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PubMed1986

Caerulein and carbamoylcholine stimulate pancreatic amylase release at resting cytosolic free Ca2+.

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.

Aminoquinolines↗
PubMed1997

A gene encoding for an alpha-amylase from thermophilic Bacillus sp. strain TS-23 and its expression in Escherichia coli.

An alpha-amylase gene from Bacillus sp. strain TS-23 was cloned and expressed by using its own promoter on the recombinant plasmid pTS917 in Escherichia coli. A cell fractionation experiment revealed that approximately 60% of the amylase activity was in the periplasmic space. Analysis and activity staining of the concentrated supernatant fraction by SDS-polyacrylamide gel electrophoresis showed an apparent protein band with a mol. wt of approximately 65,000. The amylase gene (amyA) consisted of an open reading frame of 1,845 bp encoding a protein of 613 amino acids with a calculated mol. wt of 69,543. The predicted amino acid sequence showed high homology with Bacillus species, E. coli and Salmonella typhimurium alpha-amylases. Deletion of 96 amino acids from the C-terminal portion of the amylase did not result in the loss of amylolytic activity. The truncated amylase, deletion of the first 50 amino acids from the N-terminus, was overexpressed in E. coli system and refolded to yield an activable enzyme.

Amino Acid Sequence↗
PubMed1999

Amylase and 16S rRNA genes from a hyperthermophilic archaebacterium.

A hyperthermophilic and amylolytic prokaryote, designated Rt3, was isolated from a thermal spring near Rotorua, New Zealand. The 16S rRNA gene of Rt3 was cloned and sequenced with the aim of determining its phylogenetic affiliations. The phylogenetic analysis of this sequence, which included a selection of archaebacterial and eubacterial 16S rRNA sequences, indicates that Rt3 most likely belongs to the archaebacterial order Thermococcales. An amylase gene (amyA) from Rt3, encoding a highly thermostable amylase activity, was cloned and its DNA sequence determined. Transcriptional signals typical of archaebacteria were evident in this sequence. The sequence is homologous to a broad range of enzymes from the AMY superfamily and contains a typical N-terminal signal peptide. Phylogenetic analysis and comparison of structural features with other AMY superfamily enzymes reveals that, firstly, the closest homologues of the Rt3 amylase are members of the Bacillus and Plant alpha-amylase groups; and secondly, that the Rt3 amylase is closely related to only one other currently known archaebacterial enzyme, i.e. an (AMY superfamily) alpha-amylase from Natronococcus.

Amino Acid Sequence↗
PubMed2002

The identification of the transcriptional regulator CRP in Aeromonas hydrophila JMP636 and its involvement in amylase production and the 'acidic toxicity' effect.

AIMS: The physiological examination of amylase production by Aeromonas hydrophila JMP636 and identification of the mechanism of regulation. METHODS AND RESULTS: Aeromonas hydrophila JMP636 was grown with single, then dual carbon sources; the growth cycle was followed and amylase activity throughout was monitored. The levels of cAMP, a known secondary messenger for the regulatory gene crp, were also examined. Amylase activity was regulated by catabolite repression. Physiological studies revealed that JMP636 exhibited both diauxic growth, with two carbon sources, and the 'acid toxicity' effect on glucose. The crp gene was cloned, expressed and inactivated from the JMP636 chromosome. Catabolite repression of amylase production and the 'acid toxicity' effect both require crp and were linked to cAMP levels. CONCLUSIONS: Regulation of amylase production was predicted to follow the model CRP-mediated cAMP-dependent Escherichia coli catabolite regulation system. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an understanding of the physiology of the opportunistic pathogen Aer. hydrophila through identification of the mechanism of catabolite repression of amylase production and the existence of crp within this cell. It also provides a broader knowledge of global gene regulation and suggests regulatory mechanisms of other Aer. hydrophila gene/s.

Aeromonas hydrophila↗
PubMed2002

A role for arabinogalactan proteins in gibberellin-induced alpha-amylase production in barley aleurone cells.

