Showing papers on "Amylase published in 1984"
TL;DR: The effects of phytic acid and its interactions with divalent cations (Ca++ and Mg++) on α-amylase activity were investigated in model systems and were found to be of noncompetative type.
Abstract: The effects of phytic acid and its interactions with divalent cations (Ca++ and Mg++) on α-amylase activity were investigated in model systems. Amylase activity was influenced by both preincubation time with phytate as well as phytate concentration. At 6-30 mM concentrations, Ca++ and Mg++ ions lowered the enzyme activity by 9-34% and 24-49%, respectively. When divalent cations were added simultaneously with phytate, a slight increase in enzyme activity with Ca++ and lowered enzyme activity with Mg++ were observed, as compared to when added independently. The enzyme activity was only moderately lowered when phytate was first preincubated with the divalent cations. Amylase inhibition by phytate was found to be of noncompetative type with an apparent inhibitor constant of 1.75 mM under the assay conditions.
207 citations
TL;DR: In this article, 24-hour intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high-resolution separation of proteins by two-dimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides.
Abstract: 24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high-resolution separation of proteins by two-dimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides. Beginning in the third hour of optimal hormone infusion at 0.25 microgram kg-1 h-1, changes were observed in the synthetic rates of 12 proteins, which progressed over the course of the 24-h study. Based on coordinate response patterns, exocrine proteins could be classified into four distinct groups. Group I (trypsinogen forms 1 and 2) showed progressive increases in synthetic rates reaching a combined 4.3-fold increase over control levels. Group II (amylase forms 1 and 2) showed progressive decreases in synthesis to levels 7.1- and 14.3-fold lower than control levels, respectively. Group III proteins (ribonuclease, chymotrypsinogen forms 1 and 2, procarboxypeptidase forms A and B, and proelastase 1) showed moderate increases in synthesis, 1.4-2.8-fold, and group IV proteins (trypsinogen 3, lipase, proelastase 2, and unidentified proteins 1-4) did not show changes in synthesis with hormone stimulation. Regulation of protein synthesis in response to caerulein infusion was specific for individual isoenzymic forms in the case of both trypsinogen and proelastase. The ratio of biosynthetic rates of trypsinogen forms 1 + 2 to amylase forms 1 + 2 increased from a control value of 0.56 to 24.4 after 24 h of hormonal stimulation (43.5-fold increase). Biosynthetic rates for an unidentified protein (P23) with an Mr = 23,000 and isoelectric point of 6.2 increased 14.2-fold, and the ratio of synthesis of P23 to amylase 2 increased 200-fold during caerulein infusion. During hormone stimulation the anticoordinate response in the synthesis of pancreatic glycosidases (decreased synthesis) and serine protease zymogens (increased synthesis) explain previous observations that showed little change in rates of total protein synthesis under similar conditions.
120 citations
TL;DR: Polymyxin B, a potent and selective inhibitor of PL/ Ca‐PK completely inhibited TPA‐induced amylase secretion, consistent with a role for PL/Ca‐PK in the regulation of pancreatic exocrine secretion.
101 citations
TL;DR: The combination of A23187 and TPA stimulates amylase secretion from rabbit pancreatic acini up to a level equal to, or slightly higher than when carbachol is used as stimulant.
95 citations
TL;DR: Using recombinant-DNA techniques it was possible to transfer the gene, which coded for the enzyme, to a host organism which was easier to cultivate, thus making industrial production a viable proposition.
Abstract: During a screening programme for amylase producing microorganisms a strain of Bacillus was isolated which produced a maltogenic amylase which had possible industrial potential. Unlike the microbial β-amylases from B. cereus, B. megaterium, B. polymyxa and B. circulans this enzyme was characterized by being stable at 60–70°C at pH 4.5.-5.5. Unfortunately the microorganism proved to be difficult to cultivate. Using recombinant-DNA techniques it was possible to transfer the gene, which coded for the enzyme, to a host organism which was easier to cultivate, thus making industrial production a viable proposition. The enzyme is suitable for producing high maltose syrups from liquefied starch. When used together with an amylopectin debranching enzyme such as pullulanase, more than 75% maltose can be obtained at 30% D. S.
