides of glycoproteins.

Porcine pancreatic alpha-amylase was shown by interaction analyses using a resonance mirror detector and alpha-amylase-immobilized Sepharose to bind with glycoproteins possessing N-glycans but not O-linked mucin-type glycans. Direct binding of three types of N-glycans to the alpha-amylase was demonstrated by surface plasmon resonance. Binding with biotin-polymer sugar probes revealed that the alpha-amylase has affinity to alpha-mannose, alpha-N-acetylneuraminic acid, and beta-N-acetyllactosamine, which are components of N-glycans. The binding of glycoproteins or carbohydrates enhanced the enzyme activity, indicating that the recognition site for N-glycans is different from its catalytic site. The binding activity was unique to porcine pancreatic alpha-amylase and was not observed for alpha-amylase from saliva, wheat, and fungus.     

Difference in transglycosylation between human pancreatic and salivary alpha-amylases

Transglycosylation reactions of alpha-amylases from human pancreatic juice and saliva were examined by using O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose as a substrate and O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol as an acceptor. The transfer reaction was estimated by quantitation of O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol produced by the enzymes from the transfer products, because the acceptor was not hydrolyzed. The amount of O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol in the digest with pancreatic alpha-amylase was six times that in the digest with salivary alpha-amylase at the stage when the substrate was completely consumed, and the difference increased gradually on further incubation. The phenomenon can be applied to differentiate the two alpha-amylases in human serum.     

Measurement of pancreatic and salivary alpha-amylase in serum: comparison of methods with five different substrates

Pancreatic and salivary alpha-amylase in human serum have been determined with the inhibitor method with 5 different substrates. An end concentration of 60 inhibitor units per liter reagent is sufficient for inhibition of the salivary alpha-amylase. The recovery of known amounts of pancreatic and salivary alpha-amylase was excellent. A mean +/- SD of 53.1 +/- 3.8% of pancreatic alpha-amylase as percentage of total alpha-amylase in serum has been observed for the 5 different substrates used. Moreover, it seems that standards obtained from crude saliva or duodenal fluid can be used as well as pure enzymes obtained from a commercial supplier.     

Genetic analysis of human salivary α-amylase isozymes by isoelectric focusing

The isozymes of human salivary alpha-amylase have been separated by thin-layer gel isoelectric focusing in a pH 4-8 gradient followed by a starch-iodine zymogram procedure. The normal isozyme pattern (N) consisted of five major isozymes together with a number of minor ones. In addition, seven variant isozyme phenotypes were also observed, which had a combined frequency of 11.7% in a random population of 368 individuals. Analysis of familial data is indicative of the inheritance of autosomal codominant alleles.     

Initial proteome analysis of mature barley seeds and malt

Several barley (Hordeum vulgare) cultivars are used in the production of malt for brewing. The malt quality depends on the cultivar, its growth and storage conditions, and the industrial process. To enhance studies on malt quality, we embarked on a proteome analysis approach for barley seeds and malt. The proteome analysis includes two-dimensional (2-D) gel electrophoresis, mass spectrometry, and bioinformatics for identification of selected proteins. This project initially focused on proteins in major spots in the neutral isoelectric point range (pI 4-7) including selected spots that differ between four barley cultivars. The excellent malting barley cultivar Barke was used as reference. Cultivar differences in the 2-D gel spot patterns are observed both at the seed and the malt level. In seed extracts one of the proteins causing variations has been identified as an alpha-amylase/trypsin inhibitor. In malt extracts multiple forms of the alpha-amylase isozyme 2 have been identified in varying cultivar characteristic spot patterns. The present identification of proteins in major spots from 2-D gels includes 27 different proteins from 42 spots from mature seed extract, while only three specific proteins were identified by analysing 13 different spots from the corresponding malt extract. It is suggested that post-translational processing causes the same protein to occur in different spots.     

Some aspects of the mechanism of complexation of red kidney bean alpha-amylase inhibitor and alpha-amylase

Bovine pancreatic alpha-amylase binds 1 mol of acarbose (a carbohydrate alpha-amylase inhibitor) per mol at the active site and also binds acarbose nonspecifically. The red kidney bean alpha-amylase inhibitor-bovine pancreatic alpha-amylase complex retained nonspecific binding for acarbose only. Binding of p-nitrophenyl alpha-D-maltoside to the final complex of red kidney bean alpha-amylase inhibitor and bovine pancreatic alpha-amylase has a beta Ks (Ks') value that is 3.4-fold greater than the Ks (16 mM) of alpha-amylase for p-nitrophenyl alpha-D-maltoside alone. The initial complex of alpha-amylase and inhibitor apparently hydrolyzes this substrate as rapidly as alpha-amylase alone. The complex retains affinity for substrates and competitive inhibitors, which, when present in high concentrations, cause dissociation of the complex. Maltose (0.5 M), a competitive inhibitor of alpha-amylase, caused dissociation of the red kidney bean alpha-amylase inhibitor--alpha-amylase complex. Interaction between red kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor and porcine pancreatic alpha-amylase proceeds through two steps. The first step has a Keq of 3.1 X 10(-5) M. The second step (unimolecular; first order) has a forward rate constant of 3.05 min-1 at pH 6.9 and 30 degrees C. alpha-Amylase inhibitor combines with alpha-amylase, in the presence of p-nitrophenyl alpha-D-maltoside, noncompetitively. On the basis of the data presented, it is likely that alpha-amylase is inactivated by the alpha-amylase inhibitor through a conformational change. A kinetic model, in the presence and absence of substrate, is described for noncompetitive, slow, tight-binding inhibitors that proceed through two steps.     

