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At least 1,639 records · Page 91Linked to original sources
PubMed1992

Aleurone nuclear proteins bind to similar elements in the promoter regions of two gibberellin-regulated alpha-amylase genes.

Binding of nuclear proteins from wild oat aleurone protoplasts to the promoter regions of two gibberellin-regulated wheat alpha-amylase genes (alpha-Amy1/18 and alpha-Amy2/54) has been studied by gel retardation and DNase 1 footprinting. Gel retardation studies using 300-430 bp fragments of the promoters showed similar binding characteristics with nuclear extracts from both gibberellin A1-treated and untreated protoplasts. DNase 1 footprints localised binding of nuclear proteins from gibberellin A1-treated aleurone protoplasts to regions in both promoters. Similar sequence elements in the promoter regions of both genes were protected from digestion although the location and number of footprints in each promoter region were different. Each footprint contained either a sequence similar to the cAMP and/or phorbol ester response elements, or a hyphenated palindrome sequence. The presence of cAMP and/or phorbol ester response element-like sequences in the footprints suggests that transcription factors of the bZIP type may be involved in the expression of alpha-amylase genes in aleurone cells. Footprints containing hyphenated palindrome sequences, found in the promoter regions of both genes, suggest the possible involvement of other classes of transcription factor. The conserved alpha-amylase promoter sequence TAA-CAGA was also shown to bind nuclear protein in the alpha-Amy2/54 promoter. These observations are discussed in relation to alpha-amylase gene expression in aleurone and to functional data concerning these genes.

Base Sequence↗
PubMed1993

Extracellular production of human hepatitis B virus preS2 antigen as hybrid proteins with Bacillus subtilis alpha-amylases in high-salt-concentration media.

To produce PreS2 antigen of human hepatitis B virus extra-cellularly in Bacillus subtilis, it was fused with the COOH-termini of B. subtilis alpha-amylases of 522 (Amy+), 467 (Amy+) and 443 (Amy-) amino acids. Among them, alpha-amylase-A467, which has 467 amino acids, was a relatively stable carrier when the cells were cultured in Luria-Bertani (LB) medium. The alpha-amylase-A443-PreS2 hybrid protein (Amy-) was quickly degraded. The alpha-amylase-A522-PreS2 hybrid was most efficiently produced when a B. subtilis transformant of a protease-super-deficient mutant was cultured in the presence of 0.5 M sodium sulphate. The production of A522-PreS2 hybrid protein under such conditions reached 5-10 mg/l and was eight, and 200-500 times higher than those obtained by the transformants of an alkaline/neutral protease-deficient mutant of B. subtilis, and a wild-type strain, in LB medium, respectively.

Bacillus subtilis↗
PubMed1992

Extracellular alpha-amylase from Streptomyces rimosus.

A purification procedure for an extracellular alpha-amylase from Streptomyces rimosus, oxytetracycline-producing strain, is described. The enzyme obtained was shown to be an acidic (pI 4.75) monomer with a relative molecular mass (M(r)) of 43,000, containing three cysteines involved in the catalytic activity of the enzyme. Its amino-terminal part has 57-67% homology with amylases from other Streptomyces species. S. rimosus alpha-amylase is sensitive to higher temperatures, and partially stabilized by Ca2+ ions. It hydrolyses starch (optimum at pH 5.0-6.0) in an endohydrolase manner giving rise to maltotriose, maltotetraose and higher oligosaccharides. Starch granules, except those from rice, were not significantly affected by the isolated alpha-amylase.

Amino Acid Sequence↗
PubMed1992

Variation in gene copy number and polymorphism of the human salivary amylase isoenzyme system in Caucasians.

The polymorphic patterns of human salivary amylase of a large number of individuals of Caucasian origin were determined by using isoelectric focusing and polyacrylamide gel electrophoresis. Nine different salivary amylase protein variants were found; three of them are recorded for the first time and their heredity is shown. Some of the variants are encoded by haplotypes expressing three allozymes. Most variants display low frequencies. Analysis of the relative intensities of variant-specific isozyme bands, combined with segregation analysis, show that extensive quantitative variation is present in the population. The numbers of salivary amylase genes in some families showing quantitative variation at the protein level have been estimated by the polymerase chain reaction. We present evidence that quantitative variations in amylase protein patterns do not always reflect variations in gene copy number but that other mechanisms are also involved.

Amylases↗
PubMed1975

The postnatal development of the sexually dimorphic duct system and of amylase activity in the submandibular glands of mice.

