Showing papers on "Amylase published in 1986"
TL;DR: In this paper, a rapid enzymic method for starch analysis, especially in cereal products, was presented, which includes a 15 min gelatinization step in a boiling water bath in the presence of a thermostable α-amylase, a 30 min amyloglucosidase incubation of a subsample, and subsequent determination of glucose with a glucose oxidase/peroxidase reagent.
Abstract: A rapid enzymic method for starch analysis, especially in cereal products, is presented. One person can analyze 30 samples per day. The method includes a 15 min gelatinization step in a boiling water bath in the presence of a thermostable α-amylase, a 30 min amyloglucosidase incubation of a subsample, and subsequent determination of glucose with a glucose oxidase/peroxidase reagent. The method was evaluated by analysis of the starch content in various raw and industrially processed wheat samples. The method showed high precision (CV=0.6–1.0%) and accuracy. Some factors which might affect the enzymic availability of starch and thus its recovery in the analysis are evaluated and discussed.
580 citations
TL;DR: In this paper, the authors identified three key factors that influence yields of resistant starch after heat processing, i.e. amylose content, processing temperature and water content, and found that the highest yields of RS (20−34% of total dry weight) were obtained from amylomaize starches, either raw or processed, and the lowest yields of amylopectin starches (32−46% RS) after incubation with α-(1→6)-debranching enzyme (pullulanase) followed by heat processing.
477 citations
TL;DR: High levels (2-565 units/g) of amylase activity were observed in human faeces, and mixed populations of gut bacteria rapidly fermented starch with the production of volatile fatty acids and organic acids.
Abstract: High levels (2-565 units/g) of amylase activity were observed in human faeces. Over 92% of amylase activity in faeces obtained from healthy persons was extracellular, whereas only about 9% of activity was associated with particulate material and washed cells. Bacterial cell-bound amylases were considerably more efficient in breaking down starch, however, than were the soluble enzymes which occurred in cell-free faecal supernatant fluids. Cell population densities of anaerobic starch-hydrolysing bacteria in the stools of ten persons ranged from 1.1 X 10(10) to 3.3 X 10(12)/g of faeces. Identification of 120 starch-hydrolysing colonies isolated from the stools of six subjects showed that the predominant amylolytic bacteria belonged to the genera Bifidobacterium, Bacteroides, Fusobacterium and Butyrivibrio. Mixed populations of gut bacteria rapidly fermented starch with the production of volatile fatty acids and organic acids. Lactate was observed to be a major, though transient intermediate during starch fermentation by these cultures. Approximately 60% of starch utilized was converted to volatile fatty acids, which in the human colon would be potentially available for absorption.
257 citations
TL;DR: In this article, a comparison of resistant starch (RS) and readily digestible starch (RDS) for comparison were made with cell-free supernatants from faecal suspensions and washed faecic bacterial cell suspensions.
Abstract: Cooking and processing of starch-containing foodstuffs results in a portion of the starch becoming resistant to hydrolytic enzymes secreted in the small intestine of man. In order to determine whether this resistant starch (RS) was degraded in the colon, samples of RS and readily digestible starch (RDS) for comparisons were incubated with (a) cell-free supernatants from faecal suspensions and (b) washed faecal bacterial cell suspensions. The data obtained showed that, whereas pancreatic amylase and faecal supernatants hydrolysed RDS, with the production of oligosaccharides, RS totally resisted breakdown. In contrast, both RS and RDS were completely degraded by the washed bacterial cells with the generation of volatile fatty acids (VFA) and organic acids. Hydrolysis and fermentation of RDS was extremely rapid and, as a consequence, oligosaccharides and lactate initially accumulated in the culture medium. RS was broken down more slowly, however, and oligosaccharides and lactate never accumulated. The rate of polysaccharide hydrolysis had a significant effect on the quantities of VFA produced, in that 54% of carbohydrate was fermented to VFA in cultures incubated with RDS as sole carbon source as compared to only 30% in cultures incubated with RS. However no qualitative difference was observed in the VFA produced by fermentation of RDS or RS.
