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PubMed2006

Activity and cellular localization of amylases of rabbit cecal bacteria.

Five 11-week-old rabbits, fed a commercial granulated feed, were slaughtered and cecal starch-degrading bacteria enumerated; total concentration of cultivable bacteria utilizing starch averaged 5.5 x 10(10) CFU/g. The activity and cellular localization of amylases was determined in 9 bacteria identified as Actinomyces israeli (strains AA2 and AD4), Bacteroides spp. (strain AA3), Dichelobacter nodosus (strain AA4), Mitsuokella multiacidus (strain AA6), Eubacterium spp. (strains AA7 and AB2), Clostridium spp. (strains AD1 and AA5). Four strains (AA3, AA4, AA5, AD4) produced extracellular amylases with an activity of 26-35 micromol of reducing sugars per h per mg of protein; in five strains (AA2, AA6, AA7, AB2, AD1) amylases were membrane-bound with an activity of 14-18 micromol of reducing sugars per h per mg of protein. All strains exhibited a low intracellular amylolytic activity. The pH optimum of amylases was 6.8-7.0. In strains producing extracellular amylases a substantial loss of viscosity was observed during incubations of cultivation supernatant with starch, similar to viscosity reduction in starch solutions treated with alpha-amylase; this indicates an endo-type (random cleavage) of extracellular amylase reaction in the bacteria under study. No strain possessed glucoamylase activity.

Amylases↗
PubMed2000

The influence of nitrogen sources on the alpha-amylase productivity of Aspergillus oryzae in continuous cultures.

The influence of the nitrogen source on the alpha-amylase productivity of Aspergillus oryzae was quantified in continuous cultivations. Both inorganic and complex nitrogen sources were investigated and glucose was used as the carbon and energy sources. For production of alpha-amylase, nitrate was shown to be inferior to ammonia as a nitrogen source. A mixture of ammonia and complex nitrogen sources, such as yeast extract or casein hydrolysate, was better than with ammonia as the sole nitrogen source. Even a low concentration of casein hydrolysate (0.05 g l(-1)) resulted in a 35% increase in the alpha-amylase productivity. The higher alpha-amylase productivity during growth on casein hydrolysate was not caused by increased transcription of the alpha-amylase genes but was caused by a faster secretion of alpha-amylase or by a lower binding of alpha-amylase to the biomass.

Aspergillus oryzae↗
PubMed1997

Gene knockout of the intracellular amylase gene by homologous recombination in Streptococcus bovis.

Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular alpha-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15-20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium.

Amylases↗
PubMed1997

Effect of uncoupling NO/cGMP pathways on carbachol- and CCK-stimulated Ca2+ entry and amylase secretion from the rat pancreas.

Nitric oxide (NO) production reportedly regulates guanosine 3', 5'-cyclic monophosphate (cGMP) formation and Ca2+ influx in pancreatic acini. We have investigated the functional roles of the NO/cGMP messenger system in rat pancreatic acini. In dispersed acini, the levels of amylase secretion, cytosolic [Ca2+]([Ca2+]i), NO synthase, and cGMP were measured. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 0.01-100 microM) had no effect on amylase secretion induced by various concentrations of carbachol, cholecystokinin octapeptide (CCK-8) or the high affinity CCK agonist, JMV-180. Similarly, L-NAME up to 100 microM did not affect the changes in Ca2+ spiking evoked by these secretagogues; nor was Ca2+ entry, refilling or oscillation altered by L-NAME. Sub- and supramaximal concentrations of these secretagogues did not change NO synthase activities compared with basal levels. While sodium nitroprusside (SNP), a NO donor, caused a 9.4-fold increase in cGMP levels compared with basal levels, carbachol, CCK-8 and JMV-180 had no effect. In addition, the guanylate cyclase inhibitor LY 83583 (10 nM to 10 microM) altered neither amylase secretion nor Ca2+ signaling induced by these secretagogues. These findings indicate that the stimulatory action of carbachol or CCK-8 is not mediated by NO or cGMP. To investigate whether cGMP stimulates pancreatic secretion we showed that both SNP and a cell-permeant cGMP analog at 0.1-1 mM stimulated amylase secretion and Ca2+ transients to a level equal to 10-15% and 13-24%, respectively, of those observed with maximal concentrations of secretagogues. The guanylate cyclase activator guanylin (1-10 microM), which increased cGMP levels 2.4-fold compared with basal levels, elicited a small amount of amylase secretion and a small Ca2+ transient. In conclusion, exogenous NO is capable of increasing endogenous cGMP, which results in a modest increase in the [Ca2+]i transient and pancreatic amylase secretion. However, the NO/cGMP system does not appear to be involved significantly in the mediation of Ca2+ signaling and amylase secretion stimulated by carbachol and CCK-8.

