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PubMed1986

Regulation of the accumulation of mRNA for alpha-amylase in barley aleurone.

The effect of gibberellic acid and Ca(2+) on the accumulation of alpha-amylase mRNAs in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) was studied using cDNA clones containing sequences of mRNAs for the high and low isoelectric point (pI) alpha-amylases. There is no significant hybridization between the two alpha-amylase cDNA clones under the hybridization and washing conditions employed. These clones were therefore used to monitor levels of mRNAs for high and low pI alpha-amylases. It is shown that although the synthesis of the high pI alpha-amylase proteins depends on the presence of Ca(2+) in the incubation medium, the accumulation of mRNA for this group occurs to the same degree in the presence or the absence of Ca(2+). The accumulation of low pI alpha-amylase mRNA is also not affected by the presence or absence of Ca(2+) in the incubation medium. These results establish gibberellic acid, not Ca(2+), as the principal regulator of alpha-amylase mRNA accumulation in barley aleurone, while Ca(2+) controls high pI alpha-amylase synthesis at a later step in the biosynthetic pathway.

Journal Article↗
PubMed1986

Ca-stimulated secretion of alpha-amylase during development in barley aleurone protoplasts.

The effects of gibberellic acid (GA(3)) and Ca(2+) on the synthesis and secretion of alpha-amylase from protoplasts of barley (Hordeum vulgare L. cv Himalaya) aleurone were studied. Protoplasts undergo dramatic morphological changes whether or not the incubation medium contains GA(3), CaCl(2), or both. Incubation of protoplasts in medium containing both GA(3) and Ca(2+), however, causes an increase in the alpha-amylase activity of both incubation medium and tissue extract relative to controls incubated in GA(3) or Ca(2+) alone. Isoelectric focusing shows that adding Ca(2+) to incubation media containing GA(3) increases the levels of alpha-amylase isozymes having high isoelectric points (pI). In the presence of GA(3) alone, only isozymes with low pIs accumulate. The increase in alpha-amylase activity in the incubation medium begins after 36 hours of incubation, and secretion is complete after about 72 hours. Protoplasts require continuous exposure to Ca(2+) to maintain elevated levels of alpha-amylase release. Immunoelectrophoresis shows that Ca(2+) stimulates the release of low-pI alpha-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA(3) alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolar CaCl(2). The response is not specific for Ca(2+) since Sr(2+) can substitute, although less effectively than Ca(2+). Pulse-labeling experiments show that alpha-amylase isozymes produced by aleurone protoplasts in response to GA(3) and Ca(2+) are newly synthesized. The effects of Ca(2+) on the process of enzyme synthesis and secretion is not mediated via an effect of this ion on alpha-amylase stability or on protoplast viability. We conclude that Ca(2+) directly affects the process of enzyme synthesis and transport. Experiments with protoplasts also argue against the direct involvement of the cell wall in Ca(2+)-stimulated enzyme release.

Journal Article↗
PubMed1990

Purification and Characterization of Pea Epicotyl beta-Amylase.

The most abundant beta-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this beta-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a beta-amylase by end product analysis and by its inability to hydrolyze beta-limit dextrin and to release dye from starch azure. Pea beta-amylase is an approximate 55 to 57 kilodalton monomer with a pl of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a K(m) of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromer-curiphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea beta-amylase is competitively inhibited by its end product, maltose, with a K(i) of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with alpha-cyclohexaamylose being a stronger inhibitor than beta-cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea beta-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea beta-amylase is the multichain type. Possible roles of pea beta-amylase in cellular glucan metabolism are discussed.

Journal Article↗
PubMed1990

Characterization of alpha-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings.

The most abundant alpha-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This alpha-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an alpha-amylase by polarimetry, substrate specificity, and end product analyses. The purified alpha-amylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This alpha-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein. Common cell wall constituents (e.g. beta-glucan) are not substrates of the enzyme. A very low amount of this alpha-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or alpha-amylase which is integrally associated with the chloroplast.