232 citations
TL;DR: It appears that the pig has sufficient pancreatic and gastric enzyme activity so that performance should not be limited, with the possible exception of the period shortly after weaning.
Abstract: Thirty-seven pigs were used to evaluate the effects of age and weaning on the level of protease in the gastric mucosa and trypsin, chymotrypsin, amylase and lipase in the pancreas. There was a positive allometry of the pancreas and gastric mucosa associated with age and with weaning to a solid diet. Increases with age in total activity of chymotrypsin, trypsin, amylase and gastric proteases were due to increases in both tissue weight and enzyme activity per gram of tissue. A general depression in pancreatic enzymatic activities, but not in gastric proteolytic activity, was found during the first week following weaning. Forty pigs were used in a second trial to evaluate the effects of age and weaning diet on the same digestive enzymes. Total activity of all enzymes assayed increased with time postweaning. Increases in total activity of lipase and chymotrypsin were due primarily to increased pancreatic weight postweaning. Amylase, trypsin and gastric protease increases were due both to increased tissue weight and increased activity per gram of tissue. There were no effects of diet on the weight of gastric mucosa or the level of activity of the gastric proteases. Pigs fed a diet containing 20% whey had larger pancreases (P less than .10) at slaughter and a greater, but nonsignificant, mean activity per gram of pancreas for all pancreatic enzymes. It appears that the pig has sufficient pancreatic and gastric enzyme activity so that performance should not be limited, with the possible exception of the period shortly after weaning. However diet digestibility and subsequent pig performance may be more directly related to the extent of release of these enzymes into the intestine and the conditions that exist therein.
218 citations
TL;DR: A 650-nucleotide cDNA from barley aleurone layers encoding a protein that is closely related to a known α-amylase inhibitor from Indian finger millet, and that has homologies to certain plant trypsin inhibitors is cloned and sequenced.
Abstract: We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known α-amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for α-amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to α-amylase. PAPI protein is secreted from aleurone layers into the incubation medium.
184 citations
TL;DR: It is concluded that more than 95% inhibition of amylase reduces dietary starch digestion within the small intestine and uptake of dietary starch from theSmall intestine, markedly decreases postprandial release of insulin and gastric inhibitory polypeptide, and may alter postpr andial upper gastrointestinal motor function.
158 citations
TL;DR: It was found that as the enzymes moved from duodenum to ileum, 74% of amylase activity, 22% of trypsin activity, and 1% of lipase activity survived transit, suggesting that for these enzymes the sites of enzymatic activity and immunorecognition are not identical.
Abstract: To determine survival of pancreatic enzymes during small intestinal aboral transit in humans, seven healthy volunteers were intubated with an oroileal tube. By using nonabsorbable markers we measured the cumulative amount of lipase, trypsin, and amylase activities and lipase and trypsin immunoreactivities delivered postprandially to the duodenum, midjejunum, and terminal ileum. We found that as the enzymes moved from duodenum to ileum, 74% of amylase activity, 22% of trypsin activity, and 1% of lipase activity survived transit. Enzymatic activity and immunoreactivity of trypsin and lipase disappeared at different rates, suggesting that for these enzymes the sites of enzymatic activity and immunorecognition are not identical. Since tryptic activity is present even in the absence of immunorecognizable trypsin, complete structural integrity of the trypsin molecule may not be essential for its enzymatic activity. The short intraluminal survival of lipolytic activity may partially explain why patients with progressive exocrine pancreatic insufficiency malabsorb fat earlier than other nutrients.
138 citations
TL;DR: Effects of age and diet composition on amylase, trypsin and chymotrypsin activities in the Pancreas and intestinal contents, pancreas weights and body weights were determined from birth to 56 d.
