Showing papers on "Amylase published in 1976"

Gibberellic acid enhances the level of translatable mRNA for α-amylase in barley aleurone layers

Abstract: α-AMYLASE is synthesised de novo in response to the plant hormone gibberellic acid (GA3) in the aleurone layers of barley grains1. This enzyme induction depends on RNA and protein synthesis, as determined by the use of inhibitors2–4, and it occurs after a temperature-dependent lag period5. The biochemical events of the lag period have been studied with respect to ionic requirements5–7, early protein synthesis8, RNA synthesis9–12, polysome formation13 and lipid synthesis and turnover14–17. It has been postulated that new mRNA for α-amylase synthesis might be produced in response to the hormone2,3 and there is evidence that GA3 enhances the incorporation of labelled ribonucleosides into poly(A)-containing RNA of barley aleurone layers18–20. Stimulation could be detected about 4 h after hormone treatment and although GA3 probably stimulates synthesis of a limited number of proteins, there was stimulation of a wide size range of poly(A)-containing RNAs. Thus it was not possible to say that GA3 had a selective effect on the synthesis of the mRNA for α-amylase.

Pancreatic acinar cells: the role of calcium in stimulus-secretion coupling.

Abstract: 1. Segments of mouse or rat pancreas were placed in a flow cell through which physiological salt solutions of varying composition were pumped at a constant rate. Intracellular recordings of membrane potential, resistance and electrical time constant were made from the acini using fine glass micro-electrodes. In some experiments two micro-electrodes were inserted into two acinar cells within the same acinus to assess directly cell to cell coupling. The concentration of amylase in the effluent was measured continuously. 2. Electrical coupling between two acinar cells was observed when the tips of the two micro-electrodes were less than 50 mum from each other. The coupling ratio was close to 1. Acetylcholine (ACh) always evoked depolarization of exactly the same amplitude in two coupled cells and reduced the amplitude of current-pulse induced membrane potential changes in both cell simultaneously. 3. Stimulation with ACh caused an immediate increase in amylase output. Replacement of superfusion fluid Na by Tris or Cl by sulphate abolished ACh-evoked increase in amylase release, but the subsequent reintroduction of Na or Cl caused an increase in amylase release of a magnitude similar to what was normally observed following stimulation. 4. Omitting Ca from the superfusion fluid and adding EGTA rapidly depolarized the acinar cell membrane, reduced the input resistance and caused a marked reduction in amylase secretion. During exposure to a Ca-free, EGTA containing solution a marked increase in amylase release occurred following maximal ACh stimulation. 5. Addition of small amounts of Mg, Ca or Mn to a Ca-, Mg-free solution caused an increase in membrane potential, input resistance and electrical time constant and markedly increased amylase release. The effect on the electrical parameters was reversed in the absence of extracellular Na while extracellular Na was of no importance for the effect on amylase release. 6. The effect of ACh on amylase was enhanced during superfusion with a fluid containing 20 mM-Ca. The presence of Mn (5 mM) in an otherwise normal control had no effect on ACh-evoked release. 7. These results show that ACh acts on the acinus by reducing the surface cell membrane resistance. It is suggested that the ACh-receptor interaction causes a release of Ca from the surface cell membrane and that the concentration of Ca in the surface cell membrane determines the specific membrane resistance particularly for Na. The release of Ca to the cytosol activates exocytosis while the Na influx is of importance for acinar fluid secretion. The effect of ACh on amylase secretion can be mimicked by agents displacing membrane-bound Ca (Mg, Ca, Mn).