The submandibular glands of developing and mature Strong A mice were studied by light and electron microscopy. The glands of both sexes show the same cell types during development, but during maturation the glands display a degree of sex-dimorphism. Striated ducts, which differentiate from the larger intralobular ducts present in the neonatal gland, first appear by 5 days of age and reach their mature condition by 20 days of age. Granular convoluted tubule cells, which differentiate from striated duct cells, are first seen at 15 days of age in both sexes. Subsequently, they show a more rapid development in males than in females, and are dimorphically represented by 20 days of age. Intercalated ducts in the neonatal gland contain nongranular and granular cells. With maturation the number of granular cells decreases, apparently due to their conversion into the nongranular type, with their eventual disappearance from the glands of adult males. Their retention in adult females further defines the sexual dimorphism shown by these glands. Amylase activity in gland homogenates is first detectable at 20 days of age in both sexes. During development the male glands show a rapid rise in levels of amylase activity, whereas female glands show a more gradual rise. In mature animals, male glands have higher levels of amylase activity than female glands. The developmental and adult status of amylase activity parallels that of the granular convoluted tubules.

Age Factors↗
PubMed1978

The effect of the ionophore A23187 on amylase release, cellular integrity and ultrastructure of mouse pancreatic acini.

The effects of the Ca2 + ionophore A2317 on pancreatic amylase and lactate dehydrogenase (LDH) release, cellular electrolyte balance and ultrastructure were studied with the use of incubated pancreatic fragments. A23187 (0.3 micrometer) in the presence of Ca2 +, increased amylase release but at higher concentrations (1-10 micrometer) also increased LDH release and increased uptake of 14C-sucrose with concomitant loss of tissue K + and gain in Na +. The ultrastructure of the majority of acini appeared normal and showed depletion of zymogen granules. Microtubules and microfilaments which have been implicated in the release process were normal or increased in number. In the absence of Ca + the ionophore had no effect on secretion, cellular integrity or ultrastructure. It is concluded that A23187 in the presence of Ca2 + increases amylase release by a mechanism comparable to the terminal steps in stimulus-secretion coupling induced by physiological secretagogues. This provides further evidence that amylase release is mediated by a rise in cell Ca2 + although the mechanisms of the ionophore- and physiological secretagogue-induced rise in Ca + are probably different. High concentrations of ionophore (greater than 1 micrometer) also induce Ca2 + dependent damage in a fraction of the cells.

Amylases↗
PubMed1988

Expression of a Bacillus alpha-amylase gene in yeast.

A recombinant plasmid, pSR11.3, containing the alpha-amylase gene (AMY) of Bacillus amyloliquefaciens was characterized and expressed in Bacillus subtilis. A 2.3 kilobase BamHI-BglII fragment carrying AMY was cloned into pBR322 (pEL322) and in both orientations into a multi-copy Escherichia coli-yeast shuttle vector YEp13 (pAM13) and expressed in E. coli HB101 and various Saccharomyces stains. We report on the successful secretion of an active bacterial enzyme in yeast without using yeast promoter and secretory signals. Enzyme production in B. subtilis 1A297(pSR11.3), E. coli HB101(pEL322) and Saccharomyces JM2773-15B(pAM13) transformants was measured as 125, 22 and 123 U/ml, respectively. The molecular weight of the purified alpha-amylase secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B-(pAM13) was estimated to be 55 kDa. The pH and temperature optima for the alpha-amylase activities of the transformants were 6.5 to 8.0 and 50 to 65 degrees C, respectively. Amylose hydrolysis profiles of the alpha-amylases secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) indicate effective meso-thermostable hydrolytic enzymes with maltotriose and maltose, respectively, as major end products.

Bacillus subtilis↗
PubMed1989

Regulated overproduction of alpha-amylase by transformation of the amylolytic yeast Schwanniomyces occidentalis.

High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called "SwARS1". The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5-10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned alpha-amylase gene from S. occidentalis secreted about five times more alpha-amylase than cells without additional copies of the alpha-amylase gene. Both the chromosomal copy and the plasmid-carried copies of the alpha-amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.

Blotting, Southern↗
PubMed1980

Production of amylase(s) by Schwanniomyces castellii and Endomycopsis fibuligera.

Schwanniomyces castellii and Endomycopsis fibuligera Produced extracellular amylase(s) when grown on various carbon sources and at different pH values. Both yeast species showed significant amylase synthesis in the presence of either maltose or soluble starch. On the other substrates tested (glucose, cellobiose, sucrose, trehalose, melezitose, raffinose, ethanol, glycerol) differences were found regarding growth and amylase production. Free glucose in the culture medium apparently inhibited enzyme synthesis. The pH range allowing maximal growth and amylase production was 4.5-6.0 for E. fibuligera and 5.5-7.0 for S. castellii.

Amylases↗
PubMed1975

Variability of the molecular size of extracellular amylase produced by intact cells and protoplasts of Bacillus caldolyticus.

