Showing papers on "Amylase published in 1977"

Regulation of amylase release from dispersed pancreatic acinar cells.

Abstract: 1. A study has been made of factors influencing release of amylase from dispersed pancreatic acinar cells. 2. In the basal, unstimulated, condition cells released 2-3% of the total amylase present in 30 min. 3. The rate of amylase release was stimulated 50-70% by C-terminal octapeptide of cholecystokinin (CCK-OP, maximally effective concentration, 3 X 10(-10) M); carbamylcholine (maximally effective concentration, 10(-5 M); secretin (maximally effective concentration greater than 10(-6) M); vasoactive intestinal peptide (VIP, maximally effective concentration, 10(-8) M); and adenosine 3':5' monophosphate (cyclic AMP) and guanosine 3':5' monophosphate (cyclic GMP) as well as their dibutyryl derivatives (maximally effective concentrations, 10(-3) M). 4. The responses to CCK-OP or carbamylcholine were potentiated by secretin, VIP or dibutyryl cyclic AMP. 5. The responses to secretin or VIP were potentiated by CCK-OP, carbamylcholine, or dibutyryl cyclic GMP. 6. There appear to be two pathways for the regulation of amylase release from pancreatic acinar cells: one pathway can be stimulated by cholecystokinin or cholinergic agonists, and the response to these stimuli is mediated by cyclic GMP; the other pathway can be stimulated by secretin or VIP, and the response to these stimuli is mediated by cyclic AMP.

Differential serum amylase determination by use of an inhibitor, and design of a routine procedure.

Abstract: We describe a new method for measuring pancreatic and salivary-type amylases in serum that requires no electrophoresis or chromatography. An inhibitor protein (from wheat) with a 100-fold greater specificity for human salivary than for human pancreatic amylase was used to analyze mixtures of the two enzymes. The concentration of pancreatic and salivary amyalase was determined in 141 normal sera (72 men and 69 women). Statistically significant differences were found for serum pancreatic amylase between mean and women, higher values being shown in women. No sex-related difference was found for the salivary component of serum amylase. With this method, the increase in serum amylase activity in pancreatitis was shown to be attributable to the pancreatic component. In mumps, the increase is attributable to the salivary component. In pancreatic insufficiency, serum pancreatic amylase activities were significantly lower than in the controls. Our method is simple and rapid; our results agree well with those of other authors who used chromatographic or electrophoretic methods.

Lead inhibition of enzyme synthesis in soil.

Abstract: Addition of 2 mg of Pb2+/g of soil concident with or after amendment with starch or maltose resulted in 75 and 50% decreases in net synthesis of amylase and alpha-glucosidase, respectively. Invertase synthesis in sucrose-amended soil was transiently reduced after Pb2+ addition. Amylase activity was several times less sensitive to Pb2+ inhibition than was enzyme synthesis. In most cases, the rate of enzyme synthesis returned to control (Pb2+) values 24 to 48 h after the addition of Pb. The decrease in amylase synthesis was paralleled by a decrease in the number of Pb-sensitive, amylase-producing bacteria, whereas recovery of synthesis was associated with an increase in the number of amylase-producing bacteria. The degree of inhibition of enzyme synthesis was related to the quantity of Pb added and to the specific form of lead. PbSO4 decreased amylase synthesis at concentrations of 10.2 mg of Pb2+/g of soil or more, whereas PbO did not inhibit amylase synthesis at 13 mg of Pb2+/g of soil. Lead acetate, PbCl2, and PbS reduced amylase synthesis at total Pb2+ concentrations of 0.45 mg of Pb2+/g of soil or higher. The results indicated that lead is a potent but somewhat selective inhibitor of enzyme synthesis in soil, and that highly insoluble lead compounds, such as PbS, may be potent modifiers of soil biological activity.

Adaptation of rat pancreatic amylase and chymotrypsinogen to changes in diet.

