Amylase

About: Amylase is a research topic. Over the lifetime, 14164 publications have been published within this topic receiving 296069 citations.

Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starch-binding sites and periplasmic starch-degrading enzymes.

Abstract: Bacteroides thetaiotaomicron can utilize amylose, amylopectin, and pullulan as sole sources of carbon and energy. The enzymes that degrade these polysaccharides were found to be primarily cell associated rather than extracellular. Although some activity was detected in extracellular fluid, this appeared to be the result of cell lysis. The cell-associated amylase, amylopectinase, and pullulanase activities partitioned similarly to the periplasmic marker, acid phosphatase, when cells were exposed to a cold-shock treatment. Two other enzymes associated with starch breakdown, alpha-glucosidase and maltase, appeared to be located in the cytoplasm. Intact cells of B. thetaiotaomicron were found to bind 14C-starch. Binding was probably mediated by a protein because it was saturable and was decreased by treatment of cells with proteinase K. Results of competition experiments showed that the starch-binding proteins had a preference for maltodextrins larger than maltohexaose and a low affinity for maltose and maltotriose. Both the degradative enzymes and starch binding were induced by maltose. These findings indicate that starch utilization by B. thetaiotaomicron apparently does not involve secretion of extracellular enzymes. Rather, binding of the starch molecule to the cell surface appears to be a first step to passing the molecule through the outer membrane and into the periplasmic space.

The Role of Insulin in the Regulation of α‐Amylase Synthesis in the Rat Pancreas

Abstract: . Changes in pancreatic exocrine enzyme activities were studied in different forms of experimental diabetes in rats. The effects of adrenalectomy on these changes were measured. The early actions of insulin on pancreatic enzyme activities and on incorporation of (4,5-3H)-leucine into amylase were also determined. The main results are: 1) Alloxan-diabetes leads to a decrease in amylase activity, a decreased rate of amylase synthesis and an increase in the activities of trypsinogen and chymotrypsinogen as described by Palla et al. [9]. Only the decrease in amylase activity correlates with the increase in blood glucose concentration. 2) Adrenalectomy does not reverse the changes in the activities of exocrine pancreatic enzymes induced by diabetes. 3) Diazoxide-diabetes is accompanied by an increase in all pancreatic enzyme activities, most probably due to the inhibition of enzyme secretion. 4) After treatment with streptozotozin a significant decrease in pancreatic amylase activity appears after 36 h. Amylase activity continues to fall during the following days, in contrast the increase in the activities of proteolytic enzymes is not significant until the 4th day. 5) Insulin treatment of severely diabetic, non-ketotic rats leads, as early as 90 min. after injection, to a significant increase in pancreatic amylase activity with no change in the other exocrine enzyme activities. A decrease in all enzyme activities, seen 6 h after insulin, is due to enhanced enzyme secretion. 6 As soon as 2 h after injection insulin leads to a significant increase in the incorporation of (4,5-3H)-leucine into pancreatic amylase but not into total pancreatic protein. This increase is completely abolished by actinomycin D. 7) Short-term effects of adrenalectomy (20 h) and effects of substitution with 6-α-fluoro-16α-methyl-prednisolon on the incorporation of (4,5-3H)-leucine into pancreatic amylase and total pancreatic protein result mainly from changes in the size of the cold leucine pool. According to these results insulin regulates pancreatic amylase synthesis mainly at the level of transcription. Insulin plays a permissive role in pancreatic amylase synthesis and is not involved in short-term regulation of amylase synthesis in the non-diabetic state. Clinical findings in juvenile diabetics indicate a similar type of regulation for amylase synthesis.

Production and partial characterization of thermostable and calcium-independent α-amylase of an extreme thermophile Bacillus thermooleovorans NP54

