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At least 973 records · Page 54Linked to original sources
PubMed1999

Pancreatitis induced by endoscopic balloon sphincter dilation and changes in serum amylase levels after the procedure.

BACKGROUND: The short-term safety of endoscopic balloon sphincter dilation remains to be defined. We prospectively investigated the rate of pancreatitis and changes in pancreatic enzyme values induced by endoscopic balloon sphincter dilation. METHODS: One hundred nine patients with bile duct stones were treated using endoscopic balloon sphincter dilation between July 1995 and September 1997. Serum amylase values were determined before and after the procedure in 100 patients treated 106 times, and these values were compared with the incidence of pancreatitis. Patients were classified into 3 categories according to serum amylase levels before therapy (group A, within the normal range; group B, above the normal upper limit up to 500 mU/mL; group C, above 500 mU/mL). The severity of pancreatitis was categorized into 4 grades (minimal, postprocedural abdominal pain lasting no longer than the next morning; mild, elevation of amylase values to at least three times normal accompanied by postprocedural abdominal pain necessitating treatment for 3 nights; moderate, required 4 to 10 nights of treatment; severe, required intensive care). RESULTS: Postprocedural serum amylase levels in groups A and B were significantly elevated compared with baseline levels. In contrast, these levels in group C were decreased. Overall, postprocedural amylase levels were elevated in 95 studies. Minimal or mild pancreatitis developed in 21 (15 minimal and 6 mild) patients in groups A and B only. CONCLUSIONS: Although pancreatic serum enzymes were elevated in almost all patients after endoscopic balloon sphincter dilation, procedure-related pancreatitis developed in a small percentage. There does not seem to be any contraindication to the use of endoscopic balloon sphincter dilation in patients with high amylase levels and an indication for the procedure, although such patients sometimes have stone-related pancreatitis or may be predisposed to developing pancreatitis.

Aged↗
PubMed2000

Factors regulating amylase secretion from chicken pancreatic acini in vitro.

In mammals, cholecystokinin regulates pancreatic exocrine secretion under physiological conditions. We have shown, however, that cholecystokinin at physiological concentrations does not induce pancreatic amylase secretion in birds. Therefore, we investigated the effects of various neurotransmitters and gut hormones on the pancreatic amylase secretory response in isolated chicken pancreatic acini. Acetylcholine (half-maximal stimulation at 800 nM) and vasoactive intestinal polypeptide (half-maximal stimulation at 40 pM) produced a concentration-dependent increase in amylase secretion at physiological concentrations. The combination of acetylcholine and vasoactive intestinal polypeptide produced an additive response in amylase secretion. Sodium nitroprusside, a spontaneous nitric oxide releaser, and bombesin, induced amylase secretion at concentrations greater than 10 nM and 100 nM, respectively. Gastrin and secretin increased amylase secretion at pharmacological concentrations (10 to 100 nM). Our findings suggest that neural regulation is important for pancreatic enzyme secretion in birds and the contribution of gut hormones seems to be physiologically unimportant.

Amylases↗
PubMed2003

Three alpha-amylases from malted finger millet (Ragi, Eleusine coracana, Indaf-15)--purification and partial characterization.

Three alpha-amylases (E.C. 3.2.1.1) were purified to apparent homogeneity from 72 h finger millet malt by three step purification via fractional acetone precipitation, DEAE-Sephacel ion exchange and Sephacryl S-200 gel permeation chromatographies with a recovery of 6.5, 2.9, 9.6% and fold purification of 26, 17 and 31, respectively. alpha-Nature of these amylases was identified by their ability to rapidly reduce the viscosity of starch solution and also in liberating oligosaccharides of higher D.P. and were accordingly designated as amylases alpha-1((b)), alpha-2 and alpha-3, respectively. These amylases, having a molecular weight of 45+/-2 kDa were found to be monomeric. The pH and temperature optima of these alpha-amylases were found to be in the range of 5.0-5.5 and 45-50 degrees C, respectively. K(m) values of these amylases for various cereal starches varied between 0.59 and 1.43%. Carbodiimide (50 mM) and metal ions such as Al(3+), Fe(2+), and Hg(2+) (5 mM) have completely inhibited these enzymes at 45 degrees C. Amino acid analysis of these enzymes indicated high amounts of glycine which is an unusual feature of these enzymes.

