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PubMed1985

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers.

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA(3)) with or without 5 millimolar CaCl(2) shows that alpha-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca(2+). A cDNA clone for alpha-amylase was isolated and used to measure alpha-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca(2+). No difference was observed in alpha-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA(3) with 5 millimolar CaCl(2) and layers incubated in GA(3) alone. RNA isolated from layers incubated for 12 hours in GA(3) with and without Ca(2+) was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca(2+) in the incubation medium. Immunoprecipitation of translation products showed that the RNA for alpha-amylase synthesized in Ca(2+)-deprived aleurone layers was translatable. Ca(2+) is required for the synthesis of alpha-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.

Journal Article↗
PubMed1985

Imunohistochemical Localization of alpha-Amylase in Cotyledons of Vigna mungo Seedlings.

We studied the localization of alpha-amylase with indirect fluorescence microscopy in transversely sectioned cotyledons of Vigna mungo seedlings. Tissue sections were fixed in periodate-lysine-paraformaldehyde and treated with anti-alpha-amylase immunoglobulin G followed by fluorescein isothiocyanate labeled goat anti-rabbit immunoglobulin G. alpha-Amylase appeared in the cells farthest from vascular bundles on the second day of growth and appeared gradually closer to the vascular bundles as growth progressed. The pattern of alpha-amylase appearance was similar in detached cotyledons, indicating that attachment of the embryonic axis has no effect on this pattern. However, in attached cotyledons, alpha-amylase disappeared from the regions where starch grains had been digested, but in detached cotyledons there was no disappearance of alpha-amylase, and digestion was slower than in intact cotyledons.

Journal Article↗
PubMed1989

Control of alpha-Amylase Development in Cotyledons during and following Germination of Mung Bean Seeds.

Developmental patterns of alpha-amylase in Vigna radiata cotyledons during and following germination were quite different depending on the differences in the treatments of cotyledons during the imbibitional stage. When axis-detached cotyledons were imbibed in water with seed-coats attached, alpha-amylase activity did not increase and remained low. On the other hand, when the cotyledons were imbibed in water after seed-coat removal, the enzyme activity increased markedly. If the axis was attached to the cotyledons, alpha-amylase showed a marked development even under the former imbibition conditions. These changes in the enzyme activity were in parallel with those in the enzyme content, and the content, in turn, was dependent upon the availability of mRNA for alpha-amylase. We propose that the regulation of the development of alpha-amylase in cotyledons may involve some factor(s) inhibitory to accumulation of alpha-amylase mRNA, which is present in dry cotyledons and can be removed from cotyledons by leakage or by the presence of the axis.

Journal Article↗
PubMed1990

Tobacco Plants Transformed with the Bean alphaai Gene Express an Inhibitor of Insect alpha-Amylase in Their Seeds.

Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant alpha-amylases. We recently (J Moreno, MJ Chrispeels [1989] Proc Natl Acad Sci USA 86:7885-7889) presented strong circumstantial evidence that this alpha-amylase inhibitor (alphaAI) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5' and 3' flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M(r) 10,000-18,000) that cross-react with antibodies to the bean alpha-amylase inhibitor. Most of these polypeptides bind to a pig pancreas alpha-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas alpha-amylase activity as well as the alpha-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called alphaai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.

Journal Article↗
PubMed1991

Independent Regulatory Aspects and Posttranslational Modifications of Two beta-Amylases of Rye : Use of a Mutant Inbred Line.

We have examined the occurrence/disappearance, tissue location, and posttranslational modification of beta-amylase proteins in rye (Secale cereale L.) kernels at three physiological stages (development, maturity, germination) with a normal inbred line and a mutant line exhibiting a high but incomplete beta-amylase deficiency. This deficiency corresponds to a lack of accumulation of beta-amylase activity in the endosperm and does not affect the level of activity in the outer pericarp and green tissues as compared to the normal line. Two antigenically related but distinct beta-amylases (I and II) were detected in the normal line (II being the major constituent) and only one (I) in the mutant line. I and II display very similar electrophoretic polymorphism. In both lines, I appears to be ubiquitous, although it disappears from the outer pericarp during ripening. Antigen II was present only in the normal line and appears to be specific for the endosperm and perhaps for the maternal green tissues of the seed. Posttranslational modifications occurring during germination, which are mimicked by the action of papain, affect II but not I. The two groups of beta-amylases are discussed in relation to recent reports indicating the presence of two types of beta-amylase with different functions and gene loci in barley and wheat.