Abstract: Effects of age and diet composition on amylase, trypsin and chymotrypsin activities in the pancreas and intestinal contents, pancreas weights and body weights were determined from birth to 56 d A total of 120 pigs, five to seven pigs/litter from 18 litters, were slaughtered at birth, 14, 27, 29, 31, 42 and 56 d Litters were allotted to dietary treatments (corn-soy, A; corn-soy + 20% dried whey, B; corn-soy + 5% lard, C) and offered these diets as creep feed at 14 d All pigs were weaned at 28 d, placed in elevated nursery pens and fed their respective diets Total activities of amylase, trypsin and chymotrypsin in the pancreas and small intestine increased (P less than 05) with age Both trypsin and amylase activities, measured per kilogram body weight or gram pancreas weight, were low at 29 d in the intestine and increased to 56 d Pigs on diet B had the highest level of trypsin and chymotrypsin in the intestinal contents (P less than 05) Trypsin activity in the pancreas (units/kg body weight) was lowest (P less than 05) for pigs on diet B and highest (P less than 05) for those on diet C (units/g pancreas and units/kg body weight) Amylase activity (units/kg body weight) was lower (P less than 05) in the pancreas for pigs on diet B than for those on diets A and C Pigs on diet A had lower (P less than 01) intestinal amylase activities than those on diets B and C Enzyme activities in the intestinal contents and pancreas were low following weaning In the pancreas, activities decreased at 31 d(ABSTRACT TRUNCATED AT 250 WORDS)
110 citations
TL;DR: Amylase production was higher on complex and semisynthetic medium than on synthetic medium supplemented with amino acids, and Amino acids, in particular isoleucine, cysteine, phenylalanine, and aspartic acids, were found to be vital for amylase synthesis.
Abstract: Bacillus stearothermophilus grew better on complex and semisynthetic medium than on synthetic medium supplemented with amino acids. Amylase production on the complex medium containing beef extract or corn steep liquor was higher than on semisynthetic medium containing peptone (0.4%). The synthetic medium, however, did not provide a good yield of extracellular amylase. Among the carbohydrates which favored the production of amylase are, in order starch > dextrin > glycogen > cellobiose > maltohexaose-maltopeptaose > maltotetraose and maltotriose. The monosaccharides repressed the enzyme production, whereas inositol and d-sorbitol favored amylase production. Organic and inorganic salts increased amylase production in the order of KCI > sodium malate > potassium succinate, while the yield was comparatively lower with other organic salts of Na and K. Amino acids, in particular isoleucine, cysteine, phenylalanine, and aspartic acids, were found to be vital for amylase synthesis. Medium containing CaCl2 2H2O enhanced amylase production over that on Ca2+ -deficient medium. The detergents Tween-80 and Triton X-100 increased biomass but significantly suppressed amylase synthesis. The amylase powder obtained from the culture filtrate by prechilled acetone treatment was stable over a wide pH range and liquefied thick starch slurries at 80°C. The crude amylase, after (NH4)2SO4 fractionation, had an activity of 210.6 U mg−1. The optimum temperature and pH of the enzyme were found to be 82°C and 6.9, respectively. Ca2+ was required for the thermostability of the enzyme preparation.
106 citations
TL;DR: It is reported in AR42J pancreatic acinar cells that glucocorticoids increased the synthesis, cell content, and mRNA levels for amylase, and the results indicate that this increase leads to enhanced sensitivity to CCK.
TL;DR: Comparison with human and mouse pancreatic and salivary α-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian α-AMylases.
TL;DR: The results indicate that the antimicrobial defense capacity of whole saliva is not impaired in diabetic patients and the levels of innate defense factors were similar in both study groups.
Abstract: We analyzed the flow rate and composition of paraffin-stimulated whole saliva samples from 35 adult diabetic patients and their age- and sex-matched, non-diabetic, clinically healthy controls. All patients had insulin-dependent diabetes (IDDM) with a mean (± S.D.) duration of 14.0 ± 9.1 years. The saliva analysis included the quantitation of total protein, amylase, immunoglobulins (isotypes A, G, and M), and the non-antibody, innate antimicrobial factors (lysozyme, lactoferrin, salivary peroxidase, myeloperoxidase, thiocyanate, and hypothiocyanite). The whole saliva samples from diabetic patients had significantly higher amounts of IgA (p < 0.001) and IgG (p < 0.05) than did the controls. No differences between the study groups were observed in flow rate, protein content, amylase activity, or IgM. The levels of innate defense factors were similar in both study groups except for salivary peroxidase, which was higher (p < 0.02) among diabetics than among controls. Our results indicate that the antimicrobial...