Abstract: This study represents an attempt to determine the effect of dietary protein quality and hypophysectomy on the enzymic adaptability of the pancreas in the rat. The specific enzymes studied were amylase, which was purified by immunologic techniques and chymotrypsinogen (activated), which was isolated by affinity column chromatography. Content and synthesis of each enzyme were accurately determined in relation to total pancreatic protein. When rats were fed a 64% sucrose diet (19% casein), there was a two- to three-fold increase in synthesis of amylase. However, if a poor-quality protein (gelatin, gluten, or zein) was substituted for casein, there was no increase in the synthesis of amylase in response to increased carbohydrate. When rats were fed a 19% sucrose diet (64% casein), there was a significant increase in chymotrypsinogen synthesis. Of the poor-quality proteins, gluten was the only one effective in stimulating synthesis of chymotrypsinogen. Peptides, either free or as part of a protein, were necessary for the stimulation of chymotrypsinogen synthesis. Amylase synthesis in hypophysectomized rats was considerably depressed and unresponsibe to increased carbohydrate. This effect could be partially relieved with hydrocortisone, corticosterone, or thyroxin, but not with growth hormone. Hypophysectomy had little effect on synthesis or content of chymotropsinogen.

Inhibitory effect of colchicines on amylase secretion by rat parotid glands: possible localization in the golgi area

Abstract: Colchicine inhibited amylase secretion by isolated rat parotid glands only 6 h after administration of the drug in vivo. This delayed effect was not the result of the inability of the drug to reach its reaction site. When parotid glands were emptied of their secretory granules by isoproterenol treatment, the subsequent replenishment of cells with granules was inhibited by colchicines. Colchicine concomitantly produced alterations of the Golgi complexes, the cisternae of which were reduced in size and surrounded by clusters of microvesicles. Incubation of parotid glands with colchicines for prolonged durations failed to alter stored amylase secretion as stimulated by isoproterenol, but it inhibited the release of de novo synthesized enzyme. Another colchicines-binding activity, firmly bound to the particular fraction of homogenates, was found, of which a part may represent membrane located microtubular protein. An assembly-disassembly cycle of microtubules appears to exist in the parotid gland, as in the liver. However, only 14 percent of tubulin was found to be polymerized as microtubules in parotid glands as opposed to 40 percent in the liver. The present data suggest that colchicine primarily inhibits the transfer of secretory material towards or away from the Golgi complexes but not the hormone-stimulated secretion of stored amylase.

Yields of yeast growth on starch

Abstract: A survey was made of 81 starch-assimilating yeasts, representing 59 species and varieties, with respect to their capacity for the direct conversion of starch into SCP. The extent of starch conversion by the native amylases of the strains during exponential growth, expressed as yield on starch (final amount of dry biomass formed per unit mass of starch originally supplied), varied over a wide range (0.043–0.590) The highest yields were obtained with strains ofLipomyces starkeyi andL. kononenkoae which converted on the average respectively 84% and nearly 100% of the starch supplied. The rate of starch hydrolysis byL. kononenkoae did not limit its specific rate of growth and SCP production.

Secretion of fluid and amylase in the perfused rat pancreas.