89 citations
01 Jan 1984
TL;DR: Starch—a mixture of amylose and amylopectin—serves as a food reserve for plants and provides a mechanism by which non-photosynthesizing organisms can utilize the energy provided by the sun.
Abstract: Publisher Summary Starch—a mixture of amylose and amylopectin—serves as a food reserve for plants and provides a mechanism by which non-photosynthesizing organisms can utilize the energy provided by the sun. To utilize starch, the organisms must have enzymes that catalyze the hydrolysis of the (l→4) glycosidic bonds found between the α-D-glucopyranose residues. Enzymes that are capable of catalyzing the hydrolysis of the α-D-(l→4) linkages are called amylases, which are produced by plants, bacteria, and animals. In addition to the amylases, there are isoamylases or debranching enzymes that hydrolyze the α-D-(l→6) linkages of amylopectin. A number of analytical techniques have been devised for measuring the hydrolysis of glycosidic bonds found in starch. The most quantitative procedures involve the measurement of the formation of new reducing groups, hemiacetal or aldehyde groups, that result from the hydrolysis of the glycosidic, acetal, linkage.
83 citations
TL;DR: High levels of extracellular amylolytic activity were detected in culture supernatants of these species, except for Cryptococcus flavus and Trichosporon pullulans, which are described for the first time.
76 citations
TL;DR: During adaptation to diets containing normal protein or decreased levels of protein and correspondingly increased levels of carbohydrate, amylase and the majority of protease zymogens were synthesized in inverse proportion to nutritional substrates in the diet.
Abstract: Rates of synthesis of 16 individual pancreatic exocrine proteins; tissue concentrations of amylase, trypsinogen, and chymotrypsinogen; and morphological assessment of pancreatic acinar cells were studied in the exocrine pancreas in response to inverse changes in protein and carbohydrate in the diet, administered for 12 days. Two distinct patterns of response were observed. During adaptation to diets containing normal protein (22%) or increased levels of protein (30, 45, 64, and 82% protein) and correspondingly decreased levels of carbohydrate, amylase and the majority of protease zymogens were synthesized in direct proportion to the nutritional substrates carbohydrate and protein, respectively, in the diet. With increases in dietary protein, anticoordinate patterns of response in the synthesis of exocrine isoenzymes were observed: 0.4- to 2.0-fold increases in trypsinogen forms 1 and 2, chymotrypsinogen forms 1 and 2, proelastase 1, and procarboxypeptidases A and B; 5- to 7-fold decreases in amylase forms 1 and 2; and insignificant changes in trypsinogen 3, proelastase 2, lipase, and ribonuclease. During adaptation to diets containing normal protein (22%) or decreased levels of protein (0 or 10% protein) and correspondingly increased levels of carbohydrate, amylase and the majority of protease zymogens were synthesized in inverse proportion to nutritional substrates in the diet.(ABSTRACT TRUNCATED AT 250 WORDS)
74 citations
TL;DR: The effect of extrusion cooking on availability of starch for digestion in wheat products was tested in vitro and in vivo by measuring the plasma levels of glucose and insulin after a gastric load in conscious rats as mentioned in this paper.
73 citations
TL;DR: Findings indicate that nutritional regulation in the tissue levels of pancreatic enzymes and proenzymes is mediated by changes in the content of active cytoplasmic mRNAs.