Journal Article↗
PubMed1992

Characteristics of alpha-Amylase during Germination of Two High-Sugar Sweet Corn Cultivars of Zea mays L.

The role of the scutellum and the aleurone in alpha-amylase production in the high-sugar sweet corn cultivars Illini X-tra Sweet (shrunken-2, sh2) and Illinois 677a (sugary, sugary enhancer; su se) was compared to that in the starchy (Su) hybrid Funks G4646 with the use of alpha-amylase enzyme assays, isoelectric focusing, electron microscopy, and laser scanning confocal microscopy. The scutellum of Illinois 677a had low levels of alpha-amylase activity compared to that of Funks G4646 through 10 days after imbibition, and the aleurone of Illini X-tra Sweet had negligible activity. On the isoelectric focusing gels, the Illinois 677a scutellum had fewer alpha-amylase isozymes at 7 days compared to the Funks G4646 scutellum. The Illini X-tra Sweet aleurone had no alpha-amylase isozymes. Funks G4646 scutellar epithelial and aleurone cells contained abundant rough endoplasmic reticulum, polysomes, and dictyosomes at 5 and 7 days, respectively. The scutellar epithelial cells of Illinois 677a contained fewer of these structures by 5 days, and the Illini X-tra Sweet aleurone contained mostly lipid bodies through 7 days. Few cytoplasmic membranes and little RNA were detected with laser scanning confocal microscopy in the Illini X-tra Sweet aleurone compared to Funks G4646 at 7 days. These data suggest that the scutellum of Illinois 677a and the aleurone of Illini X-tra Sweet have impaired abilities to produce alpha-amylase.

Journal Article↗
PubMed1991

Immunocytochemical Identification and Localization of Active and Inactive alpha-Amylase and Pullulanase in Cells of Clostridium thermosulfurogenes EM1.

Clostridium thermosulfurogenes EM1 formed blebs, i.e., protrusions still in contact with the cytoplasmic membrane, that originated from the cytoplasmic membrane during growth in batch culture and continuous culture. They could be observed squeezed between the cell wall and cytoplasmic membrane in cells with seemingly intact wall layers (surface layer and peptidoglycan layer) as well as in cells with wall layers in different states of degradation caused by phosphate limitation or high dilution rates. Blebs were found to turn into membrane vesicles by constriction in cases when the cell wall was heavily degraded. Bleb and vesicle formation was also observed in the absence of substrates that induce alpha-amylase and pullulanase synthesis. No correlations existed between bleb formation and the presence of active enzyme. Similar blebs could also be observed in a number of other gram-positive bacteria not producing these enzymes, but they were not observed in gram-negative bacteria. For immunoelectron-microscopic localization of alpha-amylase and pullulanase in C. thermosulfurogenes EM1, two different antisera were applied. One was raised against the enzymes isolated from the culture fluid; the other was produced against a peptide synthesized, as a defined epitope, in analogy to the N-terminal amino acid sequence (21 amino acids) of the native extracellular alpha-amylase. By using these antisera, alpha-amylase and pullulanase were localized at the cell periphery in samples taken from continuous culture or batch culture. In samples prepared for electron microscopy by freeze substitution followed by ultrathin sectioning, blebs could be seen, and the immunolabel pinpointing alpha-amylase enzyme particles was seen not only randomly distributed in the cell periphery, but also lining the surface of the cytoplasmic membrane and the blebs. Cells exhibiting high or virtually no enzyme activity were labeled similarly with both antisera. This finding strongly suggests that alpha-amylase and pullulanase may occur in both active and inactive forms, depending on growth conditions.