Amino Acids↗
PubMed1999

An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin.

The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.

Amylopectin↗
PubMed1997

Acidostable and acidophilic proteins: the example of the alpha-amylase from Alicyclobacillus acidocaldarius.

Acidophilic microorganisms grow optimally at pH values between 1-4. They have adapted to the acid condition by maintaining their cytoplasmic pH at a value close to neutrality. Hence, only those (macro)-molecules, which face the acid medium, have had to adapt to this extreme condition. Literature data show that several exoproteins from thermoacidophilic prokaryotes are characterized by a low charge density. It is proposed that this property contributes to the stability of these proteins both below and above the pKa-values of their glutamate and aspartate residues. As an example of an acidophilic protein, the alpha-amylase from the Gram-positive Alicyclobacillus acidocaldarius ATCC27009 was studied. The enzyme is thermoacidophilic, with optima of temperature and pH of 75 degrees C and pH 3, respectively. The nucleotide sequence of the cloned gene (8) indicates that the alpha-amylase belongs to a large family of starch-degrading enzymes with a characteristic catalytic (beta alpha)8-domain. Three essential and probably catalytic acidic residues have been conserved, suggesting that the acidophilic alpha-amylase degrades starch with essentially the same mechanism as do its neutrophilic relatives. Still, the acidophilic protein contains three exchanges in residues uniformally or almost uniformally conserved among all members of the enzyme family. In order to test whether these exchanges contribute to the acidic pH optimum, the alpha-amylase gene was expressed in Escherichia coli. Sonication of the enzyme-producing cells released alpha-amylase activity associated with a 140 kDa protein. The optima of temperature and pH for the protein produced in E. coli were similar to those of the native enzyme. Experiments are underway in which it is tested which residues contribute to the acid pH optimum of the alpha-amylase.

Amino Acid Sequence↗
PubMed1998

Metabolic evolution in alpha-amylases from Drosophila virilis and D. repleta, two species with different ecological niches.

alpha-Amylases from Drosophila virilis and D. repleta were partially purified by ion exchange chromatography. The two amylases share common characteristics for pH and cations effects, although with slight differences. D. virilis has optimal activity at pH 6.6 and D. repleta at pH 7.2. Calcium, sodium, and potassium cations activate amylolytic activity in both species but Ba2+ has an activation effect in D. repleta only. In contrast, there are major differences in thermal offbility and kinetics among amylases of the two species. D. virilis amylase is much more stable at high temperature and the optimal temperatures are very different between the two species, respectively, 45 degrees C and 30 degrees C for D. virilis and D. repleta. alpha-Amylase activity using different substrates is greater on starch than on glycogen in both species and still higher on amylose for D. virilis, the nonfungus feeder species. alpha-Amylase of D. repleta, the mycophagous species, has a better affinity to amylopectin and glycogen. Such differences in substrate specificity suggest adaptation to different resources in these species living in different habitats. Metabolic evolution seems to have occurred through a "tradeoff" between kinetic effectiveness and the nature of substrate, with a higher Vmax on amylose for D. virilis and a lower K(m) on glycogen for D. repleta.

Adaptation, Physiological↗
PubMedDate not supplied

Changes of salivary amylase in serum and parotid gland during pharmacological and physiological stimulation.

Although serum amylase level is an important diagnostic factor in certain salivary and pancreatic diseases, little information is available regarding the mechanism by which parotid amylase reaches the circulatory system. The present study was carried out to investigate the relationship between parotid isoamylase concentrations in blood serum and in parotid tissue in response to various stimuli. Wistar rats were fed with standard laboratory rodent chow; water was supplied ad libitum. In the first experiment, after a 16-h fasting, rats received either 5 mg/kg pilocarpine or saline (control). In the second study, after fasting, half of the rats were fed for 1 h, the other half received no food. In the third experiment, the changes in serum and tissue enzyme levels were monitored in freely fed animals during the peak-food intake phase, the first 2 h of the dark period. Amylase concentration was determined by using starch as a substrate. Pancreatic and parotid isoamylase levels in serum were separated by gel-electrophoresis utilizing differences in ionic properties of the isoenzymes. As expected, pilocarpine strongly stimulated tissue amylase discharge and serum amylase elevation. Similar, but less pronounced changes were observed not only during refeeding of fasted animals, but also in nonfasted rats during their peak-feeding period. Our data suggest that pharmacological stimulation, such as with pilocarpine or feeding in fasted state, as well as a mild stimulation of parotid function by spontaneous food intake during nonfasted state results in a decrease in parotid tissue amylase activity and a proportional increase in serum levels of parotid isoamylase.