Amylases↗
PubMed2005

Production of amylase by Arthrobacter psychrolactophilus.

Arthrobacter psychrolactophilus ATCC 700733 grew with a doubling time of 1.5-2.3 h (22 degrees C) and produced up to 0.2 units/mL (soluble starch assay) of extracellular amylase in tryptic soy broth without dextrose (TSBWD) containing 0.5% or 1.0% (w/v) soluble starch or maltose as the fermentable substrate. Time-course experiments in media containing soluble starch as substrate showed that amylolytic activity appeared in cultures at 24 h (after exponential growth had ceased), reached peak levels in 72-96 h, and declined rapidly after reaching peak levels. Peak levels were highest in TSBWD containing 1.0% soluble starch. Proteolytic activity appeared at about the same time as amylolytic activity and increased during the period of amylase production. Significant amylase production was not observed in cultures in TSBWD with 0.5% glucose or in cultures grown at 28 degrees C, but low levels of amylase were observed in TSBWD cultures grown at 19-23 degrees C which contained no added carbohydrate. A single band of activity was observed after electrophoresis of supernatant fractions in non-denaturing gels, followed by in situ staining for amylolytic activity. The amylase possessed a raw starch-binding domain and bound to uncooked corn, wheat or potato starch granules. It was active in the Phadebas assay for alpha-amylase. Activity was maximum on soluble starch at a temperature between 40 degrees C and 50 degrees C. The amylase after purification by affinity chromatography on raw starch granules exhibited two starch-binding protein bands on SDS gels of 105 kDa and 26 kDa.

Amylases↗
PubMed1982

Relationship between pancreatic and serum amylase in basal conditions and during stimulation in man. Report of a new intraoperative technique.

The correlation between serum amylase and pancreatic amylase secretion was studied in two patients. Both patients underwent sphincterotomy, and the pancreatic duct was cannulated with a polyethylene tube. The tube was left in place for 15 to 21 days. The rate of amylase secretion over 7 days was studied in response to (1) a standard meal, (2) duodenal acidification, (3) scalar doses of cholecystokinin, (4) scalar doses of secretin, and (5) scalar doses of secretin with simultaneous infusion of cholecystokinin. Blood samples were collected during the tests to measure serum amylase. No significant correlation was shown between blood concentration and output of amylase in any of the tests. Our findings show that under normal conditions serum amylase levels are not influenced by pancreatic secretion and suggest that serum amylase concentration is not related to pancreatic exocrine secretion.

Adult↗
PubMed1990

Microplate reader-based kinetic determination of alpha-amylase activity: application to quantitation of secretion from rat parotid acini.

A coupled enzyme assay for measuring alpha-amylase activity was adapted for analysis with a microplate reader. Activity was quantified by monitoring the cleavage of p-nitrophenol from a chemically defined substrate at 405 nm. Features of this assay method include: low sample volume (10 microliters); economical use of reagent (200 microliters); increased precision due to kinetic nature of assay; linearity with amylase content to 2600 U/liter; capability of processing up to 96 samples within 10 min; facilitated data analysis using readily available software. The ability to rapidly measure amylase content of a large number of samples permitted sophisticated analysis of alpha-amylase secretion patterns from dispersed rat parotid acinar cells. Examination of the dose-dependent increase in the rate of amylase secretion stimulated by carbachol revealed a biphasic response at higher concentrations, where a rapid and transient increase in amylase release was consistently observed during the first 10 min. This initial phase of amylase release was additive with the slower, sustained secretion which was stimulated by carbachol in a dose-dependent manner (Kd = 2.5 microM). Such a biphasic release pattern was not seen with other potent secretagogues (isoproterenol, dibutyryl cAMP) that are not thought to act via a Ca-dependent mechanism. These results suggest that parotid secretion patterns should be studied at a number of time points, which is feasible using the method reported herein.

Animals↗
PubMed1985

Irreversible inhibition by acetaldehyde of cholecystokinin-induced amylase secretion from isolated rat pancreatic acini.