Abstract: 1. The isolated rat pancreas was perfused with physiological salt solutions of varying composition. Flow of pancreatic juice and output of amylase during rest and after stimulation with pure secretin, pure cholecystokinin-pancreozymin (CCK-PZ), caerulein or acetylcholine (ACh) were measured. 2. Basal fluid secretion was abolished replacing perfusion fluid NA+ or Cl- by Tris+ or SO42- respectively. Readmission of Na+ or Cl- caused a transient increase above the normal control level of both fluid and amylase output. Exposure to K+-free solution severely reduced fluid output and K+ readmission resulted in a transient increase in secretory rate. 3. Maximal stimulation with ACh (10(-7) M), CCK-PZ (1-5 X 10(-10) M) or caerulein (10(-10) M) caused marked sustained fluid and amylase secretion. Maximal secretin stimulation (5-7 X 10(-9) M) caused marked sustained fluid but only a small sustained amylase secretion following an initial transient. 4. Under continuous secretin stimulation, replacement of the CO2/HCO3-buffered control fluid by a CO2/HCO3-free Tris buffered solution caused a sharp decrease in pancreatic juice flow. In the absence of extracellular CO2/HCO3-secretin did not evoke fluid or enzyme secretion. In contrast the effects of ACh, CCK-PZ or caerulein were independent on CO2/HCO3-. Monobutyryl cyclic AMP (10(-3) M) caused marked sustained fluid secretion and transient enzyme secretion. The effect was entirely dependent on the presence of CO2/HCO3-in the perfusion fluid. 5. Ouabain (10(-4)-10(-3) M) markedly inhibited both secretin- and caerulein-evoked fluid secretion while caerulein-evoked amylase secretion was hardly affected. Similar findings were made with K+-free solution. 6. The effect of maximal secretin stimulation on amylase secretion was greatly augmented in the presence of a maximally stimulating concentration of caerulein. The effects on fluid secretion of secretin and caerulein were simply additive. The effects of secretin on both amylase and fluid secretion, in the presence of caerulein, were entirely dependent on the presence of CO2/HCO3- in the perfusion fluid. 7. We conclude that two different fluid secretion processes occur in the rat exocrine pancreas. One stimulated by ACh and CCK-PZ, that is independent of extracellular CO2/HCO3- and another stimulated by secretin involving H+ or HCO3-transport. Only the effects of secretin seem to be mediated by intracellular cyclic AMP.

Digestive rhythms in the mussel Mytilus edulis

Abstract: Mytilus edulis L., collected from a mid-tide level on the shore, showed rhythmic changes in mantle fluid pH, crystalline style pH, style length and total protein, and in the amylase activity in the digestive gland. These changes were correlated with the changes in tidal height. Style size may be related to extracellular digestion in the stomach. Style size and amylase content of the style were not significantly correlated with each other. The changes in amylase activity in the digestive gland confirmed the existence of a tidal rhythm for intracellular digestion in M. edulis.

Effects of cytochalasin B on pancreatic acinar cell structure and secretion

Abstract: The effects of cytochalasin B (CB) on pancreatic structure and amylase release were studied by use of pancreatic fragments, isolated acini and isolated acinar cells. In pancreatic fragments and isolated acini CB caused the disappearance of microfilaments underlying the apical plasma membrane, loss of apical microvilli and luminal swelling, the last of which was greatly enhanced by addition of protein secretagogues. CB had no effect on basal amylase release but inhibited bethanechol-stimulated amylase in both fragments and acini. Isolated acinar cells, while retaining overall polarity, had lost most of the apical specialization including the microfilament and microvillous complex. Cells were still able to release amylase in response to bethanechol but this release was not affected by CB. The only structural effect of CB on isolated cells was margination of zymogen granules against the plasma membrane. This was, however, not accompanied by increased amylase release. It is concluded that microfilaments are important in maintaining the pancreatic acinar structure. Interference with this structure by CB leads to inhibition of bethanechol-stimulated amylase release. Microfilaments, however, may not play a direct role in secretion.

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Abstract: Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.

Intracellular uptake and α-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells

Abstract: Intracellular uptake of A23187 and the increased release of amylase and lactate dehydrogenase (LDH) accompanying ionophore uptake was studied using dissociated acinar cells prepared from mouse pancreas. Easily detected changes in the fluorescence excitation spectrum of A23187 upon transfer of the ionophore from a Tris-buffered Ringer's to cell membranes were used to monitor A23187 uptake. Uptake was rapid in the absence of extracellular Ca2+ and Mg2+ (t1/2=1 min) and much slower in the presence of Ca2+ or Mg2+ (t1/2=20 min). Cell-associated ionophore was largely intracellular as indicated by fluorescence microscopy, lack of spectral sensitivity to changes in extracellular Ca2+ and Mg2+, and by equivalent interaction of ionophore with membranes of whole and sonicated cells. A23187 (10 μm) increased amylase release 200% in the presence of extracellular Ca2+ and Mg2+. In the absence of Ca2+ (but in the presence of Mg2+) A23187 did not increase amylase release. A23187 (10 μm) also produced Ca2+-dependent cell damage, as judged by increased LDH release, increased permeability to trypan blue, and by disruption of cell morphology. The cell damaging and amylase releasing properties of A23187 were distinguished by their time course and dose-response relationship. A23187 (1 μm) increased amylase release 140% without increasing LDH release or permeability to trypan blue.