Abstract: The mechanism by which changes in diet mediate levels of exportable enzymes and proenzymes in pancreatic tissue were studied in rats. The relative levels of mRNA coding for pancreatic amylase, lipase, procarboxypeptidases A and B, and the family of serine protease zymogens have been determined by the ability of isolated RNA to direct the synthesis of these products in a high-fidelity micrococcal nuclease-treated reticulocyte-lysate translation system. Translation products synthesized in vitro correlated directly with products synthesized in vivo in pancreatic lobules. Dietary adaptation was observed when dietary carbohydrate was increased from 0 to 58% at the expense of protein (81–23%). The increase in dietary carbohydrate over this range resulted in a 2-fold increase in amylase synthesis in pancreatic lobules and a 1.8-fold increase in mRNA-directed synthesis of amylase in the translation system in vitro. Concomitant with the decrease in dietary protein, synthesis of serine protease zymogens in pancreatic lobules and in the system in vitro decreased by approximately 50%. Over this range of dietary manipulation, ratios of amylase to serine proteases showed a 3.6-fold change. When dietary carbohydrate was further increased to 81% and protein reduced to 0, non-adaptive changes were observed since there was a decrease in amylase synthesis under conditions both in vivo and in vitro. mRNAs coding for pancreatic lipase and procarboxypeptidases A and B were unaffected by the dietary changes. These findings indicate that nutritional regulation in the tissue levels of pancreatic enzymes and proenzymes is mediated by changes in the content of active cytoplasmic mRNAs.
73 citations
TL;DR: The N‐terminal amino acid sequences of two chloroform/methanol soluble globulins from barley and one form wheat are reported and are homologous with N‐ terminal sequences previously reported for α‐amylase and trypsin inhibitors from cereals and 2 S storage proteins from castor bean and rape.
TL;DR: Modifications of diet composition alter the expression of pancreatic amylase genes as a consequence of changing the level of their transcript, and that pancreaticAmylase production is mostly regulated at the pre‐translational level.
Abstract: Regulation of the expression of pancreatic amylase genes was studied by comparing groups of rats fed diets with high (75%), intermediate (20%) and low (11%) carbohydrate content. Animals on the high carbohydrate diet had nine times as much amylase mRNA as those on low carbohydrate diet, and twice as much as the intermediate group, as determined by filter hybridization of equal amounts of total pancreatic RNA to an excess of a cloned rat amylase cDNA probe. Parallel results were obtained when levels of translatable amylase RNA were compared by means of an RNA-dependent rabbit reticulocyte cell-free system. Amylase mRNA-directed synthesis represented 35% of the total in the high carbohydrate group, 4% in the low group and 14% in the intermediate group. Relative rates of amylase synthesis, determined 30 min after [3H]phenylalanine injection, followed the same pattern. While 37% of total was incorporated into amylase in the high carbohydrate group, only 8% was incorporated in the low carbohydrate group, as compared with 22% in the intermediate group. These data indicate that modifications of diet composition alter the expression of pancreatic amylase genes as a consequence of changing the level of their transcript, and that pancreatic amylase production is mostly regulated at the pre-translational level.
TL;DR: It is concluded that parasympathetic nerve stimulation of rat parotid glands after overnight starvation causes secretion of proteins in proportions similar to, but in significantly lower concentrations than those found in sympathetic saliva.
Abstract: Secretion of proteins by rat parotid glands in response to parasympathetic nerve stimulation was studied in vivo during pentobarbitone anaesthesia. Parasympathetic stimulation (3-10 Hz) via the auriculotemporal nerve resulted in a copious flow of saliva low in protein. In contrast, sympathetic stimulation (5 Hz) via the cervical sympathetic trunk evoked saliva low in volume but high in protein. Nevertheless, the specific concentrations of amylase and peroxidase (mg/mg protein) and the ratio of amylase to peroxidase remained constant. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis revealed a single, rapidly migrating protein band of unknown identity in proportionately greater amounts in parasympathetic saliva than in sympathetic saliva. Bilateral adrenalectomy led to reduced amylase and peroxidase secretion in response to parasympathetic stimulation both on a mg/ml and a mg/mg protein basis. SDS gel electrophoresis also demonstrated the decrease in specific amylase concentration following adrenalectomy. The ratio of amylase to peroxidase, however, was not significantly affected. Administration of 6-hydroxydopamine 17-72 h prior to adrenalectomy caused no further reduction in the secretion of amylase and peroxidase. Chronic sympathectomy of 2.5-4 months duration resulted in an increased protein secretion (mg/ml) by the parotid gland in response to parasympathetic stimulation. This increase was only slightly reduced by bilateral adrenalectomy. However, as observed in non-sympathectomized rats, adrenalectomy caused a significant reduction in the specific concentrations of both amylase and peroxidase, but did not affect the amylase to peroxidase ratios. We conclude that parasympathetic nerve stimulation of rat parotid glands after overnight starvation causes secretion of proteins in proportions similar to, but in significantly lower concentrations than those found in sympathetic saliva. Circulating catecholamines, however, influence the amount of amylase and peroxidase secreted by the rat parotid gland in response to parasympathetic nerve stimulation and account for most of the increased secretion of these enzymes following chronic sympathectomy.