Acetaldehyde inhibited both amylase secretion induced by maximal concentrations (300 pM) of cholecystokinin octapeptide and the binding of radioiodinated cholecystokinin to receptors on isolated rat pancreatic acini. This inhibition was concentration dependent (10 mM to 1 M for amylase secretion and 100 mM to 1 M for binding). However, a correlation between the two inhibitory effects could not be obtained. Furthermore, the inhibitory effects were not reversible. Acetaldehyde did not alter the basal amylase secretion between 6 and 45 mM concentrations. However, 60, 100 and 300 mM acetaldehyde significantly decreased basal amylase secretion; no significant change in amylase secretion was observed at 600 mM and 1 M. Higher concentrations of acetaldehyde produced a 2- to 10-fold increase in basal amylase secretion. 51Cr release from prelabeled acini revealed no significant cell membrane damage between 10 and 600 mM acetaldehyde. These data suggest that acetaldehyde inhibition of cholecystokinin-induced amylase secretion is intracellularly mediated.

Acetaldehyde↗
PubMed1987

Effect of ethanol on amylase secretion and cellular calcium homeostasis in pancreatic acini from normal and ethanol-fed rats.

The effects of ethanol on stimulus-secretion coupling were assessed by studying amylase release, Ca2+-homeostasis, and changes in physical properties of membranes in isolated rat pancreatic acini. In acini from normal rats, ethanol (50 mM and above) in vitro caused a dose-dependent stimulation of amylase release and an increase in cytosolic free Ca2+ concentration. Ethanol did not affect amylase secretion stimulated by cholecystokinin-octapeptide (CCK8), a secretagogue that acts by increasing cytosolic free Ca2+ levels, but did potentiate the secretion of amylase induced by vasoactive intestinal peptide (VIP) which raises intracellular cAMP. Ethanol also increased the rate of 45Ca2+ exchange. In acini labeled with the spin-probe 12-doxyl stearic acid, ethanol disordered the pancreatic plasma membranes. By contrast, in acini from animals that had chronically (6-7 weeks) ingested ethanol, the membranes were resistant to this disordering effect of ethanol. Chronic ethanol feeding lowered the total cellular calcium content and ionophore (A23187)-releasable pools of acinar calcium (11 and 24% respectively), and led to a 15-30% decrease in the rate of 45Ca2+ exchange. Chronic ethanol ingestion also lowered the basal rate of amylase secretion, but ethanol in vitro stimulated amylase secretion more than in control preparations. However, these differences in basal and ethanol-induced amylase secretion were not accompanied by corresponding changes in intracellular free Ca2+. The data suggest that ethanol perturbs cell membranes and also disturbs cellular Ca2+ homeostasis. These effects may explain its actions as a weak Ca2+-mediated secretagogue. However, the membrane alterations induced by chronic ethanol feeding do not prevent the ethanol-induced interference with cellular calcium homeostasis.

Amylases↗
PubMed1992

The clinical relevance of salivary amylase monitoring in bulimia nervosa.

The aim of this study was to evaluate the clinical relevance of amylase level monitoring as an objective measure in diagnosis and assessment of treatment response in bulimia nervosa. Thirty-three subjects who fulfilled DSM-111-R criteria for bulimia nervosa had serum levels of total and salivary amylase monitored during an 8-week treatment trial. At the beginning of treatment, the average total amylase level was within the upper limits of normal, whereas average salivary amylase levels were abnormally high. During the course of treatment, there was a significant reduction in the average salivary isoenzyme to within the normal range. Significant reductions in amylase levels were recorded in patients with good treatment outcome, but not in those with poor outcome. Amylase levels were not significantly correlated with severity of bulimic symptoms. These results do not justify the use of amylase assays as a routine diagnostic or monitoring test, but isoenzyme monitoring may provide useful clinical information in selected cases.

Adult↗
PubMed1978

Modification of human pancreatic amylase isozymes by peptidoglutaminase I and II.

The effects of peptidoglutaminase (PGln-ase) I and II on human pancreatic juice amylase purified as an isozyme were investigated. Several amylase isozymes were formed which corresponded to minor components of pancreatic amylase isozymes, indicating that appearance of amylase isozymes are due to enzymic deamidation. A similar result was observed when the purified amylase isozyme was incubated with the supernatant of human pancreatic juice whose amylase was previously removed by adsorption onto raw corn starch. These findings are discussed in connection with amylase isozymes in the sera of the patients suffering from pancreatic inflammation.

Amidohydrolases↗
PubMed1985

Differences in dopamine- and noradrenaline-induced amylase release from the rat parotid gland.

