Rapid method for the affinity purification of thermostable α-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Summary A rapid and efficient method the exploiting affinity of α-amylase for its substrate starch is described. α-amylase from Bacillus licheniformis was purified to homogeneity by ammonium sulphate precipitation and affinity chromatography with 230-fold purification. The α-amylase adsorption to various starches was examined in order to screen its ability for highest binding to starch. The α-amylase was bound to starch more tenaciously, hence various eluants like maltose, soluble starch and high salts could not elute the bound α-amylase. However, the bound α-amylase was instantly eluted using 2% (w/v) dextrin. The purified enzyme showed a single polypeptide on SDS-PAGE, with a molecular weight of 58 kD. Western blot analysis confirmed the specificity of antibody raised against purified α-amylase.     

Affinity chromatography of α-amylase from Bacillus licheniformis

An affinity chromatographic method with a novel eluant from Bacillus licheniformis is described. α-amylase was bound to starch, starch-celite, starch-Sepharose columns and the bound α-amylase was rapidly eluted with 2% (w/v) white dextrin. The binding capacity of α-amylase to starch column is 380 μmol/g of starch. The purified enzyme showed a single polypeptide on SDS-polyacrylamide gel electrophoresis with a molecular weight of 58 kD. The specificity of purified enzyme was confirmed by immunodiffusion, immunoelectrophoresis. Single radial immunodiffusion and western blotting studies analyzed the synthesis of enzyme at different time points.     

Polyethylene glycol as a spacer for solid-phase enzyme immobilization

With the aim to improve the performance of enzyme bound to hydrophilic solid phases, their immobilization with polyethylene glycol (PEG) tether have been studied. Sweet potato β-amylase, which hydrolyses the high molecular weight substrate starch and β-galactosidase, which acts on low molecular weight substrates, were used as model enzymes and beaded thiol–agarose as solid phase. Several two step methods for the introduction of the tether using a bis-oxirane homobifunctional PEG as well as a heterobifunctional derivative with a hydroxysuccinimide ester and a maleimide group have been evaluated. Amino groups, native and de novo thiol groups in the enzymes were utilized for immobilization.

The best approach was found to be to first introduce the PEG derivative via one of its reactive groups to the enzyme. Subsequently the formed conjugate was bound to the solid phase by the remaining reactive group.

Attempts to first introduce the PEG tether into the solid phase were not successful.

A high degree of substitution with PEG chains on the enzyme leads to high immobilization yields for both β-amylase and β-galactosidase, but relatively lower gel-bound activity for the former enzyme which is acting on a high molecular weight substrate and thus more sensitive for steric shielding effects. With optimal degree of PEG substitution (which occurred at five times molar excess of the heterobifunctional reagent) the gel-bound activity of β-amylase was increased from 12% (for the derivative without tether) to 31%.     

The binding of eosin-labeled subunit δ to the isolated chloroplast ATPase, CF1, as revealed by rotational diffusion in solution

We investigated the binding of subunit δ to solubilized chloroplast ATPase. Purified δ was covalently labeled with eosin 5-isothiocyanate and its rotational correlation time was determined by a photoselection technique as a function of added CF₁ (containing δ) and of CF₁(−δ) (lacking δ). In aqueous buffer the rotational correlation time of labeled δ was 33 ns. This is compatible with a rather elongated shape with the dimensions 2b = 100 Å/2a = 28 Å. Binding of δ to CF₁ decreased the rotational correlation time about 10-fold. The result was a biphasic decay of the laser flash-induced absorption anisotropy which was analyzed to yield the proportion of δ (bound to CF₁) relative to δ (free). CF₁(−δ), which completely lacked the δ-subunit, bound one δ (mol/mol) with high affinity (K_d ≈ 100 nM) and at least another δ with about 20-fold lower affinity. The δ-containing CF₁, revealed only the low-affinity site(s) for δ. This was compatible with a 1:1 stoichiometry of δ in isolated CF₁.     

The activation of glycogen synthase in hepatocytes from rats with a glycogen storage disorder (gsd/gsd)

A rapid high resolution method of purification of the Trp-containing S100 proteins (S100a, S100a′) and of the S100b protein has been developed. The principle of this method is based on the fact that S100b protein becomes highly hydrophobic upon Zn²⁺ binding, whereas S100a and S100a′ are not affected. On an affinity chromatography of phenyl—Sepharose column, S100b is selectively bound in presence of zinc, whereas the Trp-containing S100 patients are quickly eluted. The S100b protein is further eluted with a buffer containing EDTA.     

Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus

The amylases produced by a Bacillus stearothermophilus were purified through a series of four steps. Two separable enzyme fractions having starch hydrolysing activity were eluted from a DEAE-cellulose column by NaCl gradient elution. The homogeneity of the purified enzymes was checked on polyacrylamide gel electrophoresis. The product formation studies indicated that fraction I was an -amylase whereas fraction II was a β-amylase. The molecular weights were determined to be 48 000 and 57 000 and the carbohydrate moiety was found to be 13.2 and 0.8% for - and β-amylase, respectively. The protein digest of these enzymes indicated a total number of 15 amino acids with aspartic and glutamic acid showing the highest value. The purified amylase showed maximal activity at 80°C and pH 6.9. Fe³⁺, Cd²⁺, Pb²⁺, Hg²⁺, Ni²⁺ and Ag¹⁺ were potent inhibitors whereas Zn²⁺, Mg²⁺, Mn²⁺ and Al³⁺ were mild inhibitors. Ca²⁺, Ba²⁺, Sr²⁺ and K⁺ stimulated amylase activity in the order of Ca²⁺ > Ba²⁺ > Sr²⁺ > K⁺. PCMB, EDTA and sodium iodoacetate were inhibitory whereas glutathione (GSH) and cysteine afforded protection of enzyme activity. EDTA showed dose-dependent noncompetitive inhibition of both - as well as β-amylase activities. EDTA inhibition was reversed by the addition of Ca²⁺ and PCMB inhibition by the addition of glutathione (reduced). The K_m for - and β-amylases were found to be 1.05 and 1.25 mg starch per ml, respectively.     

Generation and analyses of the transgenic potatoes expressing heterologous thermostable β-amylase

β-Amylase hydrolyzes the -1,4-glycosidic linkages of starch resulting in the release of maltose. This reaction is of industrial importance for maltose production and for the preparation process of fermented foods and alcoholic beverages. A demand for an acceleration of the rate of enzymatic cleavage of the starch macro-molecule is a prerequisite for large-scale and highly efficient production. Increasing the temperature up to the optimum of approximately 60 °C can significantly speed up the reaction. However, at higher temperatures, the effect on protein denaturation becomes dominant, and the conversion rate decreases. The primary objective of this study was to generate transgenic plants of the “Kennebec” potato variety for production of thermostable β-amylase using Agrobacterium-mediated transformation. Four chimeric genes encoding the β-amylase with or without signal peptide sequences for targeting expression in cytoplasm, amyloplasts, or vacuoles were constructed and driven by high tuber expression promoter from Sucrose synthetase gene Sus4. Forty-two transgenic lines were selected for this study. Transgenic lines with various β-amylase constructs were verified for the existence and expression of the transgenes by PCR approaches. The expression level of the introduced β-amylase protein was estimated by immunoblot analyses using polyclonal antibodies. Recombinant β-amylase was successfully expressed in Escherichia coli B21 (DE3), and temperature ranges of these inducible recombinant proteins were found to be between 40 and 90 °C. This enzymatic complex produced in the in vitro cultured microtubers and field-grown tubers from transgenic potatoes were proved to be stable and active at 60 °C. The relative activities of β-amylase in tubers of field-grown potatoes were compared, and the maximum increase was found with transgenic line #6A of the pSUS4-AMY construct which has an 11-fold greater increase than the untransformed “Kennebec”. Variations of the chemical compositions were found in the selected transgenic lines. Results of this study suggest the feasibility of utilizing thermostable β-amylase in transgenic potatoes for the starch-processing industries.     

Glyoxyl agarose as a new chromatographic matrix

At alkaline pHs, glyoxyl agarose is able to immobilize most of the proteins contained in a crude extract. However, due to its special immobilization features, at pH 7.0 only proteins that contain at least two exposed low pK amino groups in the same plane were immobilized (β-galactosidase from Escherichia coli, catalase from bovine liver, and IgG from rabbit). However, with many other proteins, even multimeric ones, immobilization could not be achieved (e.g.: glucose oxidase from Aspergillus niger and Penicillium vitale; catalase from Micococcus sp., A. niger and bovine liver; alcohol oxidase from Pichia pastoris, Hansenula sp. and Candida boidinii, β-galactosidase from Thermus sp., etc.). Elution of the attached proteins under mild conditions was not simple, if the number of protein-support bonds was very high, only boiling in SDS allowed the elution of the proteins. However, using glyoxyl agarose 4BCL with only 20 μmol of aldheyde groups/g of support, proteins could be fully eluted by competition with amino compounds (e.g., Tris buffer). In this first approach, we have tried to take advantage of this specific immobilization at pH 7.0 to purify multimeric proteins, using a β-galactosidase from E. coli as a model. The enzyme could be eluted from the support using Tris–HCl buffer as eluting agent, with a high yield (80%) and a high purification factor (32).     