Arabinogalactan proteins (AGPs) are plant proteoglycans that have been implicated in plant growth and development. The possible involvement of AGPs in the action of gibberellin (GA), a class of plant hormones, was examined by applying beta-glucosyl Yariv reagent (beta-Glc)3Y, a synthetic phenyl glycoside that interacts selectively with AGPs, to barley aleurone protoplasts. Gibberellin induces transcription and secretion of alpha-amylases in the protoplasts. Induction of alpha-amylase was clearly inhibited by (beta-Glc)3Y but not by (alpha-Gal)3Y, a negative control of the Yariv reagent that does not interact with AGPs. Transfection analysis, using an alpha-amylase promoter-GUS fusion gene in the protoplasts, indicated that the transcriptional activation of the alpha-amylase promoter was inhibited specifically by (beta-Glc)3Y. These observations are the first indication of an involvement of AGPs in a plant hormone function. The inhibitory effect of (beta-Glc)3Y was not observed when aleurone layers or half-seed grains were used. This result, together with the fact that protoplasts do not have cell walls, suggests that the AGPs that function in alpha-amylase induction reside at the plasma membrane. An aleurone-specific AGP was detected by reversed-phase HPLC, and supported the idea that an AGP may play an important role in aleurone-specific events. The possible mechanism of AGP function in gibberellin-induced alpha-amylase production is discussed.

Cell Survival↗
PubMed1998

Engineering of factors determining alpha-amylase and cyclodextrin glycosyltransferase specificity in the cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1.

The starch-degrading enzymes alpha-amylase and cyclodextrin glycosyltransferase (CGTase) are functionally and structurally closely related, with CGTases containing two additional domains (called D and E) compared to the three domains of alpha-amylases (A, B and C). Amino acid residue 196 (Thermoanaerobacterium thermosulfurigenes EM1 CGTase numbering) occupies a dominant position in the active-site cleft. All alpha-amylases studied have a small residue at this position (Gly, Leu, Ser, Thr or Val), in contrast to CGTases which have a more bulky aromatic residue (Tyr or Phe) at this position, which is highly conserved. Characterization of the F196G mutant CGTase of T. thermosulfurigenes EM1 revealed that, for unknown reasons, apart from the F196G mutation, domain E as well as a part of domain D had become deleted [mutant F196G(delta'DE)]. This, nevertheless, did not prevent the purification of a stable and active mutant CGTase protein (62 kDa). The mutant protein was more similar to an alpha-amylase protein in terms of the identity of residue 196, and in the domain structure containing, however, some additional C-terminal structure. The mutant showed a strongly reduced temperature optimum. Due to a frameshift mutation in mutant F196G, a separate protein of 19 kDa with the DE domains was also produced. Mutant F196G(delta'DE) displayed a strongly reduced raw-starch-binding capacity, similar to the situation in most alpha-amylases that lack a raw-starch-binding E domain. Compared to wild-type CGTase, cyclization, coupling and disproportionation activities had become drastically reduced in the mutant F196G(delta'DE), but its saccharifying activity had doubled, reaching the highest level ever reported for a CGTase. Under industrial production process conditions, wild-type CGTase converted starch into 35% cyclodextrins and 11% linear oligosaccharides (glucose, maltose and maltotriose), whereas mutant F196G(delta'DE) converted starch into 21% cyclodextrins and 18% into linear oligosaccharides. These biochemical characteristics indicate a clear shift from CGTase to alpha-amylase specificity.

Amino Acid Sequence↗
PubMed2001

Evaluation of amylase and lipase in the diagnosis of acute pancreatitis.

BACKGROUND: The diagnosis of acute pancreatitis relies heavily on a raised amylase. METHODS: In the present study patients were prospectively categorized, without knowledge of pancreatic enzyme levels, into acute pancreatitis (AP; n = 51), disease controls (n = 35), indeterminate as to pancreatitis (n = 189) or exclusions (non-pancreatitis diseases where amylase may be elevated; n = 53). RESULTS: Enzyme levels were analysed by receiver operator characteristics (ROC) curves, with specificity > 80%. Day 1 serum lipase gave the greatest diagnostic accuracy (area under ROC curve = 0.128; P = 0.041 vs serum amylase). At the calculated diagnostic threshold of 208 U/L, lipase gave a sensitivity of 67% and a specificity of 97%. Other diagnostic thresholds (day 1) were: serum total amylase, 176 U/L (ROC 0.104, sensitivity 45%, specificity 97%), urinary total amylase, 550 U/L (ROC 0.108, sensitivity 62%, specificity 97%) and serum pancreatic isoamylase, 41 U/L (ROC 0.107, sensitivity 63%, specificity 85%). At delayed diagnosis (3 days) no enzyme was superior to lipase. The combination of lipase and amylase did not increase diagnostic accuracy. CONCLUSION: Serum lipase is recommended for diagnosis of AP, both early and late in the disease. Although highly specific when elevated, all pancreatic enzymes have low sensitivity for diagnosis.

Acute Disease↗
PubMed1999

Twenty-four-hour serum amylase predicting pancreatic reaction after endoscopic sphincterotomy.

