Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field

We present a simple method for efficient DNA ligation utilizing the heat generation of ferromagnetic particles subjected to an ac magnetic field. We carry out the ligation of DNA fragments with cohesive ends using T4 DNA ligase immobilized on the surface of ferromagnetic particles. When a radio frequency alternating magnetic field is applied, ferromagnetic particles dissipate heat and DNA ligase on the particles is selectively heated up and activated with little influence on the annealing of DNA ends, as a result of which the ligation efficiency increases. We show that the ligation efficiency increases with an increase in the field amplitude.     

Activity of Lipase and Chitinase Immobilized on Superparamagnetic Particles in a Rotational Magnetic Field

We immobilize hydrolases such as lipase and chitinase on superparamagnetic particles, which are subjected to a rotational magnetic field, and measure the activities of the enzymes. We find that the activities of lipase and chitinase increase in the rotational magnetic field compared to those in the absence of a magnetic field and reach maximum at certain frequencies. The present methodology may well be utilized for the design and development of efficient micro reactors and micro total analysis systems (μ-TASs).     

Starch degradation in the cotyledons of germinating lentils

Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the α type. Starch is degraded slowly in the first days; during this time, α- and β-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in α-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three α-amylases, and one β-amylase. Based on their heat lability or heat stability, two sets of α-amylase seem to exist in lentil cotyledons.     

Synthesis,X-ray diffraction analysis,quantum chemical studies and α-amylase inhibition of probenecid derived S-alkylphthalimide-oxadiazole-benzenesulfonamide hybrids

Sulphonamide and 1,3,4-oxadiazole moieties are present as integral structural parts of many drugs and pharmaceuticals. Taking into account the significance of these moieties, we herein present the synthesis, single-crystal X-ray analysis, DFT studies, and α-amylase inhibition of probenecid derived two S-alkylphthalimide-oxadiazole-benzenesulfonamide hybrids. The synthesis has been accomplished in high yields. The final structures of both hybrids have been established completely with the help of different spectro-analytical techniques, including NMR, FTIR, HR-MS, and single-crystal X-ray diffraction analyses. In an effort to confirm the experimental findings, versatile quantum mechanical calculations and Hirshfeld Surface analysis have been performed. α-Amylase inhibition assay has been executed to investigate the enzyme inhibitory potential of both hybrids. The low IC₅₀ value (76.92 ± 0.19 μg/mL) of hybrid 2 shows the good α-amylase inhibition potential of the respective compound. Ultimately, the binding affinities and features of the two hybrids are elucidated utilising a molecular docking technique against the α-amylase enzyme.     

Specific Determination of alpha-Amylase Activity in Crude Plant Extracts Containing beta-Amylase

The specific measurement of α-amylase activity in crude plant extracts is difficult because of the presence of β-amylases which directly interfere with most assay methods. Methods compared in this study include heat treatment at 70°C for 20 min, HgCl₂ treatment, and the use of the α-amylase specific substrate starch azure. In comparing alfalfa (Medicago sativa L.), soybeans (Glycine max [L.] Merr.), and malted barley (Hordeum vulgare L.), the starch azure assay was the only satisfactory method for all tissues. While β-amylase can liberate no color alone, over 10 International units per milliliter β-amylase activity has a stimulatory effect on the rate of color release. This stimulation becomes constant (about 4-fold) at β-amylase activities over 1,000 International units per milliliter. Two starch azure procedures were developed to eliminate β-amylase interference: (a) the dilution procedure, the serial dilution of samples until β-amylase levels are below levels that interfere; (b) the β-amylase saturation procedure, addition of exogenous β-amylase to increase endogenous β-amylase activity to saturating levels. Both procedures yield linear calibrations up to 0.3 International units per milliliter. These two procedures produced statistically identical results with most tissues, but not for all tissues. Differences between the two methods with some plant tissues was attributed to inaccuracy with the dilution procedure in tissues high in β-amylase activity or inhibitory effects of the commercial β-amylase. The β-amylase saturation procedure was found to be preferable with most species. The heat treatment was satisfactory only for malted barley, as α-amylases in alfalfa and soybeans are heat labile. Whereas HgCl₂ proved to be a potent inhibitor of β-amylase activity at concentrations of 10 to 100 micromolar, these concentrations also partially inhibited α-amylase in barley malt. The reported α-amylase activities in crude enzyme extracts from a number of plant species are apparently the first specific measurements reported for any plant tissues other than germinating cereals.     