One of the typical properties of the extracellular amylase produced by Bacillus caldolyticus is the tendency to disintegrate into subunits with a molecular weight (MW) of less than 10,000, when the enzyme is subjected to ultrafiltration. Disintegration is due to a loss of Ca-ions, leading to nonactive subunits. Activity can be fully restroed by addition of Ca-ions. Reactivation occurs also spontaneously if the low MW fraction is stored in glassware. Comparative ultrafiltration experiments with the subunit fraction with or without a supply of Ca revealed that in presence of this divalent cation the subunits reaggregated to the active enzyme. The different distribution patterns obtained in absence or presence of Ca showed that reactivation is directly linked to the formation of a high MW form of the protein. Substitution of Ca by other divalent cations also led to reaggregation. These aggregates are, however, inactive. The enzyme was found to be formed intracellularly in its low MW form. Experiments with protoplasts revealed that these are capable to produce and release the amylase. When the production of the enzyme by protoplasts declined, full restoration could be achieved by a recovery treatment. Normally, the enzyme released by the protoplasts consisted of equal portions of the high, medium, and low MW form of the amylase. If the cells were, however, depleted as a result of continued incubations, the extruded enzyme consisted increasingly of the low MW form, which could finally represent more than 80%. This trend could be completely reversed by the supply of carbon and nitrogen sources during the recovery treatment, whereafter the enzyme consisted again of the intially observed equal amounts of the three MW forms. Vesicles prepared from the protoplasts were also found to release amylase, but on a lower level, and only for a very limited time, with no possibility to regain activity by a recovery treatment. Subunit formation was also observed during column chromatography, which could be counteracted by a sufficient supply with Ca-ions.

Amylases↗
PubMed1977

The characteristics of amylase activity and the isoamylase pattern in serum and urine of infants and children.

Determination of amylase activity and isoamylase patterns were performed in serum and urine of normal newborns, infants and children of different ages. In the serum of newborn infants measurable amounts of amylase were present. The activity increased with the age and reached the normal adult level by approximately 8 months of age. Isoamylase analysis revealed that the low level of serum amylase in infants was mainly due to deficiency of the pancreatic-type isoamylase. The absence of the pancreatic isoamylase in newborns and young infants is a physiological and developmental phenomenon. Great caution is therefore necessary when amylase isoenzymes are used in the diagnosis of abnormal pancreatic function and such results have always to be interpreted in relation to the age of the child.

Adult↗
PubMed1982

Regulation of amylase activity in drosophila melanogaster: effects of dietary carbohydrate.

The level of amylase activity in larvae and adults of Drosophila melanogaster is dependent on the dietary carbohydrate source; flies or larvae from a food medium containing starch show higher levels of activity than individuals from a food containing simple sugars. This is shown to be due to repression of activity by sugars rather than enhancement of activity by starch. Moreover, the changes in enzyme activity reflect a change in enzyme quantity rather than a change in catalytic efficiency. The seeming stimulation of amylase activity by sucrose in some experiments is due, simply, to comparisons with "starvation" diets which cause a large nonspecific reduction in enzyme activity. Though all strains tested showed repression of enzyme activity by simple sugars, the degree of repression varies between strains. Also, in those strains which carry a duplication of the amylase structural gene, the two isozymal forms of amylase can be differentially repressed by dietary sugars.

Amylases↗
PubMed1985

Ultrastructural, immunohistochemical and biochemical studies on amylase and ACTH producing lung cancer.

Tumour tissue from a lung cancer patient who showed elevated serum amylase and adrenocorticotropin (ACTH) was studied ultrastructurally, immunohistochemically and biochemically. Histologically the tumour was a small cell carcinoma. On electron microscopic examination the tumour cells contained large zymogen-like granules within the cytoplasm. Furthermore, cells which possessed many small dense core granules of the endocrine type were also observed. It was of interest that the large zymogen-like granule-containing tumour cells had microvilli at the apical border, connected by desmosomes and forming lumina showing adenocarcinomatous differentiation. Electrophoretic analysis of the serum revealed that the major elevated amylase was of the salivary type with minor components. Immunostaining clearly demonstrated that most of the tumour cells possessed immunoreactive ACTH, whereas salivary amylase was only found in occasional clusters of the tumour cells. The results seem to indicate that the tumour showed both endocrine and exocrine characteristics--an amphicrine carcinoma, expressing amylase and ACTH simultaneously.

Adrenocorticotropic Hormone↗
PubMed1994

Effect of PT-treatment on ANP-mediated inhibition of adenylate cyclase and amylase release in rat parotid gland.