Technique for fractionation of bacteria in rumen microbial ecosystem

Abstract: Corn starch was better both in the amount of bacteria attached per unit weight of starch and in easiness of handling than other starches from different origin as adsorbent for bacterial attachment. The attachment of bacteria to starch occurred shortly after the addition of starch granules, and a maximum attachment was attained after further 5-min incubation at 38°. The amount of bacteria attached to starch granules was approximately proportional to the quantity of granules added until 2.5% in weight per volume. Effect of environmental factors on the attachment of rumen bacteria to starch was examined. The attachment of bacteria to starch was maximal in a medium containing sodium carbonate above 0.15%. Carbon dioxide as gas phase was far better in the attachment of bacteria to starch than hydrogen and nitrogen gases. There existed bacteria capable of attaching to starch even at 4° or for the first time at above 30°. The amount of bacteria attached to starch was small at 4° and more abundant at 38°.A trial was made to elute bacteria attached to starch granules first with a salt solution and subsequently with Formalin. The results obtained showed that bacteria attached to starch, excepting those attached at 4°, included the following three types of bacteria. The bacteria of thefirst type were those loosely attaching to starch, that were eluted easily with a salt solution. The bacteria of the second type were those firmly attached and were eluted for the first time with Formalin. The bacteria of the third type were those with irreversibly attaching ability, that were not eluted even with Formalin.The most characteristic properties of bacteria attached to starch were their amylase and urease activities. The specific amylase activity of bacteria attached to starch was remarkably high, compared with that of non-attached bacteria. There was little or no specific urease activity of attached bacteria, while that of non-attached bacteria was relatively high. There was no significant difference in the specific activity of other four hyrolases between bacteria attached and non-attached to starch.The actual role of bacteria which are apt to attach to starch in the rumen in situ is also discussed.

Human pancreatic secretory trypsin inhibitor.

Abstract: Human PSTI has been isolated from human pancreatic tissue and juice by procedures developed for the preparation of secretory trypsin inhibitors from bovine [2] and porcine [3] pancreatic juice. Figure 1 shows the Sephadex G-75 elution diagram of human pancreatic juice. The inhibitor activity (o—o) is retarded relative to the bulk of the secretory protein with amylase activity present in the A280nm absorbing peak eluted after the inhibitor.

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

Abstract: 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 α-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 α-amylases described in the literature, Tenebrio molitor amylase has a lower affinity for starch.

Environmental determination of selective significance or neutrality of amylase variants in drosophila melanogaster

Abstract: Strains homozygous at the amylase locus were derived from a polymorphic laboratory population of Drosophila melanogaster . The Amy 4,6 strain has higher enzyme activity than the Amy 1 strain.—Maltose has the same nutritional value as starch.—The effect of starch in pure culture depends on the yeast level. At low yeast level increasing starch increases survival, at high yeast level increasing starch increases mean dry weight. The strains do not differ in survival or mean dry weight in pure culture.—In mixed cultures at 50% input of Amy 4,6 and Amy 1 as larvae the percentage Amy 4,6 in adults increases with increasing starch at low yeast levels, but equals input frequency at high yeast levels. No increase in percentage Amy 4,6 in adults is present with increasing maltose at low yeast levels in mixed culture. The increase in percentage Amy 4,6 with increasing starch must be due to selection on the amylase locus working by competition for food in the larval stage. The single locus selection coefficient is determined by the environment and can reach quite high values.—Viability selection in the presence of starch is in the direction indicated by the enzyme activities.

The potentiating influences of insulin on pancreozymin-induced hyperpolarization and amylase release in the pancreatic acinar cell.

Abstract: 1. Insulin (1 mu./ml.) potentiated the release of amylase from isolated pancreas of rats perfused with erythrocyte-containing medium and stimulated by 0-5 m-u. pancreozymin (Pz)/ml., whereas the same concentration of insulin failed to potentiate the response evoked by or 200 m-u. PZ/ml. 2. Intracellular measurement of membrane potentials from the acinar cells of the same preparations showed that insulin (1 mu./ml.) simultaneously potentiates the hyperpolarization and amylase release in response to 0-5 m-u. PZ/ml. 3. These effects of insulin on the PZ-induced responses were inhibited by ouabain (3 X 10(-5) M). 4. The results suggest that insulin, in the concentration studied, has a potentiating action on the activity of an electrogenic Na pump, which maintains the hyperpolarization and amylase release during continuous stimulation with Pz. 5. Insulin in also potentiated the Pz-induced amylase release in the rat pancreata in situ after vagotomy and ligature of the pyloric region.

Amino sugar derivatives

Abstract: New amino sugars which are glucopyranosyl and oligoglucosidyl derivatives of 4,6-bisdesoxy-4-(4,5,6-trihydroxy-3-hydroxymethylcyclohex-2-en-1-ylamino)-α-D-glucopyranose inhibit glycoside hydrolases of the digestive tract. The compounds, of which O-{4,6-bisdesoxy-4-[1S-(1,4,6/5)-4,5,6-trihydroxy-3-hydroxymethylcyclohex-2-en-1-ylamino]-α-D-glucopyranosyl}-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose is a representative embodiment, demonstrate both saccharase and amylase inhibiting properties.

