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At least 919 records · Page 51Linked to original sources
PubMed1998

Effects of (+)-8',8',8'-trifluoroabscisic acid on alpha-amylase expression and sugar accumulation in rice cells.

The effects of (+)-8',8',8'-trifluoroabscisic acid (trifluoro-ABA) on alpha-amylase expression were studied in rice embryoless half-seeds, scutella, and suspension-cultured cells derived from the embryo, and the effects of the analog on sugar accumulation were also studied in scutella and suspension-cultured cells. Treatment with (+)-trifluoro-ABA strongly inhibited the gibberellic acid-inducible expression of alpha-amylase I-1 encoded by RAmy1A in the aleurone layers of embryoless half-seeds at the levels of transcription, protein synthesis, and enzyme activity. It was also found that (+)-trifluoro-ABA stimulated (i) the uptake of glucose from the incubation medium and (ii) the synthesis of sucrose in scutellar tissues and suspension-cultured cells of rice. The biological activity of (+)-trifluoro-ABA was found to be more potent and persistent than that of natural ABA. We further examined the effects of trifluoro-ABA on the expression of alpha-amylase I-1 in scutellar tissues and suspension-cultured cells. It was found that (+)-trifluoro-ABA did not inhibit the formation of alpha-amylase I-1 in the absence of external glucose. However, glucose and (+)-trifluoro-ABA cooperatively suppressed the formation of alpha-amylase I-1. Judging from these results, we conclude that the regulatory mechanism for the expression of alpha-amylase I-1 in the scutellar epithelium is distinguishable from that operating in the aleurone layer.

Abscisic Acid↗
PubMed2006

Expression and secretion of Bifidobacterium adolescentis amylase by Bifidobacterium longum.

Bifidobacterium adolescentis Int-57 (INT57), isolated from human feces, secretes an amylase. We have shot-gun cloned, sequence analyzed and expressed the gene encoding this amylase in B. longum. The sequenced 2477 bp fragment was homologous to other extracellular amylases. The encoded protein was predicted to be composed of 595 amino acids with a molecular weight of 64 kDa, and was designated AmyB. Highly conserved amylase domains were found in AmyB. The signal sequence and cleavage site was predicted by sequence analysis. AmyB was subcloned into pBES2, a novel E. coli-Bifidobacterium shuttle vector, to construct pYBamy59. Subsequently, B. longum, with no apparent amylase activity, was transformed with pYBamy59. More than 90% of the amylase activity was detected in the culture broth. This approach may open the way for the development of more efficient expression and secretion systems for Bifidobacterium.

Amino Acid Sequence↗
PubMed1988

Effect of methotrexate on protein and amylase secretion by rat parotid and submandibular salivary glands.

Saliva was collected from anaesthetized, methotrexate (MTX)-treated, pair-fed and control rats using either acetylcholine or bethanechol as secretagogue. Parotid saliva was analysed for protein and amylase content, and submandibular saliva for protein. Acetylcholine stimulation after MTX increased parotid protein output by 151 per cent, amylase output by 125 per cent and submandibular protein output by 229 per cent when compared to control values. There were no significant differences in either protein or amylase output between the control and pair-fed animals. With bethanechol, salivary amylase and protein secretion did not differ between MTX-treated and control animals. The significant increase in parotid protein and amylase output with acetylcholine was reduced by pretreatment with hexamethonium (5 mg/kg) or propranolol (5 mg/kg), but not by phenoxybenzamine (1 mg/kg). MTX did not increase the number or the dissociation constant of parotid beta-adrenergic receptors but did increase their number in the submandibular gland. Thus increases in protein and amylase output caused by acetylcholine MTX-treated rats may be the result of increased beta-adrenergic activity.

Amylases↗
PubMed1989

Protein synthesis and amylase messenger RNA content in rat parotid salivary glands after total or partial stimulation with isoproterenol.