地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

从地衣芽孢杆菌(Bacillus licheniformis)中克隆到耐高温α-淀粉酶基因全长, 构建了原核表达载体, 转入大肠杆菌(Escherichia coli)中, 使用IPTG于28°C诱导6小时后, 通过SDS-PAGE检测到目的蛋白, 分子量约为55 kDa, 并通过酶活力检测实验证明该蛋白具有耐高温α-淀粉酶活性。同时构建了该基因融合GFP的植物表达载体, 通过农杆菌(Agro- bacterium tumefaciens)介导瞬时转化烟草(Nicotiana tabacum)下表皮细胞并在荧光显微镜下观察, 发现在烟草下表皮细胞的细胞质和液泡中均有绿色荧光。使用I₂-KI溶液对乙醇脱色后的烟草叶片进行染色, 显色反应表明在烟草中表达的耐高温α-淀粉酶具有酶活性。最后, 采用农杆菌介导的花蕾浸泡法将重组载体转化到拟南芥(Arabidopsis thaliana)中, 筛选到稳定遗传的耐高温α-淀粉酶基因的拟南芥纯合子。研究结果为后期开展表达耐高温α-淀粉酶的转基因植物的相关研究奠定了实验基础。     

Solubilization of active GLP-1 (7–36)amide receptors from RINm5F plasma membranes

Glucagon-like peptide-1 (7–36)amide (GLP-1 (7–36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP-1 (7–36)amide have been described in RINm5F cells. We have solubilized active GLP-1 (7–36)amide receptors from RINm5F cell membranes utilizing the detergents octyl-β-glucoside and CHAPS; Triton X-100 and Lubrol PX were ineffective. Binding of radiolabeled GLP-1(7–36)amide to the solubilized receptor was inhibited conentration-dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with K_a= 0.26 ± 0.03 nM and B_max= 65.4 ± 21.24 fmol/mg of protein for the membrane-bound receptor and K_a= 22.54 ± 4.42 μM and B_max= 3.9 ± 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono- and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine-nucleotides, however neither GTP-γ-S nor GDP-β-S affected binding or labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G-protein. In conclusion, the here presented protocol allows solubilization of the GLP-1(7–36)amide receptor in a functional state and opens up the possibility for further molecular characterization of the receptor protein.     

Interactions of barley α-amylase isozymes with Ca2 , substrates and proteinaceous inhibitors

-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/)₈-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca^2 +, different substrates, and proteinaceous inhibitors for -amylases. Isozyme specific effects of Ca^2 + on the 80% sequence identical barley -amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca^2 + with identical ligands. A fully hydrated fourth Ca^2 + at the interface of the AMY2/barley -amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca^2 + occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca^2 +. Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered 'sugar tongs' site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley -amylases are reported in this review.     

The action pattern and physiological role of Tenebrio larval amylase

The larval midgut amylase of Tenebrio molitor L. may be classified as a typical -amylase on the basis of its action pattern on amylopectin, glycogen, and β-amylase limit dextrin. When the development is on the preferred diet of wheat-bran, the amylatic activity of the larvae is impaired by an -amylase inhibitor present in wheat. The inhibitor has been found to be stable to digestion by the larvae. Wheat β-amylase plays the major role in the digestion of starch by larvae. The implications of these relations are discussed.     

Release and Activity of Bound beta-Amylase in a Germinating Barley Grain

In resting grains of Triumph barley (Hordeum vulgare L. cv Triumph) about 40% of the β-amylase could be extracted with a saline solution, the remaining 60% being in a bound form. During seedling growth (20°C), the bound form was released mainly between days 1 and 3. When a preparation containing bound β-amylase was incubated with an extract made of endosperms separated from germinating grains, release of bound β-amylase took place and could be studied in vitro. The release was almost completely prevented by leupeptin and antipain, specific inhibitors of a group of SH-proteinases, but it was not inhibited by pepstatin A or EDTA, which inhibit some other barley proteinases. It is thus very likely that in a whole grain, at least the bulk of the bound β-amylase is released by the proteolytic action of one or several SH-proteinases. When the bound β-amylase was released by papain, its molecular weight was about 5000 daltons smaller than that of β-amylase released by dithiothreitol. This indicates that the release is due to removal of a sequence of β-amylase itself. A similar decrease in size took place during seedling growth. Bound β-amylase showed some activity against native starch and it hydrolyzed maltotetraose at a rate that was about 70% of the rate the same amount of bound β-amylase gave after release. Bound β-amylase is thus not inactive and it is likely that the slower rate of hydrolysis is due to steric hindrances which prevent substrates from reaching the active site.     