BACKGROUND AND STUDY AIMS: Acute pancreatitis is still the most common complication after endoscopic sphincterotomy (ES) and cholangiopancreatography (ERCP). The aim of this study was to detect the time when the peak of serum amylase was predictive for postprocedure pancreatitis or long-lasting severe hyperamylasemia, in order to plan the follow-up of patients. METHODS: Serum amylase activity was measured in a prospective series of 409 consecutive patients after ES, immediately before ES and two, four, eight and 24 hours thereafter; the two, four and eight-hour data were compared with those at 24 hours and with the outcome. Evaluation was done separately for the 198 cases with pancreatic duct opacification and for the 202 cases at high risk for postprocedure pancreatitis. RESULTS: Twenty-four hours after ES, amylase was still more than five times the upper normal limit in 26 patients, associated with pancreatic-like pain in 19 of them (mild/moderate pancreatitis) and asymptomatic in the remaining seven (long-lasting severe hyperamylasemia). There was a significant difference at all sampling times between the 26 patients with 24-hour severe hyperamylasemia and those with the lower level. Although the sensitivity of amylase measurement in detecting pancreatitis was highest at eight hours, in practice the four-hour assessment appears a reliable predictor. Almost all patients with serum amylase levels more than five times the upper normal limit at four, eight and 24 hours had had pancreatic duct opacification. In contrast, patient-related risk factors for postprocedure pancreatitis did not play a significant role in the present series. CONCLUSIONS: Serum amylase assessment four hours after ES is a reliable, cost-effective follow-up and minimizes the likelihood of underestimating the risk of post-procedure pancreatic reaction. It should be recommended particularly in out-patients and when pancreatic duct opacification has occurred.

Acute Disease↗
PubMed2002

Asymptomatic elevation of serum lipase and amylase in conjunction with Crohn's disease and ulcerative colitis.

BACKGROUND AND OBJECTIVE: Elevated serum lipase and amylase are often observed in IBD patients (Crohn's disease and ulcerative colitis) without clinical symptoms of a pancreatitis. The intention of this prospective study is to evaluate the frequency of elevated serum lipase and amylase with these patients and to try to explain this phenomenon in consideration of the existing literature. PATIENTS AND METHODS: 136 IBD patients (MC: 66; CU: 70) participated in this prospective study which lasted 3 months. All patients with increased levels of serum lipase and amylase were prospectively followed-up for another 3 months. RESULTS: We found an asymptomatic elevation of serum lipase and amylase without symptoms of a pancreatitis in 14 % of the observed IBD patients. A significance between the elevation of lipase/amylase and the activity index (CDAI,CAI) or the CRP level could not be found. A significant increase of lipase/amylase (more than twice the normal standard) was shown more often in lipase (4.4 %) than in amylase levels (0.7 %). The possible reasons for an asymptomatic increase of lipase/amylase in IBD patients (e. g. latent extra-intestinal involvement of the pancreas in IBD with pancreatitis; extra-pancreatic release of lipase/amylase from the inflammatory bowel; intestinal reabsorption of released lipase/amylase in the inflammatory bowel) are discussed in this text. CONCLUSION: An asymptomatic elevation of lipase/amylase in IBD patients is not infrequent (14 %). The increase of lipase or amylase, without typical symptoms, makes a pancreatitis with a required therapy unlikely. A specific pancreatitis therapy is not necessary in these cases. The therapy should be guided by the requirements of the IBD therapy.

Adolescent↗
PubMed1984

[Chronic increase of amylase in primary sicca syndrome].

Pancreatitis had been erroneously diagnosed in a 50-year-old female patient with primary sicca syndrome with chronic salivary-gland-caused increase of amylase of 5 years' standing. Differentiation of amylase into isoenzymes showed clearly increased salivary amylase with normal pancreatic amylase. Little is known on the increase of amylase in Sjögren's syndrome. Unresolved increase of amylase of non-pancreatic origin should thus also lead to consideration of sicca syndrome.

Amylases↗
PubMed1970

Translation-level control of amylase and protein synthesis by epinephrine.

The synthesis of RNA, protein, and amylase has been studied in normal and epinephrine-treated slices of rat parotid gland. Epinephrine stimulates synthesis of total cell protein and specifically of amylase, but amylase synthesis is preferentially increased. There is no change in either the specific radioactivity or pattern of labeling of total RNA. In the presence of actinomycin D, epinephrine stimulates secretion of amylase, as well as protein and amylase synthesis. RNA synthesis under these conditions is reduced to 10 per cent of control values. Slices obtained from animals treated with actinomycin D nine and a half hours prior to study show responses to epinephrine comparable to those from control animals, with RNA synthesis inhibited by 70 per cent. Since amylase synthesis is stimulated by epinephrine in the absence of RNA synthesis, control of the production of this enzyme is probably exerted at the level of translation.