The rate of synthesis of secretory proteins increases significantly in rat parotid glands after stimulated discharge of stored proteins. How any difference in the amount of secretory protein discharge affects the rate of subsequent protein synthesis, and whether this post-secretory synthesis is regulated at the level of messenger RNA, was now examined. One group of rats was stimulated to secrete 97% of stored secretory proteins by an intraperitoneal injection of isoproterenol. The other group received a much smaller dose to induce the discharge of about 40% of the proteins. Despite this difference in secretion, the subsequent rates of total protein synthesis, as well as of amylase, were increased to about the same extent. The amylase messenger RNA (mRNA) was identified and quantified by hybridization with a 32P-labelled amylase complementary DNA (cDNA) probe. The amylase mRNA in stimulated and unstimulated rats was of the same molecular size (Northern blot analyses). The amount of amylase mRNA, determined by dot blot analyses, were also increased in stimulated rats, although this increase was not as great as that in the rate of amylase protein synthesis. The implications of this discrepancy concern the possibility that the mechanism of regulation of secretory protein synthesis in parotid glands is at the translational level.

Amylases↗
PubMed1992

Effects of streptozotocin diabetes on amylase release and cAMP accumulation in rat parotid acinar cells.

Rat parotid responses to sympathetic nerve stimulation in vivo are impaired 2-4 weeks after the induction of streptozotocin diabetes. In this study, the effects of experimental diabetes of similar duration and severity on noradrenaline-stimulated amylase release and cAMP accumulation were examined in vitro. Amylase levels were significantly lower in acinar cells isolated from diabetic animals than in controls, and cellular amylase increased after treatment of the diabetic animals with either thyroxine (T4) or insulin. Diabetes and T4 had no apparent affect on amylase release measured as a percentage of the total. In contrast, giving insulin resulted in a significant reduction in maximal secretion (20.4 +/- 2.4% compared with 43.6 +/- 7.6%). Similar results were observed when amylase release was stimulated with forskolin. Basal cAMP levels were unaffected by diabetes or T4 (7.8 +/- 2.3 pmol/mg protein), but stimulated cAMP levels were significantly greater in diabetic acinar cells than in controls. Insulin reversed the effects of diabetes on cAMP accumulation, whereas T4 had no effect. Thus, diabetes (2-4 weeks) and insulin in vivo appear to have paradoxical effects on parotid amylase release and cAMP accumulation in vitro. Further, the effects of diabetes appear to be unrelated to thyroid status.

Amylases↗
PubMed1992

Amylase mRNA synthesis and ageing in rat parotid glands following isoproterenol-stimulated secretion.

In the parotid, as well as in other exocrine glands, secretory protein synthesis declines with age. However, whether this decline in the steady-state rate of protein synthesis reflects the reduced digestive activity of the animal or actual cellular alterations that affect synthesis is unknown. Here the ability to synthesize amylase and its mRNA during the period of enhanced protein synthesis following secretion induced by isoproterenol was compared in acinar cells of 2-and 24-month-old rats. In unstimulated glands, rates of synthesis of total protein and amylase, as well as amounts of amylase mRNA, were significantly less in the older rats than in their younger counterparts. After stimulation with isoproterenol, which induced the secretion of about 50% of stored proteins, rates of synthesis of total protein, as well as amylase, were increased by about 2.5 x the unstimulated rates in both age groups. However, the amount of amylase mRNA did not increase in parallel with the increase in the rate of amylase protein synthesis in both young and old rats. The molecular size of the mRNA was the same in stimulated and unstimulated glands of both age groups. Thus, it appears that parotid acinar cells from old rats can be stimulated to synthesize secretory proteins at an increased rate. It remains to be determined what causes the reduced rate of protein synthesis in unstimulated glands in old rats.

Aging↗
PubMed1995

Amylase secretion by cultured porcine parotid cells.