Amylases↗
PubMed2002

Influence of tableting on the enzymatic activity of different alpha-amylases using various excipients.

The purpose of the study was to show the influence of compression pressure on the enzymatic activity of different types of alpha-amylases and to analyze the loss of activity of alpha-amylase in mixtures with different excipients. Following that, the properties of excipients used for tableting enzymes were evaluated. Tablets were produced on an instrumented single punch tableting machine. The pure amylases were tableted with increasing graded compaction pressures. Mixtures were tableted to different maximum relative densities, rho(rel,max). The remaining enzymatic activity of the alpha-amylase in the tablets was determined by the starch iodine reaction. The results show a difference between different types of alpha-amylase depending on their origin and additives. Enzymatic inactivation occurs for the pure materials at all pressures used. It is initiated during and continues after compaction. It can be inhibited by freezing the tablets. Another possibility is to tablet the enzyme in a mixture with excipients, which prevent inactivation by softly embedding the enzyme. One example which even stabilizes alpha-amylase at high volume reduction is kappa-carrageenan. In conclusion, enzymatic inactivation can be markedly reduced when excipients are used for tableting, which require little compaction pressure and are able to release the mechanical stress in the form of expansion.

Carrageenan↗
PubMedDate not supplied

Evidence for two distinct members of the amylase gene family in the yellow fever mosquito, Aedes aegypti.

Genomic DNA fragments encoding a salivary gland-specific alpha-amylase gene, Amylase I (Amy I), and an additional amylase, Amylase II (AmyII) of the yellow fever mosquito, Aedes aegypti, were isolated and characterized. Two independently isolated DNA fragments, G34-F and G34-14A, encode polymorphic alleles of Amy I. A 3.2 kilobase (kb) EcoR I fragment of G34-F, F2, has been sequenced in its entirety and contains 832 base pairs (bp) of the 5'-end, non-coding and putative promoter regions that are adjacent to 2.4 kb of the Amy I coding region. One intron, 59 bp in length, is found towards the 3'-end of the clone. A third genomic clone, 3A, corresponding to Amy II, was sequenced and shown not to contain the primary DNA sequence that encodes the 260 amino acid region that uniquely characterizes the amino terminal end of the Amy I product. Amy I was assigned by restriction fragment length polymorphism (RFLP) mapping to chromosome 2 (23.0 cM) and Amy II to chromosome 1 (44.0 cM). Amy I and Amy II are highly polymorphic and there may be multiple linked copies at each locus. Comparisons between Amy I and Amy II are presented for the putative promoter and conceptual translation products. The identification of two distinct amylase genes and their separate linkage assignments provides evidence for a multigene family of alpha-amylases in Ae. aegypti.

Aedes↗
PubMedDate not supplied

Purification, biochemical characterisation and partial primary structure of a new alpha-amylase inhibitor from Secale cereale (rye).

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the alpha-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13,756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional alpha-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus alpha-amylases was observed. The inhibitor is more effective against insect alpha-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional alpha-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.

Animals↗
PubMed2003

Cross-inhibitory activity of cereal protein inhibitors against alpha-amylases and xylanases.

The purification and characterisation of a xylanase inhibitor (XIP-I) from wheat was reported previously. In our current work, XIP-I is also demonstrated to have the capacity to inhibit the two barley alpha-amylase isozymes (AMY1 and AMY2). XIP-I completely inhibited the activity of AMY1 and AMY2 towards insoluble Blue Starch and a soluble hepta-oligosaccharide derivative. A ternary complex was formed between insoluble starch, a catalytically inactive mutant of AMY1 (D180A), and XIP-I, suggesting that the substrate-XIP-I interaction is necessary for inhibition of barley alpha-amylases. K(i) values for alpha-amylase inhibition, however, could not be calculated due to the nonlinear nature of the inhibition pattern. Furthermore, surface plasmon resonance and gel electrophoresis did not indicate interaction between XIP-I and the alpha-amylases. The inhibition was abolished by CaCl(2), indicating that the driving force for the interaction is different from that of complexation between the barley alpha-amylase/subtilisin inhibitor (BASI) and AMY2. This is the first report of a proteinaceous inhibitor of AMY1. BASI, in addition, was demonstrated to partially inhibit the endo-1,4-beta-D-xylanase from Aspergillus niger (XylA) of glycoside hydrolase family 11. Taken together, the data demonstrate for the first time the dual target enzyme specificity of BASI and XIP-I inhibitors for xylanase and alpha-amylase.