Amylases from aleurone layers and starchy endosperm of barley seeds

Amylases from incubated aleurone layers or from starchy endosperm of barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated using acrylamide gel electrophoresis and analytical gel filtration with Sephadex G-200. Electrophoresis of amylase from aleurone layers yields seven visually distinct isozymes with an estimated molecular weight of 43,000. Because each isozyme hydrolyzes β-limit dextrin azure and incorporates calcium-45, they are α-amylases. On Sephadex G-200, amylase from the aleurone layers is separated into seven fractions ranging in estimated molecular weights from 45,000 to 3,000. Little or no activity is observed when six fractions are subjected to electrophoresis. Electrophoresis of only the fraction with the estimated molecular weight of 45,000 gave the seven isozymes. The amylases are heat labile and cannot be stabilized by the presence of substrate or by the protease inhibitor, phenylmethylsulfonylfluoride. Electrophoresis of amylase from the starchy endosperm yields nine β-amylases. Four of these β-amylases are isozymes with an estimated molecular weight of 43,000. The other five forms of β-amylase represent molecular aggregates of the four basic β-amylase monomers. A dimer, a tetramer, and an octamer of β-amylase can be identified with estimated molecular weights of about 86,000, 180,000 and 400,000, respectively. These estimated molecular weights were confirmed on Sephadex G-200. There are five additional fractions of β-amylase with estimated molecular weights ranging from 30,000 to 4,000. These fractions are not observed electrophoretically.     

Starch Source Influences Dietary Glucose Generation at the Mucosal α-Glucosidase Level

The quality of starch digestion, related to the rate and extent of release of dietary glucose, is associated with glycemia-related problems such as diabetes and other metabolic syndrome conditions. Here, we found that the rate of glucose generation from starch is unexpectedly associated with mucosal α-glucosidases and not just α-amylase. This understanding could lead to a new approach to regulate the glycemic response and glucose-related physiologic responses in the human body. There are six digestive enzymes for starch: salivary and pancreatic α-amylases and four mucosal α-glucosidases, including N- and C-terminal subunits of both maltase-glucoamylase and sucrase-isomaltase. Only the mucosal α-glucosidases provide the final hydrolytic activities to produce substantial free glucose. We report here the unique and shared roles of the individual α-glucosidases for α-glucans persisting after starch is extensively hydrolyzed by α-amylase (to produce α-limit dextrins (α-LDx)). All four α-glucosidases share digestion of linear regions of α-LDx, and three can hydrolyze branched fractions. The α-LDx, which were derived from different maize cultivars, were not all equally digested, revealing that the starch source influences glucose generation at the mucosal α-glucosidase level. We further discovered a fraction of α-LDx that was resistant to the extensive digestion by the mucosal α-glucosidases. Our study further challenges the conventional view that α-amylase is the only rate-determining enzyme involved in starch digestion and better defines the roles of individual and collective mucosal α-glucosidases. Strategies to control the rate of glucogenesis at the mucosal level could lead to regulation of the glycemic response and improved glucose management in the human body.     

Antiviral Cystine Knot α-Amylase Inhibitors from Alstonia scholaris

Cystine knot α-amylase inhibitors are cysteine-rich, proline-rich peptides found in the Amaranthaceae and Apocynaceae plant species. They are characterized by a pseudocyclic backbone with two to four prolines and three disulfides arranged in a knotted motif. Similar to other knottins, cystine knot α-amylase inhibitors are highly resistant to degradation by heat and protease treatments. Thus far, only the α-amylase inhibition activity has been described for members of this family. Here, we show that cystine knot α-amylase inhibitors named alstotides discovered from the Alstonia scholaris plant of the Apocynaceae family display antiviral activity. The alstotides (As1–As4) were characterized by both proteomic and genomic methods. All four alsotides are novel, heat-stable and enzyme-stable and contain 30 residues. NMR determination of As1 and As4 structures reveals their conserved structural fold and the presence of one or more cis-proline bonds, characteristics shared by other cystine knot α-amylase inhibitors. Genomic analysis showed that they contain a three-domain precursor, an arrangement common to other knottins. We also showed that alstotides are antiviral and cell-permeable to inhibit the early phase of infectious bronchitis virus and Dengue infection, in addition to their ability to inhibit α-amylase. Taken together, our results expand membership of cystine knot α-amylase inhibitors in the Apocynaceae family and their bioactivity, functional promiscuity that could be exploited as leads in developing therapeutics.     