Journal Article↗
PubMed1991

Sucrose-Induced Accumulation of beta-Amylase Occurs Concomitant with the Accumulation of Starch and Sporamin in Leaf-Petiole Cuttings of Sweet Potato.

beta-Amylase of sweet potato (Ipomoea batatas L.), which constitutes about 5% of the total soluble protein of the tuberous root, is absent or is present in only small amounts in organs other than the tuberous roots of the normal, field-grown plants. However, when leaf-petiole cuttings from such plants were supplied with a solution that contained sucrose, the accumulation of beta-amylase was induced in both leaf and petiole portions of the explants. The sucrose-induced accumulation of beta-amylase in leaf-petiole cuttings occurred concomitant with the accumulation of starch and of sporamin, the most abundant storage protein of the tuberous root. The accumulation of beta-amylase, of sporamin and of starch in the petioles showed similar dependence on the concentration of sucrose, and a 6% solution of sucrose gave the highest levels of induction when assayed after 7 days of treatment. The induction of mRNAs for beta-amylase and sporamin in the petiole could be detected after 6 hours of treatment with sucrose, and the accumulation of beta-amylase and sporamin polypeptides, as well as that of starch, continued for a further 3 weeks. In addition to sucrose, glucose or fructose, but not mannitol or sorbitol, also induced the accumulation of beta-amylase and sporamin, suggesting that metabolic effects of sucrose are important in the mechanism of this induction. Treatment of leaf-petiole cuttings with water under continuous light, but not in darkness, also caused the accumulation of small amounts of these components in the petioles, probably as a result of the endogenous supply of sucrose by photosynthesis. These results suggest that the expression of the gene for beta-amylase is under metabolic control which is coupled with the expression of sink function of cells in the sweet potato.

Journal Article↗
PubMed1996

RBI, a one-domain alpha-amylase/trypsin inhibitor with completely independent binding sites.

The bifunctional inhibitor from Ragi (Eleusine coracana Gaertneri) (RBI) is the only member of the alpha-amylase/trypsin inhibitor family that inhibits both trypsin and alpha-amylase. Here, we show that both enzymes simultaneously and independently bind to RBI. The recently solved three-dimensional NMR structure of RBI has revealed that the inhibitor possesses a hitherto unknown fold for serine proteinase and alpha-amylase inhibitors. Despite its different fold, RBI obeys the standard mechanism observed for most protein inhibitors of serine proteinases and is a strong, competitive inhibitor of bovine trypsin (Ki = 1.2 +/- 0.2 nM). RBI is also a competitive inhibitor of porcine alpha-amylase (Ki = 11 +/- 2 nM) when a disaccharide is used as a substrate of alpha-amylase. However, the inhibition mode becomes complex when larger (> or = 7 saccharide units) alpha-amylase substrates are used. A second saccharide binding site on porcine alpha-amylase may enable larger oligosaccharides to displace RBI from its binding site in an intramolecular reaction.

Amylases↗
PubMed1982

Redistribution of amylase activity accompanying its secretion by the pancreas.

Amylase activity in several tissue and body fluid compartments in the rat changed markedly when the secretion of digestive enzyme was augmented over a 3-hr period with a cholinergic agonist. As a result of stimulation, the pancreas was depleted of about one-third of its amylase activity and accounted for only 75% of the amount recovered from the animal, compared to 92% in the fasted state. Despite the continuous augmented secretion of the enzyme into the small intestine, no increase in amylase activity was detected there at the end of 3 hr. On the other hand, amylase activity in plasma and extracellular fluid increased by about an order of magnitude and accounted for 13% of the total pool, compared to approximately 1% in the fasted state. Amylase activity in several solid tissues also increased, including a 50- to 100-fold increase in parotid gland and an almost 10-fold increase in submandibular gland and kidney. The potential sources of the increased amylase activity in blood, the endocrine secretion of the enzyme by the pancreas, and its absorption from the intestine are considered. Changes in the amylase content of various tissues appear to reflect increased uptake due to increased plasma levels.

Amylases↗
PubMed2026

Multistrategy metabolic engineering of Talaromyces pinophilus for α-amylase production from lignocellulosic biomass.