Amylase in the lung.

Abstract: Although elevated amylase levels in serum, pleural fluid, and extracts of tumor tissue in primary lung cancer have been reported, electrophoretic and column-chromatographic studies have not revealed the ectopic production of amylase but have merely shown an increase of amylase activity of chiefly the salivary type in these materials The present study was designed to make clear the nature of the amylase or amylase-like substance in the serum, pleural fluid and tumor extracts, and to determine whether amylase might be produced ectopically in tumor tissues Our data not only confirmed that the hyperamylasemia in some cases of primary lung cancer was due to an increase in salivary type isoamylases, but also showed that the same isoamylase pattern occurs in serum, pleural fluids, and diseased lung tissue of patients with pneumonia However, the elution pattern of amylase in these materials in column-chromatography on Sephadex G-75 Superfine was different from that of salivary amylase On the basis of our observations, it seems reasonable to conclude that the salivary type hyperamylasemia in some cases of primary lung cancer may be due to an increase in the amylase contained in normal lung tissues, resulting from activation and release into the blood stream by some inflammatory process However, ectopic production of amylase was demonstrated in one particular case of primary lung cancer in which a high amylase content and a peculiar isoamylase were found both in the primary and metastatic lesions

Effect of diflubenzuron on growth and carbohydrate hydrolases ofTribolium castaneum

Abstract: The potency of diflubenzuron is much greater in inhibiting growth and development of 1st instar larvae ofTribolium castaneum than of 4th instar larvae, as expressed by death at the apolytic stage and retardation of larval development. A dose-dependent decrease in the activity of trehalase, invertase and amylase was obtainedin vivo with the increase in diflubenzuron concentration. At 5 ppm dietary concentration, a reduction of 37 and 27% in invertase and trehalase activity, respectively, was obtained in 4th instar larvae fed for 3 days on treated diet. The amylase activity was affected to a lesser extent. The observed disturbances of trehalase activity might hamper the supply of glucose needed for chitin build-up and those of invertase and amylase activity might affect feeding. Diflubenzuron does not inhibit these enzymesin vitro; hence, thein vivo effect seems to result from general disturbances in carbohydrate metabolism.

Action pattern, kinetical properties and electrophoretical studies of an alpha-amylase present in midgut homogenates from Rhynchosciara americana (Diptera) larvae.

Abstract: 1. 1. Rhynchosciara americana midgut amylase binds chloride ( K d = 1.6 mM at 37°C ) resulting in a 34-fold increase in the Kcat without affecting the Km, but causing a shift in the optimum pH of the enzyme from 6.8 to 8.0. 2. 2. The ions Br−, NO3− and I− also activate the amylase but with a decreasing efficiency. 3. 3. An Arrhenius plot of activity versus temperature show that the amylase display only one slope with a corresponding apparent energy of activation of 4.8 Kcal/mole. 4. 4.|The amylase is a calcium-dependent metalloenzyme which shows a pure competitive inhibition by Hg2+ and a pure non-competitive inhibition by Cu2+ with Ki respectively of 0.21 mM and 0.23 mM. 5. 5.|The resistance to the sulfhydryl reagents p-mercuribenzoate and iodoacetate together with the high Hg2+Ki suggest that sulfhydryl groups are not essential for the midgut amylase activity. 6. 6. Curves of blue-reducing values showed the midgut amylase is an α-amylase with a multiple attack degree between 1.7 and 6. 7. 7. Polyacrylamide gel electrophoresis of midgut homogenates shows the existence of two major isoamylases one of which is largely predominant. 8. 8. The results are compared with α-amylases from several sources.