The similarity of porcine and human physiology and the availability of slaughterhouse tissues suggests the use of porcine parotid cells as a model for amylase secretion. A procedure is described for the isolation of porcine parotid cells by collagenase-P/dispase digestion of the tissue. The preparation consisted of individual cells and small aggregates that were maintained in primary culture, during which the cells formed aggregates that firmly attached to the plastic substrate. The amylase content of the cultured cells remained adequate for assay of secretory activity during culture for one week after isolation. Depending upon variations in experimental treatments, the cultured cells secreted approx. 35-65% of cellular amylase in response to a carbachol challenge. The cells were slightly responsive to long exposures to isoproterenol, and were unresponsive to nicotine, elevated extracellular K+ or substance P. Secretion induced by carbachol required extracellular Ca2+, was inhibited by atropine and occurred with a nearly linear response over a 30-min period. The Ca2+ ionophore A23187 was also a potent secretagogue for amylase secretion, producing levels of secretion equal to that induced by carbachol. The ease of preparation and the retention of amylase during primary culture suggests that the preparation will be useful in studies on muscarinic receptor-mediated control of amylase secretion.

Amylases↗
PubMed1975

Purification and some properties of a novel maltohexaose-producing exo-amylase from Aerobacter aerogenes.

Maltohexaose producing amylase (EC 3.2.1.-) is the fourth known exo-amylase, the three previously known being glucoamylase, beta-amylase and Pseudomonas stutzeri maltotetraose producing amylase. The enzyme after release from Aerobacter aerogenes cells by 0.1% sodium lauryl sulfate extraction was purified by ammonium sulfate precipitation, DEAE-Sephadex column chromatography and Sephadex G-100 gel filtration to 80-fold of the original sodium lauryl sulfate extract activity, It gave a single band on disc electrophoresis, and the molecular weight by gel filtration was 54 000. This amylase showed maximal activity at 50 degrees C and pH 6.80. The pH stability range was relatively wide, the enzyme retaining more than 90% of its initial activity in the range of 6.50-9.0. 80% of the activity was retained after 15 min at 50 degrees C. This enzyme produced maltohexaose from starch, amylose and amylopectin by exo-attack, but did not act on alpha- or beta-cyclodextrin, pullulan or maltohexaitol. Also the enzyme acted on beta-limit dextrins of amylopectin and glycogen to form branched oligosaccharides. The unusual reaction of this enzyme on beta-limit dextrin is discussed from the standpoint of the stereochemistry of 1,4-alpha- and 1,6-alpha-glucosidic bonds. This is the anomalous amylase for which it is recognized that 1,6-alpha-glucosidic linkages in the substrates can mimic the effect of 1,4-alpha-bonds, as previously observed in pseudo-priming reactions of E. coli phosphorylase.

Amylases↗
PubMed1976

Purification and properties of an alpha-amylase inhibitor from wheat.

Four inhibitors of alpha-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-exchange chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60--90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract.

Amylases↗
PubMed1979

Starch metabolism in Pseudomonas stutzeri. I. Studies on maltotetraose-forming amylase.

The extracellular maltotetraose-forming amylase of Pseudomonas stutzeri was purified to homogeneity by a combination of affinity and hydroxyapatite chromatography. Sodium dodecyl sulfate-gel electrophoresis indicated that the oligomeric enzyme contains two different subunits with molecular weights of 48 000 and 58 000. Cross-linking studies using dimethyl suberimidate have demonstrated that the native enzyme consists of dimers. Seven isozymes of the amylase have been identified after polyacrylamide gel electrophoresis and amylose-digestion zymograms. The amylase of Ps. stutzeri is known to produce maltotetraose from linear and branched alpha-glucans by an exomechanism. The relatively high conversion rate of starch (75% hydrolysis), and the hydrolysis of cross-linked blue starch by this amylase indicate that the enzyme can cleave its substrates also by an endomechanism. Further strong evidence for an endomechanism was obtained from the action of the amylase on maltotetraose units which are located within the pullulan molecule. Dextran, pullulan, and maltotetraose are compeititve inhibitors. EDTA caused reversible inactivation. Amylase activity could be restored by addition of Ca2+. Heavy metals are inhibitory.

Amylases↗
PubMed1988

Mode of inhibitory action of cholecystokinin in amylase release from isolated rat pancreatic acini--inhibition of secretory process post to protein kinase C-calcium ion systems.