alpha-Amylase Isozymes in Gibberellic Acid-treated Barley Half-seeds

The presence of multiple forms of α-amylase in gibberellic acid-treated embryoless barley half-seeds was demonstrated by separation on diethylaminoethyl-Sephadex and isoelectric focusing polyacrylamide gel disc electrophoresis. Two major α-amylase fractions (A and B), each consisting of two to three isozyme components, were purified. α-Amylase fractions A and B were distinguishable in their reaction patterns. The optimal pH of fraction A α-amylase was found to reside in the acidic side (pH 5.0), as was determined by analyzing the reducing sugars formed as well as the paper chromatographic detection of reaction products. At neutral pH, 6.9, fraction A exhibited weak amylolytic activity in forming maltose. The α-amylase activity in fraction A was markedly stimulated by heat treatment (70 C/15 minutes). Fraction B, constituting a major part of amylases in the endosperm extract, was also found to be composed of α-amylase, as evidenced by the loss of enzyme activity upon allowing fractions A and B to stand at pH 3.3 for a prolonged period. The possible physiological function of the two different types of α-amylase in the carbohydrate breakdown of barley seeds is discussed.     

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

We studied the localization of α-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-α-amylase immunoglobulin G followed by fluorescein isothiocyanate labeled goat anti-rabbit immunoglobulin G. α-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 α-amylase appearance was similar in detached cotyledons, indicating that attachment of the embryonic axis has no effect on this pattern. However, in attached cotyledons, α-amylase disappeared from the regions where starch grains had been digested, but in detached cotyledons there was no disappearance of α-amylase, and digestion was slower than in intact cotyledons.     

Water stress enhances expression of an alpha-amylase gene in barley leaves

The amylases of the second leaves of barley seedlings (Hordeum vulgare L. cv Betzes) were resolved into eight isozymes by isoelectric focusing, seven of which were β-amylase and the other, α-amylase. The α-amylase had the same isoelectric point as one of the gibberellin-induced α-amylase isozymes in the aleurone layer. This and other enzyme characteristics indicated that the leaf isozyme corresponded to the type A aleurone α-amylase (low pI group). Crossing experiments indicated that leaf and type A aleurone isozymes resulted from expression of the same genes.

In unwatered seedlings, leaf α-amylase increased as leaf water potential decreased and ABA increased. Water stress had no effect on β-amylase. α-Amylase occurred uniformly along the length of the leaf but β-amylase was concentrated in the basal half of the leaf. Cell fractionation studies indicated that none of the leaf α-amylase occurred inside chloroplasts.

Leaf radiolabeling experiments followed by extraction of α-amylase by affinity chromatography and immunoprecipitation showed that increase of α-amylase activity involved synthesis of the enzyme. However, water stress caused no major change in total protein synthesis. Hybridization of a radiolabeled α-amylase-related cDNA clone to size fractionated RNA showed that water-stressed leaves contained much more α-amylase mRNA than unstressed plants. The results of these and other studies indicate that regulation of gene expression may be a component in water-stress induced metabolic changes.

     

Effect of temperature on the synthesis and secretion of alpha-amylase in barley aleurone layers

The effect of temperature on α-amylase synthesis and secretion from barley (c.v. Himalaya) half-seeds and aleurone layers is reported. Barley half-seeds incubated at 15 C in gibberellic acid (GA) concentrations of 0.5 and 5 micromolar for 16 hours do not release α-amylase. Similarly, isolated aleurone layers of barley do not release α-amylase when incubated for 2 or 4 hours at temperatures of 15 C or below following 12 hours incubation at 25 C at GA concentrations from 50 nanomolar to 50 micromolar. There is an interaction between temperature and GA concentration for the process of α-amylase release from aleurone layers; thus, with increasing GA concentration, there is an increase in the Q₁₀ of this process. A thermal gradient bar was used to resolve the temperature at which the rate of α-amylase release changes; thermal discontinuity was observed between 19 and 21 C. The time course of the response of aleurone tissue to temperature was determined using a continuous monitoring apparatus. Results show that the effect of low temperature is detectable within minutes, whereas recovery from exposure to low temperature is also rapid. Although temperature has a marked effect on the amount of α-amylase released from isolated aleurone layers, it does not significantly affect the accumulation of α-amylase within the tissue. At all GA concentrations above 0.5 nanomolar, the level of extractable α-amylase is unaffected by temperatures between 10 and 28 C. It is concluded that the effect of temperature on α-amylase production from barley aleurone layers is primarily on the process of enzyme secretion.     