Purification and some properties of an extracellular alpha-amylase from Bacteroides amylophilus.

Abstract: A medium was developed to obtain maximum yields of extracellular amylase from Bacteroides amylophilus 70. Crude enzyme preparation, obtained by ammonium sulfate precipitation of cell-free broth, contained six amylolytic isoenzymes that were detected by isoelectric focusing and polyacrylamide gel electrophoresis. One of these amylases was purified by diethylaminoethyl-Sephadex A-50 ion-exchange chromatography and Sephadex G-200 gel filtration techniques. Some properties of the purified extracellular alpha-amylase were: optimum pH, 6.3; optimum temperature, 43 degrees C: PH stability range, 5.8 to 7.5; isoelectric point, pH 4.6; molecular weight, 92,000 (by sodium dodecyl sulfatedisc gel electrophoresis); and sugars causing inhibition, cyclomaltoheptaose, cyclomaltohexaose, and alpha-d-phenylglucoside. In addition, Ca2+ and Co2+ were strong activators,and Hg2+ was a strong inhibitior; all other cations were slightly stimulatory. Dialysis against 0.01 M ethylenediaminetetraacetic acid caused a 58% loss of activity that was restored to 92% of the original by the addition of 0.04 M Ca2+. The enzyme affected a blue-value-reducing-value curve characteristic of alpha-type amylases. The relative rates of hydrolysis of amylose, soluble starch, amylopectin, and dextrin were 100, 97, 92, and 60%, respectively; Michaelis constants for these substrates were 18.2, 18.7, 18.2, and 16.7 mumol of d-glucosidic bond/liter, respectively. The enzyme degraded maize (corn) starch granules to some extent and had relatively little activity on potato starch granules.

Effect of calcitonin on the serum amylase levels after endoscopic retrograde cholangiopancreatography.

Abstract: Calcitonin or placebo was infused in a double-blind study in 30 patients undergoing endoscopic retrograde cholangiopancreatography. Calcitonin was found to have no significant effect on the hyperamylasemia following this procedure.

Production and Purification of Thermostable Amylase and Protease of Thermomonospora viridis

Abstract: Maximum yields of amylase were produced by the thermophilic actinomycete Thermomonospora viridis in a modified Simpson and McCoy medium containing 1.5% corn starch and 0.5% mycological peptone with an initial pH 7.0. Best yields of amylase were obtained after incubation for 48 h, when the pH of the medium had risen to 8.2. Amylase was purified 313-fold by precipitation with n-propyl alcohol, dialysis against tap water, adsorption on Ca3(PO4)2, and fractionation on Sephadex G-100. Protease was produced in nutrient broth containing 0.5% starch and 1.0% corn steep liquor and at an initial pH 7.0. Maximum yields of protease were produced after 42 h. The protease was purified 54-fold by precipitation with n-propyl alcohol, dialysis against tap water, adsorption on Ca3(PO4)2, and fractionation on Sephadex G-200.

Influence of heavy metals in spruce forest soil on amylase activity, CO2 evolution from starch and soil respiration

Abstract: Starch decomposition and soil respiration is partly inhibited in spruce needle mor contaminated with heavy metals (Cu, Zn, Pb and Cd) from a brass foundry at Gusum, Southern Sweden. The total decomposition of starch was measured as CO2 evolution rate and the hydrolysis of starch as increase in glucose concentration during incubation according to the methods of Hoffman and Pallauf and that of Nelson. In order to measure the effects of the Na-acetate buffer (pH 5.5) during incubation, amylase activity was also determined without buffer. Only the Hoffmann and Pallauf method without buffer was significantly different (p<0.001) from the other three methods. Application of stepwise regression showed that 43 to 62 per cent of the variability in amylase activity was accounted for by the metal- and hydrogen concentration of the soil. Corresponding figures for the total starch decomposition and soil respiration were 47 and 66 per cent respectively. re]19751213

Differentiation between the calcium-dependent effects of cholecystokinin-pancreaozymin and the bicarbonate-dependent effects of secretin in exocrine secretion of the rat pancreas.