Patent•
23 May 1986
TL;DR: In this article, a method for cooking-free saccharification of starch using an amylase produced by Corticium rolfsii AHU 9627 or its variants is described.
Abstract: A method for the cooking-free saccharification of starch using an amylase produced by Corticium rolfsii AHU 9627 or its variants. According to the method, even a high viscous suspension of 10% (w/v) or more raw-corn starch is almost completely hydrolyzed within 8 hours. The saccharification is proceeded at a higher temperature and a lower pH compared with those in known methods utilizing other amylases which are able to hydrolyze uncooked starch, so that the propagation of the infectious bacteria which would affect the saccharifying efficiency can be avoided.
TL;DR: The results, in contrast to previous proposals, suggest that selecting wheat varieties for high α-amylase inhibitory activity may not be a very reliable criterion in selecting for insect resistance.
Abstract: Protein α-amylase inhibitors were prepared from wheat and their effects tested against insect storage pests both in vitro against the insect α-amylases and in vivo in insect feeding trials. Inhibitor fraction A was found to inhibit porcine pancreatic α-amylase but not insect α-amylases, whereas fractions B, C and D (0.28) did not inhibit porcine pancreatic α-amylase but were strong inhibitors of digestive α-amylases from larvae of Tribolium confusum, a storage pest of wheat products, and Callosobruchus maculatus, a storage pest of legume seeds. Fraction D, which was a single polypeptide of Mr 13 000 was the most effective inhibitor in vitro. It would appear that the degree of inhibition by the wheat α-amylase inhibitor preparations can be correlated with the presence of the Mr 13 000 (0.28) polypeptide since the purer this polypeptide the stronger was the inhibition; fraction A which contained two polypeptides of Mr 60 000 and 58 000 caused no inhibition. The effects of fractions B and C on larval development were determined in insect feeding trials. With C. maculatus both fractions were toxic, their relative effectiveness being directly paralleled by their effectiveness observed in vitro. Only fraction C was tested against T. confusum in feeding trials. Despite this fraction being equally effective against both pests in vitro it had very little effect upon larval development of T. confusum in vivo, thus suggesting that this organism is able to detoxify the wheat α-amylase inhibitors. As far as the authors are aware, this is the first time that the effects of identified inhibitor fractions have been monitored both in vitro and in vivo. The results, in contrast to previous proposals, suggest that selecting wheat varieties for high α-amylase inhibitory activity may not be a very reliable criterion in selecting for insect resistance.
TL;DR: Adult female Calanus helgolandicus were transferred to two unialgal diets one of which, the flagellate Cryptomonas maculata, was rich in starch, while the other, the diatom Thalassiosira weissflogii, contained no starch, indicating a compensatory mechanism between digestive enzymes and the substrate ingested.
Abstract: Adult female Calanus helgolandicus were transferred, immediately after collection in the English Channel in June 1984, to two unialgal diets one of which, the flagellate Cryptomonas maculata, was rich in starch, while the other, the diatom Thalassiosira weissflogii, contained no starch. The activity of the digestive enzymes amylase, trypsin and laminarinase was measured in these two populations under foodsaturating conditions over an acclimation period of eight days. Ingestion rates were measured on a daily basis and the results confirmed, together with a constant level of body protein, that the experimental conditions were above the incipient limiting concentration. In the long-term (4 to 8 d), the activities of all three enzymes were significantly elevated in the C. maculata-fed copepods, whereas ingestion rates were lower than those on T. weissflogii. These results observed under food-saturating conditions indicate a compensatory mechanism between digestive enzymes and the substrate ingested. They are consistent with previous work on Artemia sp. suggesting that the rate of assimilation is a function of the digestive enzyme activity and ingestion rate. Enzyme activity exhibited differing shortterm responses (<48 h) on transfer to the two algal diets, which are interpreted in relation to the in situ activity of the field population.