Filamentous fungi are important hosts for industrial enzyme production. Growing demand for α-amylase has increased reliance on food-derived carbon substrates, necessitating fungal strains that efficiently utilize nongrain biomass. In this study, Talaromyces pinophilus Y117 was metabolically engineered to produce α-amylase from lignocellulosic biomass. A strong cellobiohydrolase I gene (cbh1) promoter (Pcbh1Tru) was identified to drive expression. Multiple rounds of multilocus integration of the α-amylase gene were performed using homologous multicopy genomic sequences as recombination arms with a Cre/loxP-based recyclable selection system, yielding the multicopy strain Tp4, which achieved 4124.5 U/mL α-amylase activity in shake-flask fermentation with corncob powder as the sole carbon source. To minimize enzyme degradation, the protease gene 8538 was deleted using the Cre/lox2272 system, generating Tp4Δp. This strain showed a 50% increase in shake-flask α-amylase activity (6208.4 U/mL). In 3-L bioreactor cultivation, Tp4Δp exhibited excellent production performance, achieving 26 712.2 U/mL α-amylase activity. When corncob powder was used as the sole substrate, the cellulose and hemicellulose degradation rates reached 90.00% and 70.01%, respectively, and the enzyme yield reached 213 697.5 U per gram of corncob powder. This engineered strain demonstrates strong potential for industrial applications. The synthesis-degradation synergistic optimization strategy provides a practical approach for engineering filamentous fungal cell factories to produce enzymes directly from lignocellulosic biomass. One sentence summary Metabolic engineering of Talaromyces pinophilus through promoter optimization, multicopy integration, and protease deletion enables efficient α-amylase production from lignocellulosic biomass, achieving 26 712 U/mL in bioreactor fermentation.

Talaromyces↗
PubMed1987

Primary structure and differential expression of beta-amylase in normal and mutant barleys.

The primary structure of barley endosperm beta-amylase, an enzyme which catalyses the liberation of maltose from 1,4-alpha-D-glucans, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 1754 nucleotides long [excluding the poly(A) tail] and codes for a polypeptide of 535 amino acids with a relative molecular mass of 59,663. The deduced amino acid sequence was compared with the sequences of ten peptides obtained from the purified enzyme and unambiguous identification was obtained. The N-terminal region of the deduced sequence was identical to a 12-residue cyanogen-bromide-peptide sequence, indicating that beta-amylase is synthesized as the mature protein. A graphic matrix homology plot shows four glycine-rich repeats, each of 11 residues, preceding the C-terminus. Southern blotting of genomic DNA demonstrates that beta-amylase is encoded by a small gene family, while cDNA sequence analysis indicates the presence of at least two types of mRNA in the endosperm. Dot and northern blot analysis show that Hiproly barley contains greatly increased levels of beta-amylase mRNA compared to the normal cultivar Sundance, whereas Risø mutant 1508 contains only trace amounts. These results correlate well with the deposition of beta-amylase during endosperm development in these lines. Low but similar amounts of beta-amylase mRNAs sequences were detected in leaves and shoots from normal and mutant barleys, demonstrating that the mutant lys3a (1508) and lysl (Hiproly) genes do not affect the expression of beta-amylase in these tissues.

Amino Acid Sequence↗
PubMed1983

Effects of alpha-amylase on in vitro growth of Legionella pneumophila.

Sterile parotid saliva inhibited growth of Legionella pneumophila on solid media, and the salivary component involved in this inhibition has been shown to be amylase. Disk diffusion and well plate assays were used to study possible mechanisms for this effect. The amylolytic activity of saliva copurified with inhibitory activity, and both activities were sensitive to proteinase K digestion and heat treatment. In addition, purified alpha-amylase from several sources (bacteria, fungi, porcine pancreas, and human saliva) exhibited similar activity. Incorporation of charcoal or bovine serum albumin into media blocked inhibition by amylase. Replacement of Bacto-Agar with Noble agar (both from Difco Laboratories) prevented growth inhibition in the absence of starch. However, when corn starch was present with Noble agar, amylase-induced growth inhibition occurred. Purification of starch by washing with methanol eliminated some toxic component. The toxic component from starch could be recovered from the methanol wash and inhibited growth of L. pneumophila in the absence of amylase activity. The results suggest that toxic substances exist in media components which may be unmasked during salivary amylase digestion of starch. This effect may explain, in part, the difficulty in recovery of the organism from clinical specimens containing amylase.

Amylases↗
PubMed1979

Membrane-bound and soluble extracellular alpha-amylase from Bacillus subtilis.