Animals↗
PubMed1997

Genetic factors on the second and third chromosomes responsible for the variation of amylase activity and inducibility in Drosophila melanogaster.

Using second- or third-chromosome substitution lines of Drosophila melanogaster, the genetic variation of inducibility and amylase specific activities in three media (starch, normal and glucose) were investigated. Genetic factors on both the second and third chromosomes were responsible for the variation in amylase specific activity and inducibility. In glucose medium, the genetic variance of amylase specific activity estimated for the second-chromosome substitution lines was larger than that for the third-chromosome substitution lines; however, for starch medium and inducibility, the variance was larger for the third-chromosome substitution lines. High correlations for the second-chromosome substitution lines and low correlations for the third-chromosome substitution lines were observed for amylase specific activities in different media. These results suggest that the genetic factor(s) responsible for inducibility or amylase activity variation in an induced medium such as starch should be on the third chromosome and those in the non-induced medium such as glucose should be on the second chromosome. The functional roles of the factors on the second and third chromosomes would be the repression and induction of amylase, respectively.

Animals↗
PubMed1995

Amylase activity in tracheobronchial secretions of laryngectomized patients.

The alpha-amylase activity in tracheobronchial secretions of 16 consecutive patients with a total laryngectomy was studied. None of these patients had a tracheopharyngeal fistula or pulmonary disorder which might affect the amylase activity. This study proves the presence of amylase in tracheobronchial secretions of laryngectomized patients with a normal lung at a level between x and y and the quantitative analysis of the amylase activity is discussed. The relevance of investigating laryngectomy patients is because of the nature of the surgery the lower respiratory tract is permanently and physically isolated from any other source of salivary amylase. No similar study of the analysis of amylase in normal lung tissue had been reported before. This information may be of value in order to detect salivary aspiration in patients with a tracheostomy or endotracheal intubation if the level in the aspirate is in the order of a-b times greater than that found in normal tracheobronchial secretions. (x = 35 and y = 1125 i.u./l; a = 31.8 and b = 628.6 i.u./l).

Aged↗
PubMed1994

Site-directed mutagenesis reveals critical importance of the catalytic site in the binding of alpha-amylase by wheat proteinaceous inhibitor.

A bacterial alpha-amylase from Bacillus subtilis was found to be strongly inhibited by wheat alpha-amylase inhibitors 0.53 and 0.19, which had previously been thought specific for animal alpha-amylase. Inhibition and gel filtration studies of site-directed mutants of B. subtilis alpha-amylase with the inhibitors indicated a direct correlation between the alpha-amylase activity and the inhibitory effect of inhibitor binding. A mutant enzyme His 180-->Asn, which was 20 times less active in terms of kcat than the wild type, was less sensitive to inhibition by similar degrees, while the specificity for 0.53 and 0.19 changed significantly as a result of the mutation. Catalytic-site mutants that were completely devoid of catalytic activity virtually lost the ability to bind inhibitors, even though they retained high affinities for substrates. The results show that the integrity of the catalytic site is crucial for inhibitor binding and, despite the previously observed tight binding, reveal a subtle nature of the interaction between alpha-amylase and the wheat inhibitor, which leads to a proposal of a two-step mechanism for the binding interaction.

Bacillus subtilis↗
PubMedDate not supplied

Use of co-immobilized beta-amylase and pullulanase in reduction of saccharification time of starch and increase in maltose yield.

Beta-amylase and pullulanase were co-immobilized to poly(acrylamide-acrylic acid) resin [P(AAm-AAc)] using 1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride (EDC). The combined beta-amylase and pullulanase activity was 32% relative to the nonimmobilized beta-amylase. Co-immobilization of beta-amylase and pullulanase increased the maltose yield compared to thart of the immobilized beta-amylase alone and reduced the saccharification time to about 50 h. The results showed that there is a significant increase in the thermal stability, pH stability, and stability toward gamma irradiation. The results also suggest that the co-immobilization of beta-amylase and pullulanase is a potentially useful approach for commercial starch hydrolysis.