Journal Article↗
PubMed1982

Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli.

A partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage lambda host-vector system was shown to direct the synthesis of a thermostable alpha-amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000. The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused beta-lactamase-alpha-amylase protein with amylase activity.

Amylases↗
PubMed2003

Rhizopus microsporus var. rhizopodiformis: a thermotolerant fungus with potential for production of thermostable amylases.

The effect of several nutritional and environmental parameters on growth and amylase production from Rhizopus microsporus var. rhizopodiformis was analysed. This fungus was isolated from soil of the Brazilian "cerrado" and produced high levels of amylolytic activity at 45 degrees C in liquid medium supplemented with starch, sugar cane bagasse, oat meal or cassava flour. Glucose in the culture medium drastically repressed the amylolytic activity. The products of hydrolysis were analysed by thin layer chromatography, and glucose was detected as the main component. The amylolytic activity hydrolysed several substrates, such as amylopectin, amylase, glycogen, pullulan, starch, and maltose. Glucose was always the main end product detected by high-pressure liquid chromatography analysis. These results indicated that the amylolytic activity studied is a glucoamylase, but there were also low levels of alpha-amylase. As compared to other fungi, R. microsporus var. rhizopodiformis can be considered an efficient producer of thermostable amylases, using raw residues of low cost as substrates. This information is of technological value, considering the importance of amylases for industrial hydrolysis.

Acclimatization↗
PubMed1988

Raw starch adsorption-desorption purification of a thermostable beta-amylase from Clostridium thermosulfurogenes.

The beta-amylase from Clostridium thermosulfurogenes was readily adsorbed onto raw starch. The adsorbed beta-amylase was eluted from raw starch by using boiled soluble starch solution as an elutant. The soluble starch treated beta-amylase could not adsorb onto raw starch which indicates that the soluble and insoluble substrate binding sites of the beta-amylase may be the same. The beta-amylase was purified to homogeneity by raw starch adsorption-desorption techniques and octyl-Sepharose chromatography. It had a specific activity of 4188 units/mg protein. The insoluble substrate adsorption-desorption technique may be used for the purification of other enzymes.

Adsorption↗
PubMed1983

Multiple molecular forms of the gibberellin-induced alpha-amylase from the aleurone layers of barley seeds.

A class of plant growth regulators, gibberellins, induce the synthesis of alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) in the aleurone layers of barley (Hordeum vulgare L. var. Himalaya) seeds. The purified alpha-amylase is composed of multiple isozymic forms with indistinguishable molecular weights, but different net charges. These alpha-amylase isozymes separate on isoelectric focusing gels into two groups, each containing multiple species. One group has an apparent isoelectric point (pI) of approximately 5.8 (the high pI group). The other group's pI values are around 4.5 (the low pI group). On some gels a small amount of protein focuses between the high and low pI isozymes. These proteins comigrate with the low pI isozymes upon reelectrophoresis. The synthesis of these two groups is temporally regulated. The high pI group is the dominant set of isozymes secreted from embryoless half seeds during the first two days of gibberellin administration. After four days, however, the major isozymes are those of the low pI group. This shift in isozyme pattern is due to a shift in their relative rates of synthesis. Peptide analysis of these two groups of isozymes with Staphylococcus aureus V8 protease and cyanogen bromide shows amino acid sequence differences. However, members within the same group have similar peptide patterns. Both groups of isozymes are synthesized in vitro in a wheat germ extract primed with poly(A)+ RNA isolated from gibberellin-treated aleurone layers. This indicates that the synthesis of the two groups of alpha-amylase isozymes is probably directed by two or more different populations of mature mRNA. A model that explains these observations and the available genetic information is that barley aleurone alpha-amylase isozymes are encoded by at least two sets of structural genes.

Amylases↗
PubMed1978

Association of Tenebrio molitor L. alpha-amylase with two protein inhibitors--one monomeric, one dimeric--from wheat flour. Differential scanning calorimetric comparison of heat stabilities.