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

Developmental patterns of α-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, α-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, α-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 α-amylase. We propose that the regulation of the development of α-amylase in cotyledons may involve some factor(s) inhibitory to accumulation of α-amylase mRNA, which is present in dry cotyledons and can be removed from cotyledons by leakage or by the presence of the axis.     

In Vitro Synthesis and Processing of Wheat alpha-Amylase : TRANSLATION OF GIBBERELLIC ACID-INDUCED WHEAT ALEURONE LAYER RNA BY WHEAT GERM AND XENOPUS LAEVIS OOCYTE SYSTEMS

Wheat (Triticum aestivum) RNA was used to program synthesis of the α-amylase protein by Xenopus laevis oocytes. A 41,500-dalton protein was made which was identified as α-amylase by immunoprecipitation with rabbit anti-α-amylase antiserum raised against the purified wheat protein and by its co-migration with authentic α-amylase on sodium dodecyl sulfate polyacrylamide gels. Synthesis of α-amylase was dependent upon injection of RNA extracted from gibberellic acid-induced aleurone layers from wheat. The amount of α-amylase produced was proportional to the amount of RNA injected and reached a plateau within 4 hours after injection. When the same RNA was translated in a wheat germ cell-free translation system, a 43,000-dalton protein was produced. Addition of dog pancreas microsomal membranes to the wheat germ translation system resulted in processing of the α-amylase protein to a form which co-migrated with authentic α-amylase purified from malted wheat and with the protein synthesized in oocytes.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 9. DE NOVO SYNTHESIS OF beta-AMYLASE

Germinating rice seeds were fed with [³⁵S]methionine and the incorporation of ³⁵S into β-amylase demonstrated by quantitative immunoprecipitation using rabbit anti-β-amylase immunoglobulin G fraction. Separation of the antigen-antibody complex by Na-dodecylsulfate gel electrophoresis and subsequent radioautography clearly showed the radioactive labeling of the β-amylase molecule. The specific radioactivity of β-amylase derived from scutellum by immunoprecipitation was significantly greater than that of the endosperm. The results strongly indicate that at the onset of germination of rice seeds β-amylase is synthesized de novo in the scutellum and that in later stages there occurs activation of an inactive, latent form of the enzyme associated with starch granules in the endosperm. In later stages of germination this activated form of the enzyme becomes dominant.     

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 α-amylases. We recently (J Moreno, MJ Chrispeels [1989] Proc Natl Acad Sci USA 86:7885-7889) presented strong circumstantial evidence that this α-amylase inhibitor (αAI) 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 α-amylase inhibitor. Most of these polypeptides bind to a pig pancreas α-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas α-amylase activity as well as the α-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called αai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.     

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function

Pea (Pisum sativum L.) tissues with reduced chloroplast density (e.g. petals and stems) or function (i.e. senescent leaves and leaves darkened for prolonged periods) were surveyed to determine whether tissues with genetically or environmentally reduced chloroplast density and/or function also have significantly different amylolytic enzyme activities and/or isoform patterns than leaf tissues with totally competent chloroplasts. Native PAGE followed by electrophoretically blotting through a starch or β-limit dextrin containing gel and KI/I₂ staining revealed that the primary amylases in leaves, stems, petals, and roots were the primarily vacuolar β-amylase (EC 3.2.1.2) and the primarily apoplastic α-amylase (EC 3.2.1.1). Among tissues of light grown pea plants, petals contained the highest levels of total amylolytic (primarily β-amylase) activity and considerably higher ratios of β- to α-amylase. In aerial tissues there was an inverse relationship between chlorophyll and starch concentration, and β-amylase activity. In sections of petals and stems there was a pronounced inverse relationship between chlorophyll concentration and the activity of α-amylase. Senescing leaves of pea, as determined by age, and protein and chlorophyll content, contained 3.8-fold (fresh weight basis) and 32-fold (protein basis) higher α-amylase activity than fully mature leaves. Leaves maintained in darkness for 12 days displayed a 14-fold (fresh weight basis) increase in α-amylase activity over those grown under continuous light. In senescence and prolonged darkness studies, the α-amylase that was greatly increased in activity was the primarily apoplastic α-amylase. These studies indicate that there is a pronounced inverse relationship between chloroplast function and levels of apoplastic α-amylase activity and in some cases an inverse relationship between chloroplast density and/or function and vacuolar β-amylase activity.