Rapid chemical analysis of some plant constituents

Abstract: Simple reproducible methods using a minimum of equipment are described for the routine analysis of soluble sugars, starch, total nitrogen and phosphorus in plant material. Soluble sugars are extracted with 62.5% methanol and the sugars estimated by the phenol–sulphuric acid technique. Starch in the residual tissue is digested by an amyloglucosidase and glucose is determined by a glucose oxidase method. Both nitrogen and phosphorus are assayed after wet ashing the plant tissue—nitrogen by the phenol–hypochlorite reaction and phosphorus after reduction of the phosphomolybdic acid with ascorbic acid/antimony potassium tartrate.

Starch degradation in the cotyledons of germinating lentils

Abstract: Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the alpha type. Starch is degraded slowly in the first days; during this time, alpha- and beta-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in alpha-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three alpha-amylases, and one beta-amylase. Based on their heat lability or heat stability, two sets of alpha-amylase seem to exist in lentil cotyledons.

Stable, inducible thermoacidophilic alpha-amylase from Bacillus acidocaldarius.

Abstract: Bacillus acidocaldarius Agnano 101 produces an inducible thermoacidophilic alpha-amylase. The enzyme production occurs during the stationary phase of growth in the presence of compounds with alpha-1,4-glucosidic linkages. The enzymatic activity is both present in the culture medium and associated with the cells; the enzymes purified from both sources show identical molecular and catalytic properties. The purified amylase has a single polypeptide chain of molecular weight 68,000 and behaves like an alpha-amylase with affinity constants for starch and related substances of 0.8 to 0.9 mg/ml. The pH and temperature optima for activity are 3.5 and 75degreesC, respectively. The amylase is stable at acidic pH (below 4.5). Its thermal stability is strictly dependent upon protein concentration; the half-life at 60degreesC of the amylase in a 70-mug/ml solution is about 5 days.

Effects of ions on amylase release by dissociated pancreatic acinar cells.

Abstract: Dissociated acinar cells prepared from guinea pig and mouse pancreas were intact on the basis of structure, ion content, and their ability to increase release of amylase in response to bethanechol ...

Electrophoretic pattern of amylase isoenzymes in serum and urine of normal persons.

Abstract: We separated and measured amylase isoenzymes in the serum and urine of 3036 normal persons by electrophoresis on a thin layer of polyacrylamide gel. We wished to establish the normal pattern of these isoenzymes and to evaluate the usefulness of this method of electrophoresis in clinical diagnosis. Results for patients with hyper- or hypofunctioning pancreas and salivary glands suggested that essentially all the isoamylases in human serum and urine are derived from the salivary glands and the pancreas, and revealed that isoamylases of more than 98% of normal persons consisted of two major isoenzymes and two to three minor ones. Although these observations indicate that data on changes in the proportion of amylase activity of each isoenzyme can be useful in clinical medicine, the following points should be remembered: (a) quantitative differences in the isoenzyme pattern were observed, depending upon the condition of the samples; (b) because the proportion of isoamylase activity in serum of different normal persons differs, seriatim determination of amylase isoenzymes is necessary; and (c) because five different genetically controlled types of isoamylases were observed in normal persons, genetic investigations are also necessary.

An evaluation of the usefulness of amylase isozyme differentiation in patients with hyperamylasemia.

Abstract: Amylase assays measure total activity without differentiating the relative contributions of pancreatic- and salivary-type amylase isozymes. Since polyacrylamide electrophoresis allows identification of salivary-and pancreatic-type isoxymes and their respective variants, serum and urine specimens from patients with the clinical diagnoses of mumps (4), pancreatitis (16), or undiagnosed hyperamylasemias (5) were compared with specimens from control subjects. Patients with mumps had elevations of salivary-type isozymes, while those with pancreatitis had elevations of pancreatic-type isozymes. Elevation of salivary-type isozymes was identified in the five patients who had undiagnosed hyperamylasemias; among these, the isozymes of two originated in neoplastic ovarian tissue and those of three, probably in the salivary glands. Amylase isozyme differentiation cannot unamibiguously identify the tissue source of hyperamylasemia. However, in patients whose hyperamylasemia is of unknown etiology or who respond atypically to therapy, amylase electrophoresis provides identification of the elevated isozyme type, thus providing the basis for the rational selection of further diagnostic procedures.