Effects of pertussis toxin (PT) treatment on atrial natriuretic peptide (ANP)-mediated inhibition of adenylate cyclase and amylase release were investigated in rat parotid gland. Adenylate cyclase activity stimulated by GTP gamma S in PT-treated membranes was much larger than that in normal membranes. ANP dose-dependently inhibited adenylate cyclase stimulated by GTP gamma S in control rat parotid membranes, however in membranes prepared from PT-injected (in vivo) rat parotid gland, ANP did not inhibit adenylate cyclase. ANP(10(-7)M) inhibited cAMP accumulation stimulated by forskolin (10(-6)M) in control rat parotid acinar cells by about 34%, however, in PT-treated cells, the inhibitory effect of ANP was attenuated completely. In control cells amylase release stimulated by isoproterenol (10(-6)M) and forskolin (10(-6)M) were also depressed by ANP (10(-7)M) by 27 and 30% respectively. The inhibitory response of ANP on amylase release was completely attenuated by PT-treatment. Gi was detected as a ADP-ribosylated 41-KDa protein by incubation of parotid membranes with PT and [alpha-32P]NAD. In rat parotid gland, these results suggested that ANP mediates adenylate cyclase/cAMP system and consequently reduces amylase release through ANP-C receptor coupled to Gi.

Adenosine Diphosphate Ribose↗
PubMed1992

Stability of fungal alpha-amylase in sodium dodecylsulfate.

Unfolding of a fungal alpha-amylase in aqueous sodium dodecylsulfate (SDS) solution was examined by SDS-polyacrylamide gel electrophoresis (PAGE). When the alpha-amylase was incubated with 1% SDS at room temperature and subjected to SDS-PAGE, it showed a much higher mobility than expected from the molecular weight. Circular dichroic and gel filtration analyses indicated that the protein is apparently in the native conformation upon incubation with 1% SDS. When the protein was heated in the presence of 1% SDS at 90 degrees C for 10 min, it had a lower mobility in SDS-PAGE and showed characteristics of an unfolded protein by circular dichroism and gel filtration. The melting temperatures of the protein were determined in the absence and presence of SDS by incubating it for 10 min at various temperatures. The melting temperatures were 70, 55, and 49 degrees C in the presence of 0, 1, and 2% SDS, respectively. The observed small shift of the melting temperatures by SDS suggests that the destabilizing action of SDS on the alpha-amylase is weak. However, the unfolding in SDS is not reversible process, since prolonged incubation of the protein with 1% SDS at 50 degrees C gradually increased the amount of unfolded protein. This indicates that the SDS-induced unfolding of the alpha-amylase is a slow process.

Chromatography, Liquid↗
PubMed1986

[Proteins of bee honey. VI. Isoelectric focusing of amylase in various kinds of honey].

The isolated honey amylase was characterized by preparative and analytical isoelectric focusing. An interesting aspect of the preparative isoelectric focusing was the reduction of iodine by Ampholines which were used for building up a pH-gradient, giving a false increase in apparent amylase activity as determined by the iodine/starch reaction. Dextran gels were not suited for the matrix, because interactions with the enzyme disturb the focusing. From the analytical isoelectric focusing of isolated honey amylase in ultrathin layer Polyacrylamide gels 10 protein bands resulted. A newly developed zymogram technique was positive for amylase activity in all 10 protein bands.

Amylases↗
PubMed1994

Effects of infrared radiation, solar cooking and microwave cooking on alpha-amylase inhibitor in sorghum (Sorghum bicolor L.).

Three domestic cooking methods were studied in alpha-amylase inhibitory activity in sorghum grains. In all the treatments, overnight soaked seeds lost amylase inhibitory activity much faster. All the three treatments reduced the inhibitory activity. Use of solar cooker for reducing amylase inhibitory activity works out very economically and efficiently. Microwave cooking eliminates amylase inhibitory activity within 5 minutes.

Cooking↗
PubMed1992

Histopathologic correlates of serum amylase activity in acute experimental pancreatitis.

The association of serum amylase activity with the extent of pancreatic injury in acute pancreatitis is unclear. To clarify this relationship, we induced acute pancreatitis ranging from mild to lethal in 118 Sprague-Dawley rats (350-450 g). This was achieved by controlled intraductal infusion of low- or high-dose bile salt, with or without enterokinase, followed by intravenous cerulein or saline for 6 hr. Serum amylase was measured at baseline and 6 hr. Pancreatic histopathology was evaluated by two blinded pathologists employing total surface scoring (N = 118) and morphometric 20-field documentation (N = 22). Serum amylase correlated best with edema (r = 0.61) and fat necrosis (r = 0.58), less well with acinar necrosis (r = 0.53) and inflammation (r = 0.50), and poorly with hemorrhage (r = 0.33) and perivascular infiltrate (r = 0.31). Inasmuch as edema and fat necrosis are not important determinants of severity, these observations could explain the poor prognostic value of serum amylase activity in patients with acute pancreatitis.

Acute Disease↗