Acrylic Resins↗
PubMed1972

A study of the nature of the immediate precursor of the extracellular -amylase of Bacillus amyloliquefaciens. A reappraisal.

1. A defined medium was devised for use in washed-cell experiments with post-exponential-phase cultures of Bacillus amyloliquefaciens. The medium allowed alpha-amylase to be secreted, bacterial concentration to increase and l-[U-(14)C]valine to be incorporated into protein at a linear rate, which was the same as in a post-exponential-phase culture, for up to 6h. 2. Determination of the specific radioactivity of l-[U-(14)C]valine in the medium, the intracellular amino acid pool, the cellular protein and the isolated alpha-amylase, after a 3h incubation of washed cells in the defined medium, showed that at least 76% of the alpha-amylase secreted was synthesized de novo. 3. By isolating the alpha-amylase formed during a 6h incubation in the presence of l-[U-(14)C]valine it was shown that the specific radioactivity of the N-terminal valine, within the limits of experimental error, was the same as that of the total valine residues from the complete alpha-amylase molecule. 4. A consideration of these results in relation to the whole literature on the subject strongly supports the idea that there is no reason to suppose that extracellular alpha-amylase is formed from a high-molecular-weight precursor in B. amyloliquefaciens and closely related organisms with identical characteristics of exoenzyme secretion.

Amino Acids↗
PubMed1976

Physical and catalytic properties of alpha-amylase from Tenebrio molitor L. larvae.

The amylase from Tenebrio molitor L. larvae (yellow mealworm) was characterized according to a number of its molecular and catalytic properties. The insect amylase is a single polypeptide chain with mol.wt. 68000, an isoelectric point of 4.0 and a very low content of sulphur-containing amino acids. The enzyme is a Ca2+-protein and behaves as an alpha-amylase. Removal of Ca2+ by exhaustive dialysis against water causes the irreversible inactivation of the enzyme. Moreover, the enzyme is activated by the presence in the assay mixture of Cl-, or some other inorganic anions that are less effective than Cl-, and is inhibited by F-. Optimal conditions of pH and temperature for the enzymic activity are 5.8 and 37 degrees C. The insect amylase exhibits an identical kinetic behaviour toward starch, amylose and amylopectin; the enzyme hydrolyses glycogen with a higher affinity constant. Compared with the non-insect alpha-amylases described in the literature, Tenebrio molitor amylase has a lower affinity for starch.

Amylases↗
PubMed1985

Pancreatic amylase secretion and cytoplasmic free calcium. Effects of ionomycin, phorbol dibutyrate and diacylglycerols alone and in combination.

Both protein kinase C and Ca2+ may act in concert to bring about activation of secretion. This study examined the actions on pancreatic acini of ionomycin and phorbol dibutyrate, which selectively stimulate one or the other of these pathways; their stimulatory effects were compared with those of receptor agonists, such as carbachol and caerulein, which activate phospholipase C. The Ca2+ ionophore ionomycin produced a dose-dependent increase in amylase secretion and intracellular free Ca2+ (as measured by quin-2). The increase in amylase secretion elicited by carbachol or caerulein was accompanied by a small sustained increase in intracellular free Ca2+, following an initial peak. However, the elevation in intracellular free Ca2+ produced by these receptor agonists for a given level of amylase secretion was less than that observed with ionomycin. Phorbol dibutyrate stimulated amylase secretion by a mechanism that was independent of extracellular Ca2+, and no change in intracellular free Ca2+ was observed. Synergistic stimulatory effects of phorbol dibutyrate and ionomycin were observed, whether the phorbol ester was present before, or in combination with, ionomycin. Diacylglycerols containing unsaturated fatty acids (1,2-dioleoylglycerol and 1,3-dioleoylglycerol) also stimulated amylase secretion and exhibited synergistic effects on secretion with ionomycin. These findings suggest that complete activation of amylase secretion from the pancreas requires stimulation of both Ca2+-dependent and protein kinase C-activated pathways.

Amylases↗