Amylase Synthesis Research Articles

Changes in Muscarinic Receptors and Amylase Secretion in Regenerating Acinar Cells of Rat Pancreas

Atrophic pancreatic acinar cells of copper-deficient rats recovered after copper supplementation. To clarify whether the recovered cells are regenerative and amylase secretion via muscarinic receptors alters, [3H]-thymidine uptake, [3H]-quinuclidinyl benzilate binding and amylase release were studied in these rats and compared with the values of 5-day-old and 21-week-old rats. The [3H]-thymidine uptake study indicated that the recovered acinar cells were regenerative. The number of muscarinic receptors in regenerating cells was equal to that in mature cells and was one third in cells of 5-day-old rats. The apparent dissociation constant of muscarinic receptors was about 0.73 x 10(-10) M for the regenerating cells, 0.65 x 10(-10) M for 5-day-old rats, and 1.70 x 10(-10) M for 21-week-old rats. Amylase release, as a percent of total amylase, was maintained relatively steady in spite of the low amylase content in regenerating cells. These observations suggest that the affinity of muscarinic receptors increases to compensate the impaired amylase synthesis in regenerating acinar cells and that their amylase secretion via muscarinic receptors is different from that in 5-day-old or mature rats.

Catabolite repression-resistant mutations of the Bacillus subtilis alpha-amylase promoter affect transcription levels and are in an operator-like sequence

The amyR1 locus controls the regulated transcription of amyE, the structural gene encoding alpha-amylase in Bacillus subtilis. Transcription of amyE is activated in early stationary phase cells, and can be repressed by rapidly metabolized carbon sources such as glucose. Transcription of amyE initiates in vitro from a promoter recognized by the major vegetative form of RNA polymerase, E σ 43. S 1 nuclease mapping of in-vivo amylase transcripts suggests that this promoter is also used in vivo. Two independently isolated cis-acting mutations, gra-5 and gra-10, which abolish glucose-mediated repression of amylase synthesis without altering temporal activation, were determined by DNA sequencing to result from a G · C to A · T transition at a position located five base-pairs downstream from the start site of transcription. While this is the first example of a site involved in catabolite repression of gene expression in a Gram-positive micro-organism, the region surrounding the gra mutations shows considerable homology to certain cis-acting regulatory loci in Escherichia coli, suggesting that such sequences have been evolutionarily conserved.

Mechanism of glucocorticoid-induced increase in pancreatic amylase gene transcription.

To determine the mechanism(s) responsible for glucocorticoid-induced increases in amylase content in pancreatic acinar AR42J cells, we examined the effects of dexamethasone on amylase protein biosynthesis, steady-state mRNA levels, and gene transcription. Dexamethasone treatment led to a dose-dependent increase in amylase synthesis which was one-half maximal at 2 nM and maximal at 100 nM where a 6-fold increase was achieved. This dexamethasone-induced increase in amylase synthesis was detectable after 12 h, one-half maximal after 19 h, and approached maximal after 72 h. Dexamethasone treatment also increased amylase mRNA levels in a time- and dose-dependent manner in parallel with the changes in amylase synthesis. Nuclear RNA transcript elongation (run-on) assays indicated that amylase gene transcription was also increased in a time- and dose-dependent manner. Glucocorticoid enhancement of amylase gene transcription occurred relatively slowly, with a 6-fold increase occurring after 48 h of treatment with 100 nM dexamethasone. Thus, the effects of glucocorticoids on pancreatic amylase gene transcription fully accounted for the increased levels of amylase mRNA, synthesis, and content. However, due to the slow time course of dexamethasone induction of amylase gene expression we evaluated the possibility of glucocorticoid induction of a regulatory protein. We found that inclusion of cycloheximide or puromycin during dexamethasone treatment blocked the induction of amylase mRNA. These data suggest that the glucocorticoid-induced increase in amylase gene transcription requires induction of an unidentified regulatory protein(s).