Extracellular alpha-amylase was purified to homogeneity from a Marburg strain of Bacillus subtilis. The enzyme is a single polypeptide chain of molecular weight approximately 67,000. Its NH2-terminal amino acid sequence is Leu-Thr-Ala-Pro-Ser-Ile-Lys. A membrane-derived alpha-amylase was solubilizing from membrane vesicles by treatment with Triton X-100 and was highly purified by chromatography on an anti-alpha-amylase-protein A-Sepharose column. Membrane-derived alpha-amylase was indistinguishable from the soluble extracellular enzyme by sodium dodecyl sulfate-gel electrophoresis and radioimmunoassay. The membrane-derived enzyme contains phospholipid. Approximately 30 to 80% of the phospholipid was extracted from the purified enzyme by chloroform:methanol. The extracted phospholipid was predominately phosphatidylethanolamine. Treatment with phospholipase D released phosphatidic acid. Membrane-bound alpha-amylase was latent in membrane vesicles. Release of membrane-bound alpha-amylase from vesicles by an endogenous enzyme was maximal at pH 8.5, was inhibited by metal chelators and diisopropyl fluorophosphate and was stimulated by Ca2+ and Mg2+. The amount of membrane-bound alpha-amylase was related to the level of secretion.

Amino Acid Sequence↗
PubMed1978

Albumin activation of urinary amylase as determined with the Du Pont aca.

Protein activation of urinary alpha-amylase (EC 3.2.1.1) activity was observed during an evaluation of the Du Pont aca procedure for the determination of urinary alpha-amylase. This activation effect became constant for urinary albumin concentrations exceeding 1.50 g/liter. It is recommended that urinary alpha-amylase be analyzed with sufficient albumin added to maximize this effect. The aca alpha-amylase procedure is compared to an amyloclastic method for both serum and urine analysis. Expected ranges are presented for the aca method for serum and urinary amylase, amylase clearance, and the amylase clearance/creatinine clearance ratio.

Albuminuria↗
PubMed1978

[Acid-stable and acid-unstable alpha-amylases of the mold fungi Aspergillus].

Acid-sable alpha-amylase of Asp. niger and acid-unstable, alpha-amylase of Asp. oryzae were studied. It was demonstrated, that beside being a more acid-stable properties, alpha-amylase Asp. niger has increased thermal stability as compared to alpha-amylase Asp. oryzae. The molecular weights of acid-stable alpha-amylase and acid-unstable alpha-amylase are 58 000 and 51 000, respectively. The amino acid composition, and the C- and N-terminal amino acids of both forms of alpha-amylases were determined. It was demonstrated, that the enzymes under study contain one sylfhydryl group per mole of enzyme, which in the Ca2+-bound form plays an important role in the maintenance of the catalytically active enzyme conformation.

Amino Acids↗
PubMed1981

Detection of alpha-amylase activity in unprocessed preamylase produced in the cell-free translation of porcine pancreatic RNA.

Preamylases, synthesized in the RNA-dependent rabbit reticulocyte lysate translation system supplemented with porcine pancreatic RNA were identified by their specific immunoprecipitation with anti-amylase. The preamylases have apparent Mr = 55,000 and 58,000 as compared to 52,000 and 55,000 for the purified, secreted alpha-amylase isozymes. In order to establish whether the unprocessed precursors may assume enzymatically active conformations, we have explored a highly sensitive activity gel electrophoresis technique, by which picogram quantities of enzyme can be detected. When standard alpha-amylase and translation products are subjected to electrophoresis on polyacrylamide gel containing 0.01% starch and CaCl2, active amylase which binds tightly to starch can only migrate as the starch is hydrolyzed. When the gel is subsequently stained with I2, the appearance of clear tracks, the lengths of which are roughly proportional to the logarithm of amylase concentration, signifies the presence of amylase activity. By this approach, we were able to detect amylase activity in a range corresponding to about 100 pg of pure amylase/10 microliters of translation mixture. This value agrees well with an estimate from radioactivity incorporation of total preamylase in the translation mixture, and we consequently conclude that unprocessed preamylase can assume the appropriate conformation to give enzymatic activity.

Amylases↗
PubMed1994

Urinary alpha-amylase and serum macroamylase activities in dogs with proteinuria.