The effects of dopamine and noradrenaline on amylase secretion from rat parotid gland were studied in a batch incubation system. Dopamine effectively caused amylase secretion but the concentration-response curve was shifted to the right in reserpinized animals, suggesting a minor indirect component in the action of dopamine. The noradrenaline-evoked secretion was the same in reserpinized glands as in the controls. Bromocriptine, a dopamine receptor agonist, was without effect on amylase secretion. The dopamine receptor antagonists pimozide, chlorpromazine, haloperidol and droperidol as well as SKF 38393 all effectively inhibited dopamine-induced amylase secretion without affecting the enzyme release caused by noradrenaline. The D-2 antagonist sulpiride was without effect on both dopamine- and noradrenaline-stimulated secretion. The dopamine-induced secretion was also significantly blocked by the non-selective beta-blocker propranolol as well as by beta 1- and beta 2-selective antagonists. Several alpha-antagonists were all partial blockers of dopamine-stimulated amylase secretion. In contrast, the noradrenaline-evoked amylase release was exclusively abolished by propranolol and beta 1-selective antagonists. The results suggest that the dopamine- and noradrenaline-induced amylase secretion are activated via different receptor systems, and that dopamine stimulation is mainly a postsynaptic D-1 effect and only to a minor extent due to presynaptic interaction.

Amylases↗
PubMed1994

Evidence for the involvement of protein phosphorylation in cyclic AMP-mediated amylase exocytosis from parotid acinar cells.

We evaluated the role of protein phosphorylation in cAMP-mediated amylase exocytosis from parotid acinar cells by using H89, a new protein kinase A (PKA) inhibitor, which is more lipophilic and 25 times more potent than H8. In our previous studies, H8 markedly inhibited protein phosphorylation without decreasing amylase release [Takuma, T. (1988) Biochem. J. 256, 867-871]. These findings were completely reproduced even in the small acini that were prepared by trypsin treatment before collagenase digestion. In the present study, however, H89 strongly inhibited both amylase release and protein phosphorylation in a dose-dependent manner. The inhibitory effect was specific for PKA at least up to 33 microM, since 33 microM H89 did not block amylase release stimulated by PMA. H85, a closely related compound of H89 without inhibitory effect on PKA, did not prevent amylase release or protein phosphorylation at least up to 33 microM. These results suggest that protein phosphorylation by PKA is involved in cAMP-mediated amylase exocytosis. The inhibition of protein phosphorylation by H8 might be insufficient or inadequate for blocking of amylase release.

Amylases↗
PubMed1994

Pharmacological study of gastrin-mediated amylase release in pancreatic acinar cells (AR4-2J).

In rat pancreatic acinar cells, amylase release and Ca2+ mobilization are related to the occupancy of CCKA receptor. The rat pancreatic acinar cell line (AR4-2J) possesses both CCKA (CCKA R) and CCKB (CCKB R) sub-type receptors. Using this cell line we attempted to determine the relative involvement of each sub-type in both amylase release and Ca2+ mobilization. For this purpose we used L 364718 a selective antagonist for CCKA R and PD 135158 a selective antagonist for CCKB R. We showed on AR4-2J cells that: a minority of CCKA R (Kd = 0.7 nM), a classical CCKB R (Kd = 0.93 nM) and a new high affinity gastrin binding site (Kd = 2.1 pM) coexisted; CCK through CCKA R and CCKB R, was more potent to stimulate amylase secretion (EC50 = 34 pM) and Ca2+ mobilization (EC50 = 30 pM) than to occupy its receptor. Gastrin induced a biphasic stimulation of amylase release. Gastrin through CCKB R was equally potent to stimulate amylase release (EC50 = 1.72 nM) and Ca2+ mobilization (EC50 = 3.1 nM), whereas through the high affinity gastrin binding site, gastrin-induced amylase release (EC50 = 0.73 pM) did not correlate with the Ca2+ mobilization (EC50 = 3.1 nM). These results demonstrated for the first time the existence, on AR4-2J cells, of a high affinity gastrin receptor whose occupation by gastrin induces amylase release.

Amylases↗
PubMed1983

The effect of heat on the intestinal and pancreatic levels of amylase and maltase of laying hens and broilers.