Amylase and Chymotrypsinogen Synthesis and Secretion by the Anesthetized Rat Pancreas

To investigate the origin of nonparallel secretion, pancreatic juice was collected in the anesthetized rat, during infusion of [3H]phenylalanine. In the basal state, the amylase to chymotrypsinogen ratios of activities and of [3H] incorporations were 3.5 and 2.5 times higher than in the homogenate, respectively. Both ratios decreased to the value in the homogenate upon caerulein stimulation (600 ng/kg). Inhibition of protein synthesis by cycloheximide (15 ng/kg) did not alter enzyme secretion ratios, and depressed basal protein output only partly, suggesting similar secretory pathways for basal and stimulated secretion. Finally, when [3H]phenylalanine was given before anesthesia, the amylase to chymotrypsinogen ratio of incorporations was again higher in basal secretion than in homogenate, even when further protein synthesis was blocked by cycloheximide before urethane injection. Hence, basal secretion comes from a pancreatic compartment which is functional, although minor, in the conscious animal, and shows a higher rate of amylase synthesis, compared to chymotrypsinogen, than the rest of the gland. It could consist of a subpopulation of acinar cells.

Catabolite repression of amylase synthesis in yeast

Amylase synthesis by the yeasts Saccharomycopsis fibuligera and Schwanniomyces castellii and alluvius is repressed by glucose. Steady state continuous culture data for amylase activity, E, biomass concentration, X, and reducing sugar concentration, S, were fitted to the three-parameter catabolite repression model E/X = [1 + a(S/X)]/[1 + b(S/X)], and biomass productivity, DX, and amylase productivity, DE, were determined for S. castellii and S. alluvius.

Synthesis and secretion of amylase in the rat parotid gland following autonomic nerve stimulation in vivo

Parasympathetic and sympathetic nerve stimulation in vivo, individually and in combination, was used to study secretion and synthesis of amylase in the rat parotid gland. After 30 min with sympathetic nerve stimulation (3 Hz) a decrease in glandular amylase was seen, which corresponded approximately to the salivary output. On the other hand, after parasympathetic stimulation (10 Hz), chosen to obtain comparable amylase output, there was no decrease in glandular amylase, which points to synthesis during such activation. Experiments with incorporation of [3H]leucine, reflecting amylase synthesis, showed that both types of nerve stimulations increased such uptake in parotid protein. These results indicate that beside sympathetic activity, which is the main stimulus for granular amylase secretion, parasympathetic nerve impulses can evoke considerable amylase secretion because amylase synthesis is stimulated and amylase is rapidly available from a special, possibly non-granular pool. As expected from previous experiments an augmentation of amylase secretion was found, and the present experiments also indicated an augmentation at the level of synthesis when the two nerves were stimulated at the same time.

Decrease in amylase (EC3.4.21.4) synthesis in lactating rats

The amylase (EC 3.4.21.4) and trypsin (EC 3.2.1.1) activities in the pancreas in rats during pregnancy, lactation and after the weaning period, and the secretory responses to a secretagogue (caerulein) in the exocrine pancreas of lactating rats were measured. Trypsin activity increased as lactation progressed and reached twice that of unmated rats in the second half of the lactation period. The amylase activity fell before parturition and failed to recover even after the start of lactation and was significantly decreased throughout the lactation period. The total amount of pancreatic juice produced in the lactating rats was significantly greater than that of unmated rats; the amylase output was significantly less than that of unmated rats. When the pups were removed, amylase activity in the pancreas returned to the value in unmated rats. Furthermore, the amylase activity in lactating rats receiving a daily injection of insulin significantly exceeded that of normal lactating rats. These results indicate that the decrease in amylase activity in lactating rats is due to the reduction of amylase synthesis and there is a possibility that insulin is required for normal or elevated rates of amylase synthesis in lactating rats.

Do insulin and the insulin like growth factors (IGFs) stimulate growth of the exocrine pancreas?

Previous in vivo studies have suggested a long term regulatory role for insulin in the exocrine pancreas. Furthermore, we reported that pancreatic acini have specific receptors for IGF I and II, and using different techniques (acid washing, trypsinisation, electron microscope autoradiography), that CCK8 reduces the internalisation of IGF II. To now directly study the long term role for IGF and insulin in the exocrine pancreas we used AR42J cells, a rat cell line that is derived from a transplantable tumour of the acinar pancreas. Hormone binding studies with 125I-labelled hormones indicated that those cells have insulin receptors, relatively fewer receptors for IGF II but in contrast with normal acini no detectable IGF I receptors. Insulin at concentrations as low as 1 nm stimulated the growth of AR42J cells, as measured by an increase in cell number, DNA and protein content. At 100 nM insulin had maximal effects stimulating the growth by about 50%. IGF I and II had only very weak growth promoting effects probably due to their interaction with the insulin receptor. Additionally insulin increased amylase synthesis over the same concentration range that it stimulated growth. But immunoprecipitation studies revealed that insulin induced a selective increase of amylase synthesis over general protein synthesis. These studies indicate, therefore, that insulin is a growth promoting hormone for AR42J cells and that additionally it seems to specifically regulate amylase synthesis. The role for the IGFs in the exocrine pancreas, however, still remains to be determined.