Activities of urinary alpha-amylase and serum macroamylase; concentrations of serum creatinine, immunocomplexes, and urinary protein; and patterns of proteinuria were determined in 35 dogs with proteinuria. Urinary alpha-amylase activity ranged from 37 to 4,031 U/L. Macroamylasemia was detected in 77.14% of dogs and the percentage of alpha-amylase precipitated ranged from 4.68 to 61.63. Serum alpha-amylase activity after immunoglobulin precipitation ranged from 654 to 6,390 U/L in 51.42% of the dogs; the values were higher than the reference limits. Concentrations of serum creatinine and immunocomplexes were higher than reference limits for 25.71 and 60% of dogs, respectively. Urinary protein concentrations ranged from 0.1 to 8.9 g/L. All the patterns of proteinuria were represented. Linear regression indicated correlations between urinary alpha-amylase activities, serum creatinine concentrations (P < 0.01), and concentration of immunocomplexes (P < 0.05). Mann-Whitney test indicated significantly higher urinary alpha-amylase activity (P < 0.01) and percentage of alpha-amylase precipitated (P < 0.05) in dogs with renal insufficiency.

Amylases↗
PubMed1982

Aged amylase: a valuable test for detecting and tracking pancreatic pseudocysts.

Amylase-rich fluid that incubates ("ages") within a pancreatic pseudocyst undergoes a change that can be detected by isoenzyme analysis of amylase from the serum. This aging is a result of deamination of the asparagine and glutamine residues on the amylase molecule. Eighteen of 20 patients with surgically proved pseudocysts had greater than 15% aged (deaminated) amylase in their serum. Levels of aged amylase returned to normal following treatment of their pseudocysts. Twenty of 23 patients with acute pancreatitis had levels of aged amylase below 15% (P less than .05). A criterion of 15% aged amylase resulted in 87% specificity, and 91% sensitivity for the diagnosis of pseudocysts. Because this test is noninvasive and easy to perform, it should become the ideal screen for patients at risk of development of pseudocysts, Endoscopic retrograde pancreatography, ultrasonography, and abdominal computed tomographic scanning should be reserved for confirmation of the diagnosis when the result of isoenzyme analysis is positive.

Amylases↗
PubMed1981

Serous ovarian neoplastic amylase (SONA): a potentially useful marker for serous ovarian tumors.

A series of cystic ovarian tumors was studied with regard to amylase activity in the cyst fluid. Large amounts of amylase activity were present only in those patients with endosalpingeal-type lining epithelium (serous ovarian neoplasms). The amylase activity from two such patients was further characterized using G75 Sephadex chromatography, DEAE Sephadex chromatography, agarose gel chromatography, isoelectric focusing, and heat inactivation studies. Serous ovarian neoplastic amylase (SONA) exhibited properties different from amylases of pancreatic and salivary gland origin. Serous ovarian neoplastic amylase (SONA) may be a pancreatic and salivary gland origin. Serous ovarian neoplastic amylase (SONA) may be a useful biochemical marker in diagnosing and managing patients with serous ovarian neoplasms.

Amylases↗
PubMed2000

PKC and ERK1/2 regulate amylase promoter activity during differentiation of a salivary gland cell line.

The addition of transforming growth factor alpha (TGFalpha) to a human submandibular gland cell line (HSG) cultured on basement membrane extract Matrigel, synergistically activates the acinar cell-specific salivary amylase promoter. Signaling through beta1 integrins and increased phosphorylation of ERK1/2 are involved in the increased promoter activity. Phorbol-12-myristate-13-acetate (PMA) and thapsigargin increase amylase promoter activity, suggesting that phorbol ester and calcium-dependent protein kinase C (PKC) pathways are also involved. The combination of specific inhibitors of PKC and MEK1 inhibits the amylase promoter. Inhibitors of the calcium-dependent PKC isoforms alpha, beta, and gamma decrease the promoter activity; however, PKCbeta is not detectable in HSG cells. TGFalpha alters the cellular localization of PKCalpha but not -gamma, suggesting PKCalpha is involved in TGFalpha upregulation of the amylase promoter. Furthermore, rottlerin, a PKCdelta-specific inhibitor, increases the promoter activity, suggesting PKC isoforms differentially regulate the amylase promoter. In conclusion, beta1-integrin and TGFalpha signaling pathways regulate the amylase promoter activity in HSG cells. In response to Matrigel and TGFalpha, the activation of both PKCalpha and phosphorylation of ERK1/2 results in synergistic activation of the amylase promoter. Published 2000 Wiley-Liss, Inc.

Amylases↗