Abstract: AIM An investigation was carried out on the production of alpha-amylase by Bacillus thermooleovorans NP54, its partial purification and characterization. METHODS AND RESULTS The thermophilic bacterium was grown in shake flasks and a laboratory fermenter containing 2% soluble starch, 0.3% tryptone, 0.3% yeast extract and 0.1% K2HPO4 at 70 degrees C and pH 7.0, agitated at 200 rev min(-1) with 6-h-old inoculum (2% v/v) for 12 h. When the enzyme was partially purified using acetone (80%[v/v] saturation), a 43.7% recovery of enzyme with 6.2-fold purification was recorded. The KM and Vmax (soluble starch) values were 0.83 mg ml(-1) and 250 micromol mg(-1) protein min(-1), respectively. The enzyme was optimally active at 100 degrees C and pH 8.0 with a half-life of 3 h at 100 degrees C. Both alpha-amylase activity and production were Ca2+ independent. CONCLUSIONS Bacillus thermooleovorans NP54 produced calcium-independent and thermostable alpha-amylase. SIGNIFICANCE AND IMPACT OF THE STUDY The calcium-independent and thermostable alpha-amylase of B. thermooleovorans NP54 will be extremely useful in starch saccharification since the alpha-amylases used in the starch industry are calcium dependent. The use of this enzyme in starch hydrolysis eliminates the use of calcium in starch liquefaction and subsequent removal by ion exchange.

Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization.

Abstract: The pancreatic duct of anesthetized rabbits was cannulated and, in some animals, flow of pancreatic exocrine secretions was blocked by raising the cannula to a vertical position. Blockage for 3-7 h caused a rapid and significant rise in serum amylase activity and an increase in amylase activity within the pancreas. The concentration of lysosomal enzymes in the pancreas was not altered but they became redistributed among subcellular fractions and, as a result, an increased amount was recovered in the 1,000-g, 15-min pellet, which was enriched in zymogen granules. Immunofluorescence studies indicated that lysosomal enzymes become localized within organelles which, in size and distribution, resemble zymogen granules. They also contain digestive enzyme zymogens. Blockage of pancreatic secretions also caused lysosomal enzyme-containing organelles to become more fragile and subject to in vitro rupture. These changes noted after short-term pancreatic duct obstruction are remarkably similar to those previously noted to occur during the early stages of diet and secretagogue-induced experimental pancreatitis, observations that have suggested that colocalization of digestive enzyme zymogens and lysosomal hydrolases might result in intracellular digestive enzyme activation and be an important early event in the evolution of those forms of experimental acute pancreatitis.

Pancreatic acinar cells: the role of calcium in stimulus-secretion coupling.

Abstract: 1. Segments of mouse or rat pancreas were placed in a flow cell through which physiological salt solutions of varying composition were pumped at a constant rate. Intracellular recordings of membrane potential, resistance and electrical time constant were made from the acini using fine glass micro-electrodes. In some experiments two micro-electrodes were inserted into two acinar cells within the same acinus to assess directly cell to cell coupling. The concentration of amylase in the effluent was measured continuously. 2. Electrical coupling between two acinar cells was observed when the tips of the two micro-electrodes were less than 50 mum from each other. The coupling ratio was close to 1. Acetylcholine (ACh) always evoked depolarization of exactly the same amplitude in two coupled cells and reduced the amplitude of current-pulse induced membrane potential changes in both cell simultaneously. 3. Stimulation with ACh caused an immediate increase in amylase output. Replacement of superfusion fluid Na by Tris or Cl by sulphate abolished ACh-evoked increase in amylase release, but the subsequent reintroduction of Na or Cl caused an increase in amylase release of a magnitude similar to what was normally observed following stimulation. 4. Omitting Ca from the superfusion fluid and adding EGTA rapidly depolarized the acinar cell membrane, reduced the input resistance and caused a marked reduction in amylase secretion. During exposure to a Ca-free, EGTA containing solution a marked increase in amylase release occurred following maximal ACh stimulation. 5. Addition of small amounts of Mg, Ca or Mn to a Ca-, Mg-free solution caused an increase in membrane potential, input resistance and electrical time constant and markedly increased amylase release. The effect on the electrical parameters was reversed in the absence of extracellular Na while extracellular Na was of no importance for the effect on amylase release. 6. The effect of ACh on amylase was enhanced during superfusion with a fluid containing 20 mM-Ca. The presence of Mn (5 mM) in an otherwise normal control had no effect on ACh-evoked release. 7. These results show that ACh acts on the acinus by reducing the surface cell membrane resistance. It is suggested that the ACh-receptor interaction causes a release of Ca from the surface cell membrane and that the concentration of Ca in the surface cell membrane determines the specific membrane resistance particularly for Na. The release of Ca to the cytosol activates exocytosis while the Na influx is of importance for acinar fluid secretion. The effect of ACh on amylase secretion can be mimicked by agents displacing membrane-bound Ca (Mg, Ca, Mn).