Amylases↗
PubMed1981

Phosphorylation of the same specific protein during amylase release evoked by beta-adrenergic or cholinergic agonists in rat and mouse parotid glands.

Stimulation of amylase secretion from the rat parotid gland by beta-adrenergic agonists is associated with a specific phosphorylation of three membrane-bound proteins designated as proteins I, II, and III [Jahn, R., Unger, C. & Söling, H. D. (1980) Eur. J. Biochem. 112, 345-352]. In contrast, stimuliation by carbachol induced significant phosphorylation of only protein I. This phosphorylation was low compared to isoproterenol-induced phosphorylation but corresponded to the smaller enhancement of amylase secretion. The mouse organ, however, is almost equally sensitive to beta-adrenergic and to cholinergic agonists. Incubation of mouse parotid gland slices with either 20 microM isoproterenol or 10 microM carbachol resulted in strong and comparable releases of amylase, which were accompanied by comparable phosphorylations of protein I. Proteins II and III were phosphorylated only in the presence of isoproterenol. Removal of external calcium by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate abolished the carbachol-induced release of amylase but not the phosphorylation of protein I. Isoproterenol-induced secretion of amylase and phosphorylation of proteins I, II, and III were not inhibited under these conditions. Amylase release stimulated by the ionophore A-23187 was accompanied by the phosphorylation of protein I. Two-dimensional electrophoresis revealed that the radioactive spot corresponding to protein I was located at the same position after cholinergic and after beta-adrenergic stimulation, indicating that both stimuli led to the phosphorylation of the same membrane-associated protein. These findings strongly support the view that the phosphorylation of protein I is an important step in the sequence of events leading from receptor activation to exocytosis.

Amylases↗
PubMed1988

Translational control of anionic trypsinogen and amylase synthesis in rat pancreas in response to caerulein stimulation.

Infusion of rats with optimal doses of caerulein for up to 24 hr resulted in divergent changes in protein synthesis in the exocrine pancreas: a 3-fold increase in synthesis of anionic trypsinogen and a 75% decrease in synthesis of amylase. Lipase synthesis showed no change. Rates of total protein synthesis increased 2-fold, while DNA, RNA, and poly(A)+ mRNA concentrations were unchanged during hormonal stimulation. mRNA concentrations for anionic trypsinogen, lipase, and amylase were determined by dot blot hybridization analysis with cDNA and cRNA probes. Despite 12-fold changes in the ratio of synthesis of anionic trypsinogen to amylase at 24 hr of caerulein stimulation, changes in levels of mRNA encoding these two proteins were not observed. The slight decreases observed in amylase mRNA concentrations were found in both hormone and saline-infused animals. In vitro pulse-chase experiments after 12 hr of saline or caerulein infusion indicated that differential turnover of anionic trypsinogen and amylase did not occur during hormone stimulation. These data demonstrate that the differential regulation observed in protein synthesis that results from a single period of hormone stimulation is mediated by differential regulation of mRNA translation. The high degree of conservation observed in the 5' terminal sequences of both amylase and anionic trypsinogen mRNAs between mouse, rat, and dog suggests that sequence-specific mechanisms and secondary structure may play a role in the translational control of these two mRNAs.

Amylases↗
PubMed1989

A lectin gene encodes the alpha-amylase inhibitor of the common bean.

An alpha-amylase inhibitor that inhibits insect and mammalian alpha-amylases but not plant alpha-amylases, is present in seeds of the common bean (Phaseolus vulgaris). We have purified the alpha-amylase inhibitor by using a selective heat treatment in acidic medium and affinity chromatography with porcine pancreas alpha-amylase coupled to agarose. Under sodium dodecyl sulfate gel electrophoresis, the purified inhibitor gave rise to five bands with mobilities corresponding to molecular masses ranging from 14 to 19 kDa. N-terminal sequencing (up to 15 amino acids) of the polypeptides obtained from these bands resulted in only two different sequences matching two stretches of the amino acid sequence deduced from an already described lectin gene [Hoffman, L. M. (1984) J. Mol. Appl. Gen. 2,447-453]. This gene is different from but closely related to the genes that code for phytohemagglutinin, the major lectin of bean. Further evidence based on amino acid composition, identification of a precursor, and recognition of the product of the gene (expressed in Escherichia coli) by an anti-alpha-amylase inhibitor serum confirms that the inhibitor is encoded by this or a closely related lectin gene. This finding assigns a biological function, which has been described at the molecular level, to a plant lectin gene product and supports the defense role postulated for seed lectins. The lack of homology with other families of enzyme inhibitors suggests that this may be the first member of a new family of plant enzyme inhibitors.