Infant human pancreas. A potential source of islet tissue for transplantation.

Abstract: Twelve pancreases from human infants one year old or less were analyzed for tissue insulin and amylase content before and after dispersal of pancreatic fragments by mincing and collagenase digestion. Tissue insulin and amylase content provide an index of pancreatic islet mass and exocrine digestive enzyme content, respectively. The results were compared with similar anaylses performed on juvenile and adult human pancreases before and after islet isolation and on intact and dispersed neonatal rat and adult rat pancreas. Infant human pancreas has an average tissue insulin concentration of 1,128 μg./gm. of tissue and a total insulin content of 1,718 μg/pancreas, as against values of 140 μg./gm. of tissue and 7,209 μg./pancreas for adult human pancreas. Average tissue amylase concentration is 0.24 mg./gm. of tissue in infant human pancreas and 3.0 mg./gm. of tissue in adult human pancreas. The insulin/amylase ratio in infant pancreas is 4,800, as against 46 in the adult pancreas. Neonatal rat pancreas, which can be dissociated and transplanted without separation of islet and exocrine components, has a similarly high tissue insulin and low tissue amylase content when compared with adult rat pancreases. Infant human pancreas has a total islet mass 24 per cent that of an adult human pancreas, and neonatal rat pancreas has a total islet mass 11 per cent of that of an adult rat pancreas. One neonatal rat pancreas prepared by minimal collagenase digestion can cure diabetes when transplanted via the portal vein to a rat. Following dispersal of infant human pancreas by collagenase digestion, the islet content and the insulin/amylase ratio of the recovered tissue equals or exceeds that which usually can be isolated from adult cadaver pancreases. Infant human pancreas is a rich source of islet tissue that is relatively uncontaminated by exocrine digestive enzymes. After dispersal, infant human pancreas may be ideal for transplantation to selected diabetic patients.

Enzymic Activities in the Pancreas, Digestive Tract and Feces of Rats Fed Raw or Heated Soy Flour

Abstract: Trypsin, chymotrypsin and amylase levels were determined in the pancreas, all along the intestinal tract, and in the feces of rats fed raw or heated soy flour diets. The levels of all enzymes measured in the pancreas in the non-fasted state were lower in the raw than in the heated soy flour-fed rats. Fasting equalized these levels. Trypsin and amylase tended to be lower, and chymotrypsin was significantly higher in the intestinal tracts of raw soy flour-fed rats than in the group fed heated soy flour; the greatest differences were found in the ileum. Trypsin and chymotrypsin levels in the feces were higher in the group fed raw soy flour than in the group fed heated soy flour. Amylase in the feces of the raw soy flour-fed rats was higher at the beginning of the experiment and dropped sharply to be even lower than in the heated soy flour-fed rats at days 13 to 14 of the experiment. It was concluded that measuring the enzymic levels in the feces is a very sensitive method for determining whether a test diet induces hypersecretion of digestive enzymes in rats. This method can be used from the start of feeding the experimental diet. As the animal need not be killed, the effect of the test diet upon enzymic secretion can be studied as a function of time, and it might be suitable to studies with large animals.

Purifications and Enzymatic Properties of β-Amylase and Pullulanase from Bacillus cereus var. mycoides

Abstract: A β-amylase and a pullulanase produced by Bacillus cereus var. mycoides were purified by means of ammonium sulfate fractionation, adsorption on starch and celite and Sephadex G–100 column chromatography. The purified enzymes were homogeneous in disc electrophoresis.The β-amylase released only maltose from amylose, amylopectin, starch and glycogen, and the released maltose was in β-form. The pullulanase released maltose, maltotriose and maltotetraose from β-limit dextrin and maltotriose from pullulan, but not amylose-like substance from amylopectin.The optimum pHs of β-amylase and pullulanase were about 7 and 6~6.5, respectively. The optimum temperatures of the enzymes were about 50°C. The enzymes were inhibited by the sulfhydryl reagents such as mercuric chloride and p-chloromercuribenzoate, and the inhibitions with p-chloromercuribenzoate were restored by the addition of cysteine. The molecular weights of β-amylase and pullulanase were estimated to be 35,000±5,000 and 110,000±20,000, respectively.