TL;DR: A convenient method for detecting these genes (STA genes) in replica plates containing large numbers of meiotic progeny was developed and should be suitable for direct commercial application in manioc-producing regions in Brazil and elsewhere.
Abstract: Alcoholic fermentation, growth, and glucoamylase production by 12 strains of Saccharomyces diastaticus were compared by using starch and dextrins as substrates. Haploid progeny produced from a rapidly fermenting strain, SD2, were used for hybridization with other S. diastaticus and Saccharomyces cerevisiae haploids. Alcoholic fermentation and enzyme production by hybrid diploids and their haploid parents were evaluated. Although the dosage of the STA or DEX (starch or dextrin fermentation) genes may enhance ethanol production, epistatic effects in certain strain combinations caused decreases in starch-fermenting activity. Both the nature of the starch or dextrin used and the fermentation medium pH had substantial effects on alcohol production. Commercial dextrin was not as good a substrate as dextrins prepared by digesting starch with alpha-amylase. Crude manioc starch digested by alpha-amylase was fermented directly by selected hybrids with almost 100% conversion efficiency. The manioc preparation contained adequate minerals and growth factors. This procedure should be suitable for direct commercial application in manioc-producing regions in Brazil and elsewhere. A rapidly fermenting haploid strain, SD2-A8, descended from strain SD2, contains two unlinked genes controlling formation of extracellular amylase. A convenient method for detecting these genes (STA genes) in replica plates containing large numbers of meiotic progeny was developed.
TL;DR: Results suggest that when "functionally normal" isolated acinar cells are rendered permeable, Ca2+-but not cyclic nucleotides-acts as a second messenger for amylase secretion, and furthermore that protein kinase C may be involved in the secretory process.
TL;DR: The composition of secretory proteins in the parotid saliva of rats can be altered by experimental conditions which affect gland secretory activity, and the mechanisms by which these changes occur are not known.
TL;DR: In this paper, the α-amylase isoenzymes were detected in the two barley varieties "Ingrid" and "Pomo" and the effect of different pH and temperatures on the activities of the α amylase I and II was investigated.
Abstract: In this study we have detected the α-amylase isoenzymes in the two barley varieties ‘Ingrid’ and ‘Pomo’. We further report on the effect of different pH and temperatures on the activities of the α-amylase I and II (following the nomenclature of MacGregor & Daussant18) in ‘Pomo’ malt, as well as the stabilising effect of Ca2+ under such different conditions. The importance of enzyme concentration and the inhibition of Hg2+ is also considered.
TL;DR: Serial measurements of these three enzyme levels in patients recovering from acute pancreatitis indicated that pancreatic isoamylase and lipase were elevated above normal to a greater extent and remained elevated much longer than did the total amylase.
Abstract: We compared results of measurements of total serum amylase, pancreatic isoamylase, and lipase measurements in patients with hyperamylasemia. Serial measurements of these three enzyme levels in patients recovering from acute pancreatitis indicated that pancreatic isoamylase and lipase were elevated above normal to a greater extent and remained elevated much longer than did the total amylase. This finding indicates an appreciable sensitivity advantage of the pancreatic isoamylase and lipase over total amylase measurement during the recovery phase of pancreatitis. Comparison of pancreatic isoamylase and lipase levels in selected sera indicated a good correlation (r=0.84) between these two measurements in patients who did not have macroamylasemia. Lipase was normal in sera with amylase elevations due solely to salivary isoamylase. Thus, in nonmacroamylsemic sera, pancreatic isoamylase and lipase appear to be roughly interchangeable markers of the level of pancreatic enzymes in the blood. An advantage of the lipase assay is that this enzyme is normal in hyperamylasemia caused by macroamylasemia, whereas the inhibitor assay indicates that the pancreatic isoamylase is elevated. Development of automated assays for either pancreatic isoamylase or lipase should lead to the routine use of one of these assays in place of the present reliance on total amylase measurements in the diagnosis of pancreatitis.