Thermal stabilization resulting from protein . protein association between two protein inhibitors (coded as 0.19, a dimer, and 0.28, a monomer) from wheat flour and the alpha-amylase from Tenebrio molitor L. (yellow mealworm) larvae was investigated by differential scanning calorimetry (heating rate 10 degrees C/min). Thermograms (plots of heat flow vs. temperature) for the two inhibitors showed broad endothermic peaks with the same extrema (denaturation temperatures) at 93 degrees C, and equal, small enthalpies of denaturation (2 cal/g). The amylase produced a sharp endotherm at 70.5 degrees C, but a larger enthalpy change on denaturation (6 cal/g). The amylase . inhibitor complexes differed in thermal stability, but both showed significant stabilization relative to free enzyme. The complex formed with monomeric inhibitor 0.28 showed a higher denaturation temperature (85.0 degrees C) than that formed with dimeric inhibitor 0.19 (80.5 degrees C). This order of stabilization agrees with the relative affinities of the inhibitors for the amylase. These thermograms are consistent with previous results which indicated that 1 mol of amylase binds 1 mol of inhibitor 0.19.

Albumins↗
PubMed1981

A modified amylase assay, using a fluorescent substrate, and its application to a study of the rat parotid gland in vitro.

A modified fluorescence assay for alpha-amylase activity is described. The method employs amylopectin anthranilate as substrate and offers the advantages of economy of time and resources over a previously described technique using the same substrate. The sample containing alpha-amylase is incubated with the substrate for 5 min at 30 degree C in a final volume of 750 microliter. The fluorescent products of the reaction are separated from the substrate by the addition of methanol, and the methanol-soluble fluorescence is measured in a fluorescence spectrometer. A highly reproducible linear relationship between fluorescence and alpha-amylase activity is obtained for enzyme activities up to 2 units. The absolute sensitivity of the assay under these conditions was estimated to be 0.02 EU (= 0.08 EU ml-1). The application of the assay method to a study of the effects of isoprenaline and substance P-like peptides on the release of alpha-amylase from rat parotid gland slices is described. The assay is particularly suitable for studies on agonists, such as substance P, which have a low ceiling effect in terms of amylase release.

Amylases↗
PubMed1982

Isolation and characterization of Taka-amylase A apoprotein deglycosylated by digestion with almond glycopeptidase immobilized on Sepharose.

Taka-amylase A (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), which contains a single asparagine-linked oligosaccharide unit, was digested with almond glycopeptidase immobilized on Sepharose 6B at 20 degrees C for 4 h. A maximum of 10% of the parent protein was isolated as apoprotein by column chromatography on Con-A Sepharose. The characteristics of the apoprotein were compared to those of the native Taka-amylase A. The removal of the sugar chain from Taka-amylase. A caused no change in the pH-activity profile or in kinetic parameters of the hydrolysis of soluble starch. The stability of the apoprotein toward changing pH and digestion by proteases did not show any appreciable difference from that of the native Taka-amylase. These results suggest that the carbohydrate moiety of Taka-amylase A is not an essential participant in the catalysis.

Amidohydrolases↗
PubMed1983

Complete amino acid sequence of an alpha-amylase inhibitor in wheat kernel.

We recently purified a new alpha-amylase inhibitor (called 0.53-inhibitor according to its mobility on polyacrylamide gel electrophoresis, at pH 8.5, relative to Bromophenol blue (taken as 1), under the conditions described by O'Donnell, M.D. and McGeeney, K.F. (1976) Biochim. Biophys. Acta 422, 159-169)) from wheat kernel, which has 500-times greater inhibitory activity towards human salivary amylase than towards human pancreatic amylase (Maeda, K., Takemori, Y. and Oka, O. (1982) Agric. Biol. Chem. 41, 2873-2875). Elucidation of the primary structure and structural comparison with other amylase inhibitors are essential to understand the mechanism of the selective inhibitory behavior of 0.53-inhibitor. The complete amino acid sequence of 0.53-inhibitor has been determined after cleaving the protein with CNBr and trypsin separately. 0.53-Inhibitor is composed of two identical subunits with 124 amino acid residues and contains nine cysteine residues in each subunit. Comparison of the sequence of 0.53- and 0.28-inhibitor, which is a major alpha-amylase inhibitor in wheat, with Mr 12 000, of monomeric form, shows high sequence homologies of nine cysteine regions, but significant differences are evident.