Abstract: 1. Differentiation between the secretory effects of pure natural cholecystokinin-pancreozymin (CCK-PZ) and those of synthetic secretin was studied in the isolated pancreas of the rat perfused with a standard solution containing both Ca2+ and HCO2-, a Ca2+ deficient solution, or a HCO3-deficient solution. 2. Secretin induced a dose-dependent increase in the flow of pancreatic juice and a slight but definite increase in amylase output which was also dependent upon the dose of secretin. 3. The increase in the flow of pancreatic juice, induced by continuous stimulation with secretin (5 Ivy dog m-u./ml.), was completely abolished during perfusion with a CHO3- deficient solution, but was only slightly suppressed during perfusion with a Ca2+-deficient solution. 4. The increase in flow, induced by continuous stimulation with CCK-PZ (5 Ivy dog m-u./ml.), was partly affected by the deprivation of HCO3-, but was strongly inhibited by the deprivation of Ca2+. The CCK-PZ-induced amylase output was not affected by the deprivation of HCO3-, but was significantly inhibited by the deprivation of Ca2+. 5. The present results favour the view that CCK-PZ acts on the acinar cells to increase both amylase output and juice flow, whereas secretin acts on centro-acinar and terminal duct cells to increase mainly juice flow.

Secretory behaviour of hypertrophic and hyperplastic salivary gland.

Abstract: The enzyme content and the secretory behaviour of normal rat salivary glands were compared with these properties in glands made hypertrophic and hyperplastic by the chronic administration of isoproterenol. The enlarged glands displayed reductions in the concentrations of ribonuclease, deoxyribonuclease and amylase. The secretory behaviourin vivo was similar for all enzymes in both types of glands, but the enlarged glands secreted a lower percentage of their contentin vitro. The reduction in amylase activity was shown by immunological techniques to be due to a reduction in the number of enzyme molecules. The reduction in ribonuclease activity was not due to changes in the level of ribonuclease inhibitors.

Macroamylasaemia after treatment with hydroxyethyl starch.

Abstract: After infusion of 500 ml of 6% hydroxyethyl starch into fifty-four patients an increase of serum amylase was observed which in fifty-one cases exceeded the upper limit of normal (190 U/l). In most cases serum amylase reached twice the basal value. Renal function influenced the duration of the increase in serum amylase, but not the maximum increase (201+/-15 U/l; mean+/-SEM). In patients with advanced renal failure (glomerular filtration rate (GFR) = 2-10 ml/min) serum amylase was still markedly elevated after 72 h (298+/-24 U/l; mean+/-SEM). In patients with normal renal function (GFR greater than 90 ml/min) serum amylase decreased to 183+/-40 U/l (mean+/-SEM) within 72 h without reaching basal values. After infusion of HES no changes were observed in serum lipase or in amylase or lipase activities in duodenal secretion. Amylase excretion in the urine decreased. The assumption of a macroamylasaemia caused by formation of an HES-amylase complex was confirmed by gel filtration. The elimination from plasma of this high molecular enzyme-substrate complex is slow and causes hyperamylasaemia. In no case was the macroamylasaemia associated with signs or symptoms. An awareness of this causal relationship seems to be important, to avoid the erroneous diagnosis of a pancreatic disease.

The Human α-Amylases

Abstract: About 12 years ago in 1965, Kamaryt and Laxova105,106 suggested a genetic basis for the inheritance of salivary and pancreatic amylase in human serum. Although results from these and other early isozyme studies of α-amylase differed when obtained from saliva, pancreas, serum, and urine,157 it was not until extensive studies performed after 1969 that the multiple isozymes demonstrable in various tissue fluids and extracts were shown to be the heterogeneous products of two loci, Amy 1 (salivary-type amylase) and Amy 2 (pancreatic-type amylase).