TL;DR: Data support the proposition that amylase is initially an integral protein in the membrane of cytoplasmic vesicles at the apex of the epithelial cells and is subsequently processed to become soluble and is set free into the midgut lumen by exocytosis.
TL;DR: Overall results show that the periphery of starch granules is the major site of deposition for bound β-amylase in dry seeds, resulting in the conversion of free β-Amylase into a bound form during the desiccation phase of seed development.
Abstract: Association of β-amylase with starch granules in the starchy endosperm of barley (Hordeum vulgare L. cv. Menuet) grains was characterized biochemically. In whole homogenates of dry seeds, two forms of β-amylase were detected: one is free β-amylase extractable with saline solution and the other is bound β-amylase extractable with saline solution containing a reducing agent. The two forms of β-amylase were shown to be identical in terms of mobility on disc gels, antigenicity, and molecular specific activity, indicating that the β-amylase molecules of the two forms are identical. The starch granules were isolated from either dry seeds or mature seeds harvested before the desiccation phase. Both starch granule preparations were morphologically identical by microscopic inspection. The bound β-amylase was predominantly associated with starch granules isolated from dry seeds, whereas it was not associated with starch granules from mature seeds harvested before desiccation. Overall results show that the periphery of starch granules is the major site of deposition for bound β-amylase in dry seeds. The association of β-amylase with starch granules occurs during the desiccation phase of seed development, resulting in the conversion of free β-amylase into a bound form.
TL;DR: A basic protein was isolated from aqueous extracts of barley seeds and NMR-spectroscopy revealed that it possesses tertiary structure and Amino acid composition and N-terminal sequence show that it corresponds to the probable amylase/protease inhibitor (PAPI) encoded by a formerly described cDNA.
Abstract: A basic protein was isolated from aqueous extracts of barley seeds. It contains 91 amino acid residues including 8 half-cystines, but no phenylalanine or tryptophan; the calculated molecular weight is 9.694. Amino acid composition and N-terminal sequence show that the protein corresponds to the probable amylase/protease inhibitor (PAPI) encoded by a formerly described cDNA. The protein is 50% homologous with an α-amylase inhibitor I-2 from seeds of Indian finger millet and it shows a distant homology with two Bowman Birk type protease inhibitors. However, the barley protein does not inhibit any of the enzymes tested. They include α-amylases, a β-amylase, β-glucanases, serine proteases, a sulfhydryl protease, aspartate proteases, and serine carboxypeptidases. Nevertheless, difference UV-spectroscopy indicated that the protein interacts with porcine pancreatic α-amylase. NMR-spectroscopy revealed that it possesses tertiary structure.
TL;DR: End-product analysis of amylopectin degradation by chloroplast and crude extracts indicates that maltose is the major product of both, and the possible role of these enzymes in starch degradation by pea chloroplasts is discussed.
Abstract: Starch-degrading enzymes in isolated pea (Pisum sativum L. cv. Laxton's Progress No. 9) chloroplasts were investigated and compared with those in crude pea leaf and stipule preparations. End-product analysis of amylopectin degradation by chloroplast and crude extracts indicates that maltose is the major product of both. Two multiforms of β-amylase (EC 3.2.1.2) were detected in pea chloroplasts using an electrophoretic transfer technique. A starch-debranching enzyme (EC 3.2.1.10) was detected in chloroplasts by electrophoretic transfer and the degradation of pullulan. A different multiform of debranching enzyme was found in crude preparations. α-Amylases (EC 3.2.1.1) were detected by electrophoretic transfer through gels containing starch and starch azure, and by change in viscosity of a starch solution, but were only found in crude preparations indicating an extrachloroplastic location. Incubation of maltotriose with chloroplast extracts gave high levels of glucose production and formation of oligosaccharides with degrees of polymerization larger than that of maltotriose indicating transglycosylase (EC 2.4.1.25) activity. Neither α-glucosidase (EC 3.2.1.20) nor maltose-phosphorylase (EC 2.4.1.1) activity were found in either chloroplast or crude preparations, whereas starch-phosphorylase (EC 2.4.1.1) activity was in both. The possible role of these enzymes in starch degradation by pea chloroplasts is discussed.