TL;DR: In this article, a simple and precise method suitable for the routine determination of starch and β-glucan in barley and malt is described, where perchloric acid (50 mM) was used to effect rapid (3 min) and exhaustive extraction of both glucans which were then measured directly from this single extract by specific enzymic hydrolysis of the individual glucans to glucose.
Abstract: A simple and precise method suitable for the routine determination of starch and β-glucan in barley and malt is described. Perchloric acid (50 mM) was used to effect rapid (3 min) and exhaustive extraction of both glucans which were then measured directly from this single extract by specific enzymic hydrolysis of the individual glucans to glucose. The glucose was also measured enzymically. Little or no acid hydrolysis of starch or β-glucan was observed under the extraction conditions used; most or all of the free glucose could be attributed to hydrolysis of sucrose. Complete solubilisation of the gum and hemicellulosic components of β-glucan was achieved. Preincubation of the acid extracts with protease prior to amyloglucosidase digestion resulted in higher measurements (approximately 4% w/w) of starch. The method was used to measure the levels of starch and β-glucan in five varieties of barley with contrasting malting quality, in micro-malts prepared from these samples and in commercial lager and ale malts.
TL;DR: Dietary fibre was higher in wheat/rye bread than in the corresponding flours and the increase was most pronounced in crumbs from bread baked with mainly low-extraction-rate flour, and could be accounted for to a large extent as “resistant starch”.
Abstract: Dietary fibre, assayed with an enzymatic/gravimetric method, was higher in wheat/rye bread than in the corresponding flours. The increase was most pronounced in crumbs from bread baked with mainly low-extraction-rate flour, and could be accounted for to a large extent as “resistant starch”, i. e. a starch fraction available to amyloglucosidase only after solubilization with 2m-KOH. The resistant starch was formed at dough-making and/or baking and did not increase further during freezing or storage at room temperature. The chemical modifications leading to resistant starch formation remain to be investigated. Starch-lipid complexes are probably not involved, since these are hydrolyzed by the heat-stable amylase used in the dietary fibre assay.
TL;DR: In this article, the effect of drum-drying on starch availability in white wheat flour was studied and compared with boiling and pressure cooking, and the results showed that the starch in two drumdried products was less susceptible to salivary amylase digestion in vitro than in the corresponding boiled or pressure-cooked materials.
TL;DR: The results support the proposal that intermediate and final digestion in Erinnyis ello occur under the action of glycocalyx-associated hydrolases and of plasma membrane-bound enzymes (aminopeptidase, and perhaps also amylase and trypsin).
TL;DR: The α-amylase inhibitor is identified as an ABA-induced protein in barley aleurone layers and it is suggested that it functions as an active mediator of amylase activity during the development and germination of barley seeds.
Abstract: The in vivo synthesis of α-amylase and of a bifunctional inhibitor of endogenous α-amylase and subtilisin was studied in embryoless barley half grains treated with abscisic (ABA) and gibberellic (GA3) acids. Fluorography of newly synthesized proteins and of immunoprecipitated α-amylase and its inhibitor separated by SDS-PAGE showed that GA3 induces the production of large amounts of α-amylase while the synthesis of other proteins, including the α-amylase inhibitor, is reduced. In contrast, synthesis and/or accumulation of the inhibitor is induced in embryoless half seeds incubated with ABA. These results identify the α-amylase inhibitor as an ABA-induced protein in barley aleurone layers and suggest that it functions as an active mediator of amylase activity during the development and germination of barley seeds.
TL;DR: A new group of amylases are proposed to be classified as “exo-alpha amylase”, which does not fit the criterion of Reese who proposed inversion of configuration by exo-glucanases.
TL;DR: The ovarian tumor amylase was similar to the salivary and distinct from the pancreatic enzyme by apparent molecular mass and doublet formation on sodium dodecyl sulfate--polyacrylamide electrophoresis, specific activity of pure enzyme, and sensitivity to specific alpha-amylase inhibitors.