Amino Acid Sequence↗
PubMed1979

Substance P stimulation of amylase release by isolated parotid cells and inhibition of substance P induction of salivation by vasoactive peptides.

Substance P stimulation of salivation in rats has been studied as has its in vitro enhancement of amylase release by isolated parotid cells. The extent of the stimulation on amylase release by isolated parotid cells was dependent upon the concentration of substance P, with the minimum effective concentration being 1 nM. The substance P effect was detectable within 1 min after incubation and lasted for at least 50 min. Substance P stimulation was demonstrable at 25--37 degrees C but not at 0 degrees C. Adrenocorticotropic hormone (ACTH), thyrotropin-releasing hormone (TRH), vasopressin and neurotensin had no effect on amylase release. These results suggest that substance P may act directly on the parotid cells. Examination of the salivary-stimulating activity of fragments of substance P showed that the C-terminal octapeptide and (pyroglutamyl)hexapeptide were active, although less potent than substance P, whereas its free acid, C-terminal tetra- and tri-peptides were inactive. Vasopressin, angiotensin II and neurotensin could inhibit substance P induced salivation, whereas TRH, ACTH and somatostatin had no effect. Amylase activity per unit volume of saliva was not changed by the injection of vasopressin, angiotensin II or neurotensin. These vasoactive peptides did not affect substance P stimulation of amylase release by isolated parotid cells. The results indicate that vasopressin, angiotensin II and neurotensin inhibit the action of substance P on salivation at sites other than the parotid cells.

Acetylcholine↗
PubMed1981

Nucleotide sequence of the promoter and NH2-terminal signal peptide region of the alpha-amylase gene from Bacillus amyloliquefaciens.

We have isolated and partially sequenced the gene coding for alpha-amylase (EC 3.2.1.1) from Bacillus amyloliquefaciens by molecular cloning in the plasmid pUB110 using Bacillus subtilis as a host. The nucleotide sequence of the NH2-terminal region of the cloned gene was determined and found to contain a 31-residue-long stretch of amino acids preceding the NH2-terminal sequence of the extracellular alpha-amylase. Within this sequence there is a 15-residue-long stretch of uncharged amino acids similar to that found at the NH2 terminus of other precursors to exported proteins. This "signal sequence" is probably removed in conjunction with the translocation of alpha-amylase through the cytoplasmic membrane. In vitro labeling of alpha-amylase with radioactive amino acids in a coupled transcription-translation system followed by partial sequencing established the exact location of the NH2 terminus of the alpha-amylase gene. The nucleotide sequence preceding the NH2 terminus has properties resembling the RNA-polymerase- and ribosome-binding sites found at the 5' terminus of many prokaryotic genes.

Amylases↗
PubMed1977

The action pattern of human salivary alpha-amylase in the vicinity of the branch points of amylopectin.

Salivary alpha-amylase hydrolyses amylopectin in stages. At the end of the so-called second stage, there are present glucose, maltose, and a series of alpha-limit dextrins containing (1 leads to 4)- and (1 leads to 6)-alpha-D-glucosidic bonds. The structures of the limit dextrins containing a single (1 leads to 6)-bond were examined. Six such dextrins were found. Of these, two were capable of being further hydrolysed by alpha-amylase, whereas the remaining four were true, amylase-resistant alpha-limit dextrins. The structures of the limit dextrins afforded information about those (1 leads to 4)-alpha-D-glucosidic bonds of amylopectin that are capable of being cleaved by salivary alpha-amylase and those that are resistant. In order to define further the action of alpha-amylase, the alpha-amylolytic products of 6-alpha-maltotriosyl-D-glucose, 6(3)-alpha-maltotriosylmaltotriose, and 6(3)-alpha-maltotriosylmaltotetraose were examined.

Amylases↗