Effects of inverse changes in dietary lipid and carbohydrate on the synthesis of some pancreatic secretory proteins.

The effect of ingesting isocaloric and isonitrogenous diets with increasing amounts of lipid (0-30%) and consequently decreasing amounts of carbohydrates (68.7-1.25%) on the exocrine pancreas was studied in adult male Wistar rats. Pancreatic contents of chymotrypsin, lipase and colipase activity, as well as synthesis of amylase, lipase, procarboxypeptidases and individual serine proteases were examined. Lipid-free diets and diets containing 1% lipid were found to have little effect on pancreatic proteins as compared with lipid-rich diets where two distinct patterns of response were observed. Ingestion of diets containing 3-20% lipid resulted in a progressive increase in the activity of lipase, colipase and chymotrypsin up to 2-fold in the first case and 1.6-fold in the two other cases when animals were fed the 20% fat diet. Under the latter conditions, the relative synthesis of secretory proteins, as expressed as percentage of the radioactivity incorporated into individual proteins compared to that incorporated into the total mixture of exocrine proteins, was unchanged for procarboxypeptidases, whereas it was stimulated for lipase (2-fold) and serine proteases (1.6-fold). Amylase relative synthesis progressively decreased as the lipid content of diets increased. Consumption of hyperlipidic diets containing 25% and 30% fat resulted in a further enhancement in the activity of lipase and colipase in the gland in contrast with chymotrypsin activity which was unchanged as compared to the control diet (3% lipid). As far as biosynthesis was concerned, a plateau in the relative synthesis of lipase and serine protease was reached. Amylase relative synthesis further decreased down to 2.2-fold when rats were fed the 30% fat-rich diet whereas that of procarboxypeptidases was markedly increased (about 1.7-fold). Absolute rates of synthesis of total pancreatic secretory proteins, as expressed with regard to the DNA content of the tissue, indicated that biosynthesis of all secretory pancreatic proteins was stimulated by hyperlipidic diets (at least 2-fold with the 30% lipid diet). Consequently, when such an increase was taken into consideration, the absolute synthesis of amylase was found to be unchanged throughout the dietary manipulations, whereas that of lipase, procarboxypeptidases and serine proteases were stimulated by 4.0-fold, 3.4-fold and 3.2-fold, respectively.

Regulation of amylase synthesis in Bacillus circulans F-2.

In the usual batch cultivation, Bacillus circulans F-2 produced amylase only when granular carbon sources such as raw starch or crosslinked starches (CLP) were added. In the dialysis cultivation, where CLP and partially purified amylase were incubated inside the dialysis tubing, the bacterium inoculated outside of the tubing grew and produced the amylase. Amylase production of this bacterium was further investigated in feeding cultivation, in which maltose was fed to the cultivation medium at various rates. The bacterial growth increased with the increase of the feeding rate of maltose, but maximum amylase production was observed at a feeding rate of 45 mg/hr/1. No amylase was produced on the media containing monosaccharides, sucrose, lactose, or isomaltose in the feeding cultivation although bacterial growth was observed. The amylase of this bacterium was found to be inducible. Replacement of 20% of the maltose with glucose resulted in a great decrease (70%) in the amylase production. This shows that the amylase synthesis of B. circulans F-2 is severely repressed by glucose.

Effect of amino compounds on alkaline amylase production by alkalophilic Bacillus sp.

Of the amino compounds investigated, β-alanine, dl-norvaline and d-methionine were effective for the production of alkaline amylase by alkalophilic Bacillus no. A-40-2. The addition of 0.5% dl-norvaline and 0.5% d-methionine to the culture medium increased amylase production 1.7-fold, while they repressed growth slightly or strongly. There was no relation between amylase yield and the extracellular protein amount. Because alkaline protease activity was negligible in the culture fluid, these compounds might change the regulation of amylase synthesis and/or make amylase excretion easier.

Yeast cells may use AUC or AAG as initiation codon for protein synthesis

A yeast expression plasmid without an ATG codon for initiation of mouse α-amylase protein synthesis directs the synthesis and secretion of active enzyme indistinguishable from both native mouse α-amylase and amylase synthesized from plasmids with normal ATG initiation codons. The initiation of amylase synthesis directed by this plasmid is at either an AUC or an AAG codon. In either case the amino acid sequence of the hydrophobic core and peptidase cleaving region of the signal peptide are normal, and the protein translation remains in frame with the structural gene of the mouse α-amylase.