Abstract: Human serous-type ovarian tumors contain an acidic isoenzyme of amylase. Previous attempts at purification of tumor amylases have yielded preparations contaminated with other proteins. The purification scheme presented here incorporates an affinity-chromatography procedure, with use of cycloheptaamylose linked to epoxy-activated Sepharose, that is specific for alpha-amylase (EC 3.2.1.1). Purified amylase isoenzyme from a human serous ovarian tumor was characterized and compared with the purified salivary and pancreatic isoenzymes. All three were similar in amino acid composition, pH optimum, substrate specificity, calcium requirement, heat inactivation, and Km for maltotetraose substrate. The ovarian tumor amylase was similar to the salivary and distinct from the pancreatic enzyme by apparent molecular mass and doublet formation on sodium dodecyl sulfate--polyacrylamide electrophoresis, specific activity of pure enzyme, and sensitivity to specific alpha-amylase inhibitors. All three isoenzymes differed in net electrical charge as evidenced by diethylaminoethyl-Sephadex ion-exchange chromatography and isoelectric focusing. The tumor amylase is clearly distinct from the pancreatic and differs from the salivary enzyme in net electrical charge. Evidence is presented that this charge difference may reflect, at least in part, deamidation of an amylase that is similar to or identical with salivary amylase.
TL;DR: An exploration was conducted as to whether the relative concentration of two intracellular proteins could be evaluated quantitatively from their labeling densities in ultrathin cryosections labeled with the immunogold technique, indicating that the intensity of the immunoreaction is a reliable reflection of antigen concentration in this system.
Abstract: An exploration was conducted as to whether the relative concentration of two intracellular proteins could be evaluated quantitatively from their labeling densities in ultrathin cryosections labeled with the immunogold technique. As a model rat pancreatic cells were used in which the content of amylase (Am) and chymotrypsinogen (Ch) was experimentally altered. Rats were fed either normal laboratory chow or food containing soybean trypsin inhibitor (STI), which affects the Am/Ch ratio in the tissues. The changes in Am and Ch protein levels and enzyme activities were measured biochemically in cell suspension homogenates or in zymogen granule fractions. Within 5 days a maximal change in the Am/Ch was observed as a result of adaptation to the STI diet. The Am/Ch ratio determined biochemically was compared with that from counts of gold particles bound to the respective protein in immunogold-labeled cryosections. The two data sets matched fairly well, indicating that the intensity of the immunoreaction is a reli...
TL;DR: During the second half of the year, activity of the starch-mobilising enzymes increases parallel to increases in starch content, although starch mobilisation has not yet commenced, and glucose, fructose and maltose attain maximum values during the summer months.
TL;DR: In this paper, electrical field stimulation (FS) evoked a marked, tetrodotoxin-sensitive increase in the amylase output from in vitro segments of rat pancreas.
Abstract: In this study of nervous control of exocrine secretion, electrical field stimulation (FS) evoked a marked, tetrodotoxin-sensitive increase in the amylase output from in vitro segments of rat pancre...
TL;DR: A negative correlation between the age of the infants and the concentrations of Na, K, Cl and total protein was observed and a positive correlation was found for phosphate and amylase as a function of age.
TL;DR: It was suggested that the higher production of α-amylase in the continuous culture using starch as the inducer was partly related to the predominance of some conditional non-sporulating variants with a higher amylase forming activity and to derepression of the enzyme at a low glucose concentration.
Abstract: The concentration and productivity of α-amylase increased remarkably, 15- and 11-fold respectively, in a continuous culture of Bacillus caldolyticus DSM 405 compared with batch culture, provided starch was used as the sugar source in a casitone medium. In the casitone medium with or without glucose hardly any improvement of enzyme production was observed in continuous culture. The addition of a small amount of starch to the glucose-casitone medium had a marked effect in stimulating amylase formation in continuous culture but no effect in batch culture. It was suggested that the higher production of α-amylase in the continuous culture using starch as the inducer was partly related to the predominance of some conditional non-sporulating variants with a higher amylase forming activity and to derepression of the enzyme at a low glucose concentration.