The incubation of isolated rat pancreatic acini with low doses (1 x 10(-11)-1 x 10(-10) M) of cholecystokinin-octapeptide (CCK8) induced amylase release. This CCK8-induced amylase release has been shown to be mediated through the protein kinase C activation and the Ca2+ mobilization which are linked to the phospholipase C-mediated hydrolysis of phosphoinositides. However, the incubation of the acini with high doses (1 x 10(-9)-1 x 10(-7) M) of CCK8 reduced amylase release to the level less than that induced by the maximally effective dose (1 x 10(-10) M) of this secretagogue. Under the same conditions, the high doses of this secretagogue did not inhibit the phospholipase C-mediated hydrolysis of phosphoinositides. The stimulatory action of the maximally effective dose of CCK8 in amylase release was mimicked by the simultaneous addition of protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ca2+ ionophore A23187. A high dose (1 x 10(-7) M) of CCK8 reduced the amylase release induced by the combination of TPA and A23187. These results suggest that the high doses of CCK8 inhibit the secretory process post to the protein kinase C-Ca2+ systems and thereby reduce the amylase release induced by the maximally effective dose of CCK8 in rat pancreatic acini.

Amylases↗
PubMed1992

Effects of calyculin A on amylase release in streptolysin-O permeabilized acinar cells.

The effects of the phosphatase inhibitors calyculin A and okadaic acid on amylase release from streptolysin-O permeabilized rat pancreatic acini were investigated. Both agents induced similar biphasic effects with moderate potentiation of calcium-stimulated amylase release at medium and strong inhibition at higher concentrations. Calyculin A was thirty times more potent than okadaic acid and at 100 nM totally inhibited calcium-induced amylase release while 3 microM okadaic acid reduced amylase release by 78%. 100nM calyculin A also completely inhibited GTP gamma S-potentiated amylase release and partially inhibited phorbol ester potentiated secretion. The data indicate that inhibition of a serine/threonine phosphatase, probably a type 1 phosphatase, leads to inhibition of calcium-induced amylase release in permeabilized pancreatic acini.

Amylases↗
PubMed1977

Immunoreactivities of alpha-amylase of humans and rats.

The immunoreactivities of amylase from human saliva and pancreatic juice and rat parotid and pancreas were investigated. Antisera were prepared in rabbits against each of the human and rat amylase. Human salivary and pancreatic amylases reacted similarly with the antibodies to both human salivary and pancreatic amylases. Rat parotid and pancreatic amylases reacted differently with the antibodies to both rat parotid and pancreatic amylases.

Amylases↗
PubMed1979

Action of human pancreatic and salivary alpha-amylases on maltooligosaccharides: evaluation of kinetic parameters.

The kinetic studies on the reactions of human pancreatic and salivary alpha-amylases with several maltooligosaccharides (maltotetraose, maltopentaose, maltohexaose, and maltoheptaose) were carried out. The susceptibility to hydrolysis with human pancreatic alpha-amylase decreased in the order of maltopentaose, maltohexaose, maltotetraose, and maltoheptaose, while with human salivary alpha-amylase maltopentaose was hydrolysed slightly slower than maltohexaose but fairly faster than maltotetraose or maltoheptaose from a viewpoint of the rates of reactions based on the amount of substrate changed. The relative rates of production of substrates, utilized in the coupled yeast alpha-glucosidase reaction, increased in the order of maltoheptaose, maltohexaose, maltotetraose, and maltopentaose with human pancreatic alpha-amylase, while with human salivary alpha-amylase in the order of maltoheptaose, maltotetraose, maltohexaose, and maltopentaose. Thus, maltopentaose was considered to be the best substrate over maltotetraose, maltohexaose or maltoheptaose for the alpha-glucosidase coupled method of alpha-amylase determination.

Adult↗
PubMed1984

Differential assay of human pancreatic and salivary alpha-amylases in serum using a new fluorogenic substrate.

The difference in the mode action of human pancreatic and salivary alpha-amylases on O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D - glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucitol FG6R), a fluorogenic derivative of maltohexaitol, was found. The products of the enzymatic hydrolysis were analyzed by high-performance liquid chromatography (HPLC) in 8 min. FG6R was hydrolyzed by these enzymes to O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose (FG3) and maltotriitol, or O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-D-glucose (FG4) and maltitol. Pancreatic alpha-amylase produced more FG4 than salivary alpha-amylase. Taking advantage of the differences in action of the two amylases, a differential alpha-amylase assay in serum was performed. The method is simple and rapid and can be used for routine clinical assays of alpha-amylases.