Crosslinked potato starch as an affinity adsorbent for bacterial α-amylase

Crosslinked potato starch was prepared as an affinity adsorbent for bacterial α-amylase. To this end, reaction parameters for crosslinking in an ethanol/water solvent were investigated. The degree of crosslinking, and consequently the suitability of crosslinked starch as an adsorbent for α-amylase, changed by altering these parameters. An increase in the degree of crosslinking of the adsorbent caused lower affinity for bacterial α-amylase which resulted in an unfavourable decrease in adsorption capacity and a favourable decrease in the degradation of the adsorbent by the enzyme. 1 g of a suitable adsorbent for bacterial α-amylase, prepared with an epichlorohydrin/glucose monomer ratio of 0·65 (starch concentration 150 mg/ml, ethanol/water ratio 2·0, sodium hydroxide/epichlorohydrin ratio 1·0), can adsorb 9·8 mg of an α-amylase from B. licheniformis at 4°C in 20 h.The equilibrium constant between bound and unbound α-amylase is dependent on the temperature. An effective desorption was possible by a shift to higher temperatures. Degradation values smaller than 0·1% were measured after an incubation of 1 h at 70°C in a desorption buffer with 20% glycerol.It was concluded that coulombic interactions and hydrogen bonds are of no or little importance in enzyme adsorption. Van der Waals forces, which are responsible for the large temperature effect, are the main forces in the interaction between α-amylase and crosslinked starch.     

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F₁) as the triphosphate, either in the absence or presence of Mg²⁺, label covalently bound is not hydrolysed.

2. In the presence of Mg²⁺ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits.

3. After successive photolabelling of F₁ with 8-azido-ATP (no Mg²⁺) and 8-azido-ADP (with Mg²⁺) 4 mol label is bound to F₁, 2 mol to the and 2 mol to the β subunits.

4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F₁ previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits.

5. F₁ that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F₁.

6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F₁.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

An efficient purification process for sweet potato beta-amylase by affinity precipitation with alginate

β-amylases are used in production of maltose syrup. It is shown that sweet potato β-amylase can be purified by affinity precipitation with alginate with 80% activity yield and 44 fold purification. SDS-PAGE of the purified protein showed a single band and a subunit weight of 50 kDa. Preliminary data with soybean and barley enzymes indicate that this may be a general method for purification of β-amylases.     

A Slow ATP-induced Conformational Change Limits the Rate of DNA Binding but Not the Rate of �� Clamp Binding by the Escherichia coli �� Complex Clamp Loader

In Escherichia coli, the γ complex clamp loader loads the β-sliding clamp onto DNA. The β clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, γ complex binds β and DNA to form a ternary complex. Binding to primed template DNA triggers γ complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of γ complex binding β and DNA. A fluorescence intensity-based β binding assay was developed in which the fluorescence of pyrene covalently attached to β increases when bound by γ complex. Using this assay, an association rate constant of 2.3 × 10⁷ m⁻¹ s⁻¹ for γ complex binding β was determined. The rate of β binding was the same in experiments in which γ complex was preincubated with ATP before adding β or added directly to β and ATP. In contrast, when γ complex is preincubated with ATP, DNA binding is faster than when γ complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote β binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for γ complex to bind β before DNA during the clamp loading reaction cycle.     

Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2–GDPNP–tRNA complex.     

Interaction of sialosyl cholesterol with the cell surface of rat astrocytes and its biological activities

The interaction between sialosyl cholesterol (- or neuraminyl cholesterol, - or β-SC) and the plasma membrane of astrocytes was investigated by the use of ¹⁴C-labeled - or β-SC. Both - and β-SC were dose-dependently and time-dependently bound to rat astrocytes. The Scatchard plot analyses showed that rat astrocytes bound apparently 9.69 × 10⁹ molecules of both -SC/cell (apparent K_d = 2.29 × 10⁻⁵ M) and β-SC/cell (apparent K_d = 5.39 × 10⁻⁵ M) at 37°C. Both the binding of -SC to astrocytes and the subsequent inhibition of DNA synthesis were decreased at the low temperature (4°C), and also suppressed by serum proteins including albumin. One molecule of bovine serum albumin (BSA) bound 2.3 molecules of -SC with the slightly lower K_d-value (8.03 × 10⁻⁶ M) than that for the binding site on astrocytes. BSA not only suppressed the -SC-binding to astrocytes but also increased its release from the cells to the culture media. Gangliosides such as GM1 and GM3 unaffected the -SC-binding, promoted the small release of -SC from the cell surface, and inhibited the morphological changes of astrocytes induced by -SC. The mechanism of -SC-binding to cultured astrocytes with reference to the effects of serum or gangliosides is discussed.