TL;DR: It is hypothesized that a catalytic or small amount of any amylase-rich food (ARF) should be able to decrease the viscosity of a traditional weaning rice gruel, and if this could be substantiated, it would be a useful and simple way to reduce the dietary bulk of starchy gruels.
Abstract: In developing countries of Asia, including India, rice is widely used in gruel or cooked form as a first or early introductory food for infants and toddlers [1,2]. The starch granules in rice flour or rice grains not only swell on cooking but show a great propensity for taking up water (water-holding and binding). This contributes to the typical dietary bulk of a rice gruel with low caloric density per unit volume of food consumed. Older infants (over 6 months of age) and young toddlers (13 to 36 months of age) just cannot ingest sufficient amounts of such preparations to fulfil their daily energy requirements [3-6] . The formulation of high-energy (nutrient density) and low-bulk foods for young children utilizing simple and traditional processing methods, such as germination and malting, was recently proposed [7-11]. A series of studies conducted in our laboratory on malted and roasted mixes clearly indicated that the viscosity of a 10 per cent hot-paste slurry made of a mixture of individually malted and powdered wheat, bengal gram (Cicer arietinum), and groundnut (Arachis hypogaea), in the proportion 4:1:1, was significantly lower than that of the mixture in which each food ingredient was roasted [8,11, 12]. In a mixture consisting of cereals and legumes, the malted cereal constituent contributes substantially to the reduction in viscosity of a hot paste [9,13]. Although fully malted ready-to-mix (RTM) or ready-to-eat (RTE) mixes had decidedly higher nutrient density per unit volume of food than the roasted variety, the major and overriding constraints were the time, labour, and space required for their production. These, we felt, would make it too inappropriate and unfeasible a procedure to be readily adopted by rural, tribal, or slumd-welling mothers. Because of its high amylase content, 5 per cent malted barley (Hordeum vulgare) flour substantially reduces the viscosity of 15 per cent hot-paste slurries of weaning foods on the Indian market, such as Nestum, Farex, Cerelac, and Balamul [9] . We therefore hypothesized that a catalytic or small amount of any amylase-rich food (ARF) should be able to decrease the viscosity of a traditional weaning rice gruel, If this could be substantiated, it would be a useful and simple way to reduce the dietary bulk of starchy gruels. In the Gujarat region, barley is unknown. Among cereal grains, malted ragi (Eleusine coracana), bajra (Pennisetum typhoideum), sorghum (Sorghum vulgare), and maize (Zeas mays) flours have high amylase activity [9]. Since ragi (finger millet) was also unknown in this region and since bajra is a commonly consumed staple in the low-income groups, it was decided to develop an ARF from bajra and study its bulk-reducing properties on traditional weaning foods such as gruels prepared from rice flour, or flaked or puffed rice. The specific objectives of this study were: (i) to develop and standardize the method of preparation of ARF and to establish its organoleptic acceptability; (ii) to study the shelf life of the ARF; (iii) to compare the amylase activity and the viscosity-reducing properties of the ARF against those of a pure enzyme, namely, takadiastase; and (iv) to evaluate the acceptability of the rice gruels with and without ARF among mothers, and the intake of the same by infants.
TL;DR: N-terminal sequences of proteins CMa, CMb and C Mc have been determined and found to be homologous to those previously determined for CMd and CMc, an observation which confirms that their structural genes are members of a dispersed multi-gene family.
TL;DR: The distribution of each enzyme activity in a population of granules suggests quantal packaging of amylase and chymotrypsinogen into the granules.
Abstract: The activities of both chymotrypsin and amylase in individual zymogen granules of rat pancreas were measured by means of micromanipulation and microfluorometric methods. The enzyme content and the ratio of amylase to chymotrypsin varied widely among granules taken from the same animal. These results are compatible with short-term nonparallel bulk secretion of the two enzymes through exocytosis. The distribution of each enzyme activity in a population of granules suggests quantal packaging of amylase and chymotrypsinogen into the granules.