Chromatography, High Pressure Liquid↗
PubMed1984

Kinetics of human pancreatic and salivary alpha-amylases with carboxymethylamyloses as substrates.

The enzymatic measurement of alpha-amylase in human serum with chemically modified amylose as substrate was studied. Several substitutions of carboxymethylamylose were prepared from amylose, suitable amounts of monochloroacetic acid, and sodium hydroxide. The relationship between the degree of substitution of the carboxymethylamylose and the kinetic parameters of human pancreatic, salivary and porcine pancreatic alpha-amylases was determined. The action of alpha-amylases on such modified amyloses in the presence of glucoamylase was measured by a specific enzymatic assay having glucose as the product. The kinetic parameters of human pancreatic alpha-amylase were similar to those of human salivary alpha-amylase. It was possible to determine suitable sensitivities for the alpha-amylase assay using this substrate. These results suggested a subsite model.

Amylose↗
PubMed1986

Effects of decreasing intraluminal amylase activity on starch digestion and postprandial gastrointestinal function in humans.

We used an amylase inhibitor preparation that markedly improves postprandial carbohydrate tolerance in humans to investigate the effects of decreased intraluminal amylase activity on digestion of starch and postprandial gastrointestinal and hormonal responses. Four fasting volunteers were intubated with an oroileal tube to obtain duodenal, jejunal, and terminal ileal samples. After intubation, subjects ingested 50 g of rice starch given with placebo; on the second day, starch was given with the amylase inhibitor. Compared with placebo, the amylase inhibitor significantly (p less than 0.05) reduced duodenal, jejunal, and ileal intraluminal amylase activity by more than 95% for 1-2 h; increased postprandial delivery of total carbohydrate (glucose polymers in particular) to the distal small bowel; increased breath hydrogen concentrations; decreased intestinal water absorption and increased distal intestinal volume delivery to the distal bowel; shortened duodenoileal transit time but doubled postprandial gastric emptying time; reduced the early postprandial plasma glucose rise by 85% and eliminated the late postprandial glucose fall to below fasting levels; and abolished postprandial plasma concentrations of insulin, C-peptide, and gastric inhibitory polypeptide. Postprandial trypsin output was not influenced. We conclude that more than 95% inhibition of amylase reduces dietary starch digestion within the small intestine and uptake of dietary starch from the small intestine, markedly decreases postprandial release of insulin and gastric inhibitory polypeptide, and may alter postprandial upper gastrointestinal motor function.

Adult↗
PubMed1995

Biliary amylase and congenital choledochal dilatation.

The relationship between levels of biliary amylase measured at operation and clinical features was studied in a series of 55 children with congenital biliary dilatation (choledochal cyst) who presented between 1976 and 1993. There were 36 cystic and 19 fusiforms dilatations in the series. The most common modes of presentation were painless jaundice (n = 23) and pancreatitis (n = 22). Five infants presented with abnormal antenatal ultrasound examinations. Children with pancreatitis were older than those with painless jaundice (4.2 versus 1.5 years; P = .005), and a higher proportion had raised levels of biliary amylase (100% versus 44%; P < .0001). There was no difference in the age at presentation (P = .32), clinical mode of presentation (P = .3), or the level of biliary amylase (P = .25) between cystic and fusiform dilatations. A correlation was found between age at surgery and biliary amylase in the cystic (rs = 0.55; P = .001) but not in the fusiform group (P = .22). All infants with antenatal diagnoses were cystic dilatations. Choledochal cystic dilatations that were diagnosed antenatally did not have significant amylase reflux, suggesting that the aetiology of this subgroup is truly congenital. Children who present at a later age with pancreatitis invariably have high levels of biliary amylase, which is presumed to occur because of a common channel and reflux of biliary and pancreatic secretions.

Adolescent↗