The effects of exposing laying hens and broilers daily to intermittent periods of 4 hr heating at 42 degrees C on the intestinal and pancreatic levels of amylase and maltase were investigated. The initial exposure to heat, characterized by heat stress, brought about a significant increase in the duodenum and jejunum parts of the tetra breed hen and only in the duodenum of broilers. The levels of amylase in the distal parts of the intestine of both breeds sharply decreased. The increase in amylase levels in the proximal parts of the intestine under the conditions of initial heating vanished after 3 days of heating; its levels continued to fall in the distal parts. In heat acclimatized laying hens the levels of amylase were lower than those of the control hens both in the intestine and pancreas. The pancreatic level of amylase was reversely related to the levels in the intestine. It is assumed that the intestinal level of amylase is regulated by the pancreas. These findings indicate that the pancreas plays an important role during the adaptation of chickens to heat, through the regulation of intestinal level of amylase. The response in maltase level to heat stress and heat acclimatization was insignificant.

Amylases↗
PubMed1990

Effect of phenylalanine on pancreatic amylase secretion in chicks (Gallus domesticus).

1. Effect of phenylalanine (Phe) on pancreatic amylase secretion in growing chicks was investigated in four experiments. 2. In Experiment 1, birds were injected through a wing vein with 0.25 ml Phe at 0, 0.1, 0.5, 2.5 and 12.5 mM in physiological saline. No significant difference was observed in amylase secretion among treatments. 3. Effect of various concentrations of Phe with cholecystokinin (CCK, 0.31 Crick unit) on amylase secretion was investigated in Experiment 2. Amylase secretion increased with time, although no significant effect was detected in Phe treatment. 4. Efficacy of Phe and tyrosine (Tyr) injection with CCK on amylase secretion was compared. There was no significant difference between Phe and Tyr treatments. 5. Birds were injected intraperitoneally with dl-p-chlorophenylalanine (p-CP), which is an inhibitor of phenylalanine hydroxylase, or saline 1 day before the collection of pancreatic amylase in Experiment 4. Both chicks showed increased amylase secretion with CCK (0.31 Crick unit), whereas the response was at a drastically reduced rate in chicks with the p-CP treatment.

Amylases↗
PubMed1994

Thyroxine control of pancreatic amylase gene expression: modulation of PTF1 binding activity.

The role of pancreas specific transcription factor (PTF1) in thyroxine (T4) modulation of amylase gene expression in suckling rats was evaluated. Electrophoretic mobility shift assay (EMSA) was used to determine the PTF1 binding activity by the amount of a synthetic oligonucleotide containing the amylase enhancer sequence bound by nuclear protein extracts. Nuclear protein from rat pancreata showed a developmental increase of PTFI activity correlated with age. To study the action of T4, pups were made hyperthyroid by T4 injection and hypothyroid by feeding propylthiouracil (PTU) to the lactating dams. EMSA of nuclear proteins isolated from these groups showed an increase in PTF1 binding activity in the T4 group and a decrease in the PTU group. Concomitantly, T4 increased, while PTU decreased both amylase enzyme and mRNA concentrations. T4 replacement reversed the effect of PTU on PTF1 binding, amylase enzyme activity and mRNA levels. To examine the age dependence of T4 effects, T4 was injected to pups for 5 days prior to killing at the age of 15, and 25 days. T4 was effective when given at an earlier age (15 days) but not at a later stage (25 days) in increasing amylase activity and amylase mRNA levels. Nuclear proteins isolated from pancreata of these groups showed an increase in PTF1 binding activity in the T4-treated 15-day-olds but not in the 25-day-olds in comparison to their corresponding age matched littermates. These results suggest that PTF1 is an important intermediary in T4 modulation of amylase gene expression during ontogeny of the rat exocrine pancreas.

Amylases↗
PubMed1991

Interpretation of polymorphic DNA patterns in the human alpha-amylase multigene family.

Previous molecular studies have clearly shown that the human amylase locus has a very complicated structure. Multiple salivary and pancreatic amylase genes are present on haplotypes with variable numbers of genes. To study the population heterogeneity, human genomic DNA from family members and random individuals was digested with a number of different restriction enzymes and hybridized with probes representing various parts of the human pancreatic amylase cDNA. The complex patterns obtained were, in most cases, compatible with predictions from the restriction enzyme maps of cloned human amylase genes. With some enzymes deviations from the predicted intensities of the bands associated with the pancreatic amylase gene AMY2A were observed. These findings can be explained by unequal homologous crossovers between AMY2A and AMY1A, resulting in haplotypes with one gene less or one gene more than the haplotypes described thus far. Moreover, a very complicated TaqI polymorphism was found that can be explained by homologous crossovers between different salivary amylase genes. Because some salivary amylase genes have an inverted orientation with respect to the others, these data provide evidence for the occurrence of intrachromosomal, homologous crossovers, as proposed by us previously (P. C. Groot et al., 1990, Genomics 8: 97-105).

Blotting, Southern↗