Lipase synthesis in the rat pancreas is regulated by secretin.

Conscious rats were infused with optimal doses of secretin (16 clinical units [CU]/kg/h), cerulein (0.25 microgram/kg/h), and both for varying periods of time over 24 h. The presence of zymogen granules in acinar cells and the tissue content of enzymes showed progressive decreases over 3 and 12 h for cerulein and secretin stimulation, respectively. Stimulation with either hormone resulted in a two-fold increase in protein synthesis at 6 and 12 h. Kinetically, the increases observed in protein synthesis were not directly coupled to the observed decreases in tissue content of enzymes. Cerulein stimulation selectively increased the synthesis of anionic trypsinogen forms 1 and 2 and chymotrypsinogen forms 1 (anionic) and 2 (cationic) and decreased the synthesis of amylase. Secretin stimulation selectively promoted the synthesis of lipase and proelastase 2. Lipase synthesis was increased 2.8-, 5.8-, and 4.8-fold at 6, 12, and 24 h, respectively. Proelastase 2 synthesis was increased 2.3-, 3.0-, and 3.4-fold at the same time points. In combination, secretin and cerulein stimulation resulted largely in competitive effects, suggesting that the two hormones exert effects on protein synthesis through different mechanisms. The findings suggest that the anticoordinate changes observed in the synthesis of functional groups of pancreatic enzymes by nutritional substrates in the diet are modulated by specific hormones. Synthesis of the majority of protease zymogens but not cationic trypsinogen or proelastase 2 is modulated by cholecystokinin and its peptide analogs. In contrast, the synthesis of pancreatic lipase and proelastase 2 is regulated by secretin. The findings indicate for the first time that secretin regulates the synthesis of specific proteins in the exocrine pancreas.

Translational Control of Protein Synthesis in Isolated Pancreatic Acini

We wished to determine whether the stimulation of protein synthesis by CCK8, carbachol, and insulin in isolated rat pancreatic acini resulted from translational or transcriptional induction of protein synthesis, and whether these hormones had similar or different effects on the rates of synthesis of individual enzymes. Isolated pancreatic acini were prepared from streptozocin-treated rats by collagenase digestion, mechanical dissociation, and centrifugation through a bovine serum albumin (BSA) cushion. Sixty-minute incubations, with maximally effective doses of CCK8, carbachol, and insulin, produced a 50, 90, and 100% increase, respectively, in the rate of protein synthesis. After inhibition of transcription with actinomycin D, the hormones still produced a 23, 50, and 61% increase, respectively, in the rate of protein synthesis. The study of the effect of the three hormones and the combination of CCK8 and insulin on the rate of synthesis of trypsinogen, chymotrypsinogen, lipase, and amylase, purified by isoelectric focusing, demonstrated that the hormones induced similar effects on the pattern of enzyme synthesis, and that they all induced the rate of synthesis of chymotrypsinogen slightly more than that of the other enzymes studied. We conclude that the hormones studied exert similar posttranscriptional influences in the regulation of protein synthesis in the pancreatic acinar cell.

AXIAL CONTROL OF AMYLASE SYNTHESIS AND SECRETION IN COTYLEDONS OF AGROSTEMMA GITHAGO

SUMMARY In Agrostemma githago (Caryophyllaceae) starch reserves are located outside the embryo in the perisperm. One of the main degradative enzymes, α-amylase, is synthesized in and secreted by the cotyledons. The synthesis and secretion of amylase were shown to be under axial control. Benzylaminopurine (BAP)1 or gibberellic acid (GA3) replaced axis requirement. BAP probably acted by a general enhancement of metabolism, whereas GA3 had, like the axis, a specific effect on the cotyledons. The degradation of storage proteins in the cotyledonary tissue was controlled in a similar way. Two arguments raised in the literature against hormonal regulation of reserve breakdown in dicots are 1) that the axis withdraws otherwise inhibitory hydrolysis products from the cotyledons and 2) that reduced activity of hydrolytic enzymes in detached cotyledons is an artefact due to lack of oxygen. These arguments were shown to be most likely invalid.