TL;DR: In this article, a rat pancreatic acini loaded with the intracellularly trapped fluorescent indicator quin2 was used to measure cytosolic free calcium concentrations (Ca2+]i) and amylase secretion.
Abstract: Cytosolic free calcium concentrations ([Ca2+]i) and amylase secretion were measured in isolated rat pancreatic acini loaded with the intracellularly trapped fluorescent indicator quin2. Both caerulein and carbamoylcholine caused a rapid increase in [Ca2+]i, with a maximal 3-fold increase at 10(-9) M-caerulein and 10(-4) M-carbamoylcholine. However, caerulein (10(-12) M and 10(-11) M) as well as carbamoylcholine (10(-7) M) caused a significant stimulation of amylase release, while not inducing any detectable rise in [Ca2+]i. Changes in [Ca2+]i after addition of either secretagogue were transient and did not last more than 2-3 min. By contrast, when amylase secretion was monitored as a function of time, two distinct secretory phases could be observed upon addition of either carbamoylcholine (10(-5) M) or caerulein (10(-10) M). An initial, rapid phase (0-5 min) which caused a 6-7-fold increase above basal, followed by a sustained (5-30 min), but less marked, secretory rate (2-3-fold above basal). Addition of atropine (10(-4) M) 5 min after carbamoylcholine (10(-5) M) (i.e. after termination of the rise in [Ca2+]i and of the first secretory phase) did not cause any significant change in [Ca2+]i, while significantly inhibiting amylase secretion from 5 to 30 min to the same rate observed in the absence of the secretagogue. These results show that caerulein and carbamoylcholine, two agents thought to activate secretion mainly through mobilization of Ca2+ from intracellular stores, are capable of eliciting amylase secretion independently of a concomitant rise in [Ca2+]i. Furthermore, with both secretagogues the rise in [Ca2+]i, when observed, was only transient, while the stimulation of amylase release was sustained.
TL;DR: There is a direct correlation between changes in amylase activity and changes in the amount of translatable mRNA as assayed in microinjected Xenopus oocytes, which means that the glucose repression is occurring at a pretranslational stage.
Abstract: We have previously shown that dietary glucose can reduce amylase activity in both adults and larvae of Drosophila; this reduction in enzyme activity reflects a reduction in the quantity of amylase protein, rather than an inhibition of enzyme activity. Here, we report that we have now defined conditions in which the repressive effect of glucose can be greater than 100-fold. Moreover, this repression is partially counteracted by the addition of exogenous cyclic AMP. We also show that there is a direct correlation between changes in amylase activity and changes in the amount of translatable mRNA as assayed in microinjected Xenopus oocytes. This means that the glucose repression is occurring at a pretranslational stage.
01 Jun 1986
TL;DR: It is concluded that a purified amylase inhibitor is effective and potentially beneficial in the treatment of diabetes mellitus and the validity of the "starch blockade" concept is tested.
Abstract: Slowing starch digestion by inhibiting amylase activity in the intestinal lumen should improve postprandial carbohydrate tolerance in patients with diabetes mellitus. Crude bean-derived amylase inhibitor ("starch blocker") that contains only minimal antiamylase activity, however, does not modify carbohydrate assimilation. To test the validity of the "starch blockade" concept, we assessed the effect of a partially purified bean-derived amylase inhibitor with increased antiamylase activity on carbohydrate tolerance in normal subjects and in patients with non-insulin-dependent diabetes mellitus. In comparison with a placebo, ingestion of this inhibitor with 50 g of starch substantially reduced postprandial increases in plasma concentrations of glucose and insulin in both normal subjects and those with diabetes. We conclude that a purified amylase inhibitor is effective and potentially beneficial in the treatment of diabetes mellitus.
TL;DR: High levels of amylase in fluid saliva resulted in high levels being detected in saliva stains, and Interpretations are made as to the possible sources ofAmylase activity found in stains from laboratory casework based on both the amyl enzyme concentration and the elapsed time between collection and analysis.