Multiple molecular forms of the gibberellin-induced alpha-amylase from the aleurone layers of barley seeds

A class of plant growth regulators, gibberellins, induce the synthesis of alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) in the aleurone layers of barley (Hordeum vulgare L. var. Himalaya) seeds. The purified alpha-amylase is composed of multiple isozymic forms with indistinguishable molecular weights, but different net charges. These alpha-amylase isozymes separate on isoelectric focusing gels into two groups, each containing multiple species. One group has an apparent isoelectric point (pI) of approximately 5.8 (the high pI group). The other group's pI values are around 4.5 (the low pI group). On some gels a small amount of protein focuses between the high and low pI isozymes. These proteins comigrate with the low pI isozymes upon reelectrophoresis. The synthesis of these two groups is temporally regulated. The high pI group is the dominant set of isozymes secreted from embryoless half seeds during the first two days of gibberellin administration. After four days, however, the major isozymes are those of the low pI group. This shift in isozyme pattern is due to a shift in their relative rates of synthesis. Peptide analysis of these two groups of isozymes with Staphylococcus aureus V8 protease and cyanogen bromide shows amino acid sequence differences. However, members within the same group have similar peptide patterns. Both groups of isozymes are synthesized in vitro in a wheat germ extract primed with poly(A)+ RNA isolated from gibberellin-treated aleurone layers. This indicates that the synthesis of the two groups of alpha-amylase isozymes is probably directed by two or more different populations of mature mRNA. A model that explains these observations and the available genetic information is that barley aleurone alpha-amylase isozymes are encoded by at least two sets of structural genes.     

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.     

Primary structure of human pancreatic alpha-amylase gene: its comparison with human salivary alpha-amylase gene

We have determined the entire structure of the human pancreatic alpha-amylase (Amy2) gene. It is approx. 9 kb long and is separated into ten exons. This gene (amy2) has a structure very similar to that of human salivary alpha-amylase (Amy1) gene [Nishide et al. Gene 41 (1986a) 299-304] in the nucleotide sequence and the size and location of the exons. The major difference lies in the fact that amy1 has one extra exon on the 5' side. Other differences are at the 5' border of exon 1 and the 3' border of exon 10. The close similarity of these two genes, as compared with mouse pancreatic and salivary amylase genes, suggests that during evolution, the divergence into the two amylase genes may have occurred after the divergence of mice and man.     

Interaction of human alpha-amylases with inhibitors from wheat flour

The interaction of four purified alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) inhibitors with human salivary and pancreatic alpha-amylases was investigated. The inhibitory activity of the four proteins towards salivary alpha-amylase was significantly increased by pre-incubation of the enzyme with inhibitor before adding substrate. This effect was not observed with the inhibition of pancreatic alpha-amylase by inhibitors 1 and 2. Inhibition of both amylases was affected to different degrees by incubating starch with inhibitor prior to the addition of enzyme. Maltose, at concentrations which only slightly affected amylase activity, prevented the inhibition of both enzymes by all four inhibitors. Gel filtration studies on salivary amylase-inhibitor mixtures showed the formation of EI complexes on a mol-to-mol ratio. A similar complex between pancreatic alpha-amylase and inhibitor 4 was observed, though complex formation between pancreatic alpha-amylase and the other inhibitors was not clearly demonstrated.     

Enzymes in the mucosal layer of the small intestine

It has been demonstrated by the methods of histochemical and biochemical examination of the activity of the enzymes that the mucus layer covering the small intestinal wall contains active enzymes (alkaline phosphatase, leucin aminopeptidase IV, saccharase, lactase) and pancreatic enzymes (alpha-amylase and trypsin). Emphasis is laid on the enrichment of the mucus layer with pancreatic enzymes as compared with small intestinal juice. A hypothesis has been advanced according to which the mucus layer undergoes degradation of polymeric and oligomeric substrates, which plays a physiological part in the digestion of nutritive substances and protection of the internal medium against immunoactive biopolymers. The digestion occurring in the mucus layer is proposed to be called mucus digestion.