Culture Conditions for Production of Thermostable Amylase by Bacillus stearothermophilus

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 CaCl(2) 2H(2)O enhanced amylase production over that on Ca -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 degrees C. The crude amylase, after (NH(4))(2)SO(4) fractionation, had an activity of 210.6 U mg. The optimum temperature and pH of the enzyme were found to be 82 degrees C and 6.9, respectively. Ca was required for the thermostability of the enzyme preparation.

Interactions between Ethylene, CO2, and ABA on GA3-Induced Amylase Synthesis in Barley Aleurone Tissue

Gibberellic acid-induced synthesis and release of alpha-amylase in barley aleurone tissue was inhibited by abscisic acid. This inhibition was relieved by simultaneous application of ethylene ranging in concentration from 0.1 to 100 microliters per liter. When CO(2) was applied, it eliminated the effect of 0.1 microliter per liter ethylene and reimposed the abscisic acid inhibition. All concentrations of CO(2) tested from 400 to 10(5) microliters per liter counteracted the effect of 0.1 microliter per liter ethylene, but had no observable effect on any higher concentration of ethylene. The results indicate that some processes necessary for embryo growth may be subject to regulation by ethylene and carbon dioxide at naturally occurring concentrations of the gases.

Extracellular amylase synthesis by Streptomyces limosus

A strain of Streptomyces limosus was superior to 97 strains of various Streptomyces species for the hydrolysis of starch granules. Extracellular amylase activity was induced by maltose, malt extract or starch but not by lactose, sucrose or d-xylose. The enzyme attacked starch endo-actively producing mainly maltose. It was insensitive to EDTA and 4-chloromercuribenzoate. The enzyme was unstable above 45°C but at 25°C attacked raw starch granules more efficiently than the α-amylase (1,4-α- d-glucan glucanohydrolase, EC 3.2.1.1) from Bacillus amyloliquefaciens

Regulation and genetic enhancement of glucoamylase and pullulanase production in Clostridium thermohydrosulfuricum

We studied the general mechanism for regulation of glucoamylase and pullulanase synthesis in Clostridium thermohydrosulfuricum. These amylases were expressed only when the organism was grown on maltose or other carbohydrates containing maltose units. Amylase synthesis was more severely repressed by glucose than by xylose. Catabolite repression-resistant mutants were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained glucose-starch agar plates. Amylases were produced in both wild-type and mutant strains when starch was added to cells growing on xylose but not when starch was added to cells growing on glucose. In both wild-type and mutant strains, glucoamylase and pullulanase were produced at high levels in starch-limited chemostats but not in glucose- or xylose-limited chemostats. Therefore, we concluded that amylase synthesis in C. thermohydrosulfuricum was inducible and subject to catabolite repression. The mutants produced about twofold more glucoamylase and pullulanase, and they were catabolite repression resistant for production of glucose isomerase, lactase, and isomaltase. The mutants displayed improved starch metabolism features in terms of enhanced rates of growth, ethanol production, and starch consumption.

Cholecystokinin-induced secretion and synthesis of amylase and cationic trypsinogen by pancreatic acini isolated from rats given an ethanol diet

Effects of chronic alcohol intake on secretion and synthesis of amylase and cationic trypsinogen (CT) were studied with pancreatic acini isolated from male Sprague-Dawley rats fed a Lieber-DeCarli ethanol or control diet for 30 days. Pancreatic acini were incubated in a media containing increasing concentrations of cholecystokinin octapeptide (CCK-8: 0-1000pM) followed by addition of [ 3H]leucine. Amylase and CT secreted in the media and those labeled in acini were quantitated. Basal and CCK-stimulated secretion of CT was not different in alcoholic and control groups. On the other hand, a dose-response curve of CCK-stimulated amylase secretion from alcoholic acini was markedly reduced with both basal and maximal secretion decreased to only 40% of controls. Basal incorporation of [ 3H]leucine into amylase was reduced by 70% in alcoholic acini compared to controls while that into CT was not different in the two groups. CCK-8 exhibited a biphasic effect on [ 3H]leucine incorporation into both enzymes in alcoholic acini: low concentrations of CCK-8 (<100pM) increased the incorporation whereas high concentrations (>100pM) decreased it. However, in control acini, CCK-8 induced progressive suppression of the incorporation into these enzymes, the pattern of which was similar to that previously observed in fasted rats (Am. J. Physiol. 241:G116–G112, 1981). This difference in the response pattern resulted in significantly higher rates of CCK-8 induced incorporation into CT in alcoholic acini. These results suggest that the differences observed may possibly be attributable not only to alcohol intake but also to the differences in carbohydrate intake and in temporal patterns of diet consumption.