Abstract: Amylase levels were determined for 148 semen samples and 20 saliva samples as well as for their corresponding stains. The effect of aging on the detectability of amylase activity in these stains was also investigated. The Phadebas® amylase test was used for the quantitative assay of amylase. High levels of amylase in fluid saliva resulted in high levels being detected in saliva stains. Lower levels present in most seminal fluids produce little or no detectable amounts of amylase in stains. Interpretations are made as to the possible sources of amylase activity found in stains from laboratory casework based on both the amylase concentration and the elapsed time between collection and analysis. The evidential value of the presence or absence of amylase activity in casework stains is also discussed.
TL;DR: It is suggested that CCK-receptor interaction in the fetal pancreas triggers intracellular Ca++ mobilization and elevated cytosolic [Ca++], and that the onset of secretory response to CCK that occurs postnatally may depend on amplification of these transduction events.
Abstract: We have studied the onset of secretory responsiveness to cholecystokinin (CCK) during development of the rat exocrine pancreas. Although acinar cells of the fetal pancreas (1 d before birth) are filled with zymogen granules containing the secretory protein, alpha-amylase, the rate of amylase secretion from pancreatic lobules incubated in vitro was not increased in response to CCK. In contrast, the rate of CCK-stimulated amylase discharge from the neonatal pancreas (1 d after birth) was increased four- to eightfold above that of the fetal gland. The postnatal amplification of secretory responsiveness was not associated with an increase in the number or cell surface expression of 125I-CCK binding sites. When 125I-CCK-33 binding proteins were analyzed by affinity crosslinking, two proteins of Mr 210,000 and 100,000-160,000 were labeled specifically in both fetal and neonatal pancreas. To determine if cell surface receptors for CCK in the fetal pancreas are functional and able to generate a rise in the cytosolic [Ca++], we measured 45Ca++ efflux from tracer-loaded lobules. 45Ca++ efflux from both fetal and neonatal pancreas was comparably increased by CCK, indicating CCK-induced Ca++ mobilization and elevated cytosolic [Ca++]. The Ca++ ionophore A23187 also stimulated the rate of 45Ca++ extrusion from pancreas of both ages. Increased amylase secretion occurred concurrently with A23187-stimulated 45Ca++ efflux in neonatal pancreas, but not in the fetal gland. A23187 in combination with dibutyryl cAMP potentiated amylase release from the neonatal gland, but not from fetal pancreas. Similarly, the protein kinase C activator, phorbol dibutyrate, did not increase the rate of secretion from the fetal gland when added alone or in combination with A23187 or CCK. We suggest that CCK-receptor interaction in the fetal pancreas triggers intracellular Ca++ mobilization. However, one or more signal transduction events distal to Ca++ mobilization have not yet matured. The onset of secretory response to CCK that occurs postnatally may depend on amplification of these transduction events.
TL;DR: Inhibition of secretion in vivo and lack of stimulation of amylase secretion in isolated acini suggest that the somatostatin effect in vivo is mediated by an indirect effect similar to other peptides, for example, opiates and neurotensin.
Abstract: In the present study the effect of somatostatin on amylase secretion was determined usingin vivo cannulation and isolated acini from rat pancreasIn vivo somatostatin-14 inhibited amylase secretion in basal state and that stimulated with CCK8 and acetylcholine Somatostatin-14 and somatostatin-28 failed to inhibit amylase secretion from isolated acini in basal state and that stimulated with CCK8 and bethanechol Somatostatin-14 did not increase45Ca uptake or efflux of label from acini preloaded with45Ca Cellular cyclic AMP levels were not significantly increased Somatostatin-14 did not alter the synthesis of proteinsin vitro, as judged by incorporation of a mixture of fifteen14C-labeled amino acids Somatostatin-14 stimulated phosphoprotein phosphatase in higher doses, whereas no effect was observed at lower doses Inhibition of secretionin vivo and lack of stimulation of amylase secretion in isolated acini suggest that the somatostatin effectin vivo is mediated by an indirect effect similar to other peptides, for example, opiates and neurotensin Stimulation of phosphoprotein phosphatase suggests that somatostatin may bind to the acinar cells and affect functions other than secretion and synthesis of enzymes