Amino Acid Sequence↗
PubMed1997

Cloning, sequencing, characterization, and expression of an extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis.

A gene encoding a highly thermostable extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus was identified. The gene was cloned, sequenced, and expressed in Escherichia coli and Bacillus subtilis. The gene is 1383 base pairs long and encodes a protein of 461 amino acids. The open reading frame of the gene was verified by microsequencing of the recombinant purified enzyme. The deduced amino acid sequence is 25 amino acids longer at the N terminus than that determined by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the membrane. The recombinant alpha-amylase was biochemically characterized and shows an activity optimum at pH 4.5, whereas the optimun temperature for enzymatic activity is close to 100 degrees C. alpha-Amylase shows sequence homology to the other known alpha-amylases and belongs to family 13 of glycosyl hydrolases. This extracellular alpha-amylase is not homologous to the subcellular alpha-amylase previously isolated from the same organism.

Amino Acid Sequence↗
PubMed2001

Porcine pancreatic alpha-amylase shows binding activity toward N-linked oligosaccharides of glycoproteins.

Porcine pancreatic alpha-amylase was shown by interaction analyses using a resonance mirror detector and alpha-amylase-immobilized Sepharose to bind with glycoproteins possessing N-glycans but not O-linked mucin-type glycans. Direct binding of three types of N-glycans to the alpha-amylase was demonstrated by surface plasmon resonance. Binding with biotin-polymer sugar probes revealed that the alpha-amylase has affinity to alpha-mannose, alpha-N-acetylneuraminic acid, and beta-N-acetyllactosamine, which are components of N-glycans. The binding of glycoproteins or carbohydrates enhanced the enzyme activity, indicating that the recognition site for N-glycans is different from its catalytic site. The binding activity was unique to porcine pancreatic alpha-amylase and was not observed for alpha-amylase from saliva, wheat, and fungus.

Animals↗
PubMed1994

Plasma amylase levels as a marker of disease severity in an isogenic murine model of paracoccidioidomycosis.

Survival patterns after peritoneal infection with Paracoccidioides brasiliensis vary according to the mouse strain and to the virulence of the fungal isolate. It has previously been observed that a significant increase in plasma amylase levels occurs only when susceptible mice (B10.A) were infected with a virulent isolate (Pb18). In order to verify if increased amylase levels correlate with susceptibility to P. brasiliensis infection, 12 mouse strains with different susceptibility patterns to this fungus were investigated after infection with Pb18. When compared with their respective controls, C57BI/6, B10D2/oSn, B10D2/nSn, C3H/HeJ, B10.A and BALB/c mice showed a conspicuous amylase increase and AKR, (NZB x NZW)F1, CBA/J, (A/Sn x B10.A)F1, A/Sn and DBA/2 absence of alteration. The influence of the infecting fungal isolate on this enzymatic parameter was investigated using B10.A mice and fungal isolates with diverse degrees of virulence. When compared with their non-infected controls, mice infected with Pb45 or Pb47 showed a very high amylase increase, with Pb44 or Pb18 a high one and with Pb50 or Pb265 a discrete increase. On the whole, there is an inverse correlation between survival times after infection and the increase in amylase levels. Thus, measurement of plasma amylase is a satisfactory parameter to evaluate the severity of paracoccidioidomycosis in mice.

Amylases↗
PubMed1996

Secretion of Bacillus alpha-amylase from yeast directed by glucoamylase I signal sequence of Saccharomyces diastaticus.

For the secretion of Bacillus stearothermophilus alpha-amylase from yeast, a recombinant plasmid pGAT17 was constructed by fusing B. stearothermophilus alpha-amylase structural gene in frame to the promoter and signal sequence of Saccharomyces diastaticus glucoamylase I gene (STA1). The secretion of the heterologous alpha-amylase from S. diastaticus transformed with pGAT17 was confirmed by the halo formation around colonies on selective starch agar medium. About 80% of the total alpha-amylase activity was detected in the extracellular culture medium. The secreted alpha-amylase was glycosylated and its molecular weight increased from 61 kDa to 75 kDa. The thermostability of the the glycosylated alpha-amylase was markedly enhanced, compared with that of the non-glycosylated enzyme from E. coli.

Amino Acid Sequence↗