Turku sugar studies VII:Principal biochemical findings on whole saliva and plaque

Abstract: Plaque and whole saliva samples of the subjects of the Turku sugar studies were analyzed for several enzymes and biochemical compounds. Strict xylitol diet maintained throughout the study a 50% lower quantity of plaque than the sucrose of fructose diets. Decreased plaque and whole saliva lactate concentration, diminished activity of salivary amylase, and reduced hydrolysis rate of sucrose in plaque and whole saliva were observed in relation to xylitol consumption. The xylitol diet also reduced the ratio of glucose to proteins in plaque. On the other hand, increased activity in plaque of alpha- and beta-glycosidases (against p- and o-nitrophenyl derivatives), fucosidase and aspartate transaminase, as well as increased activity of proteinases and lactoperoxidase in saliva were found in connection with xylitol consumption. The fructose diet caused less clear differences when compared to sucrose, but the experiments indicated a selectivity of the effects of dietary carbohydrates on the biochemistry of whole saliva, plaque and salivary glands. The results contribute in explaining the cariostatic effects of xylitol and the lower coriogenicity of fructose when compared to sucrose.

Salivary amylase in duodenal aspirates.

Abstract: Salivary and pancreatic isoamylases in duodenal aspirates obtained during assessment of pancreatic function after test meal stimulation were separated by agarose gel electrophoresis. Salivary amylase was found to be a constituent of the duodenal aspirates in more than 75% of the tests. The mean relative contribution of salivary amylase to the total amylase activity of the aspirates varied from about 15% in normals to about 40% in patients with chronic pancreatitis and pancreatic carcinoma. The amount of salivary amylase varied widely not only between the individuals but also within the samples of the same test series. Specific determination of the pancreatic isoamylases instead of determination of the total amylase increased the discrimination between normals and patients with pancreatic dysfunction.

Effects of oxygen supply on electrical and secretory responses of humorally stimulated acinar cells in isolated rat pancreas

Abstract: A technique is described for perfusing isolated rat pancreas with a modified Krebs-Henseleit solution This preparation made feasible the simultaneous measurement of transmembrane potential, effective membrane resistance, rate of flow of pancreatic juice and amylase output The hyperpolarizing effect of cholecystokinin-pancreozymin (CCK-PZ) was confirmed by perfusion with a solution containing 5 mU CCK-PZ/ml The hyperpolarizing efffect of CCK-PZ was enhanced when the pancreas was perfused with an oxygenated solution containing dog erythrocytes This effect was inhibited by ouabain and anaerobic conditions It is suggested that the oxidative metabolism supplies energy for a mechanism which is responsible for the hyperpolarizing effect of CCK-PZ The amylase output and the rate of flow of pancreatic juice were enhanced concurrently under conditions that augumented the hyperpolarizing effect, and they were inhibited under conditions that suppressed the effect Cellular events in stimulus-secretion coupling in the pancreatic acinar cell are discussed by correlating the electrophysiological responses and the secretory responses under various conditions

Stimulus-secretion coupling in pancreatic acinar cells: inhibitory effects of calcium removal and manganese addition on pancreozymin-induced amylase release.

Abstract: The role of Ca ions in stimulus-secretion coupling has been analysed in the isolated and perfused rat pancreas. 2. The omission of [Ca2+]O diminished but did not abolish the release of amylase in response to continuous stimulation with 5 m-u. pancreozymin (Pz)/ml. The addition of Mn2+ (1-0 mM) to this Ca-deficient environment abolished the residual release of amylase. This was followed by a complete recovery of amylase output when the control [Ca2+]O was reestablished. 3. The addition of Mn2+ (1-0 mM) to the extracellular environment containing 2-5 mM-Ca2+ reversibly inhibited the Pz-induced release of amylase. 4. A kinetic scheme based on competition of Ca and Mn at a carrier in the acinar cell membrane could quantitatively explain the effects of Ca and Mn upon the Pz-induced amylase release. 5. These results support the view that the Ca2+ influx into the acinar cells is the major contributor to the rise in [Ca2+]i which, in turn, mediates the processes in the stimulus-secretion coupling in the exocrine pancreas, and suggest that the mode of Ca influx is a facilitated diffusion.