Properties and synthesis de novo of auxin-induced α-amylase in pea cotyledons

Analysis of starch-degrading enzymes in a crude extract of detached cotyledons of Pisum sativum L. by polyacrylamide gel electrophoresis (PAGE) demonstrated the presence of one band of -amylase (EC 3.2.1.1) activity. The activity of only this amylase was promoted in cotyledons incubated with 2,4-dichlorophenoxyacetic acid (2,4-D). The auxin-induced -amylase from pea cotyledons was purified to homogeneity, as judged by the criterion of a single band after PAGE. The relative molecular mass (M_r), estimated by gel filtration, was approx. 42 000 and the enzyme contained no carbohydrate moiety. Sodium dodecylsulfate-PAGE yielded a single band that corresponded to an M_r of 41 000. The isoelectric point was 5.85 and the aminoacid composition was similar to that of -amylase from other plants. When [³H]leucine was fed to detached dry cotyledons prior to incubation, the radioactivity in -amylase from cotyledons incubated in the presence of 2,4-D was found to be approx. 10-fold higher than that from cotyledons incubated in distilled water. When -amylase from cotyledons incubated with ²H₂O that contained 2,4-D and the tritiated amylase were centrifuged together in a CsCl density gradient, the peak of enzymatic activity of deuterated -amylase was shifted to a denser fraction than the peak of radioactivity of the tritiated enzyme. These results show that auxin-induced -amylase in pea cotyledons is synthesized de novo.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - pI isoelectric point - SDS sodium dodecyl sulfate We are very grateful to Mr. Kazuo Itoh and Mrs. Matsumi Doe for carrying out the analysis of amino-acid composition.     

Structure of α-α-Hairpins with Short Connections in Globular Proteins

The analysis of conformations of more than 100 --hairpins with closely packed helical segments and connections up to four amino acid residues in length was carried out. Five types of the connections were revealed, and their and values on the Ramachandran map were found. Each type of --hairpins was shown to have a unique sequence pattern for hydrophobic and hydrophilic residues.     

The α-helix dipole in membranes: a new gating mechanism for ion channels

Electric dipoles placed side by side attract each other if antiparallel and repel each other if parallel. The hydrophobic -helical sections of proteins that span membranes are known to possess large electric dipole moments. The first part of the paper consists of a calculation of the interaction energies between such helices including screening effects. Interaction energies remain comparable with a typical thermal energy of KT up to separations of order 20 Å. In addition it is shown that, due solely to its dipole moment, an -helix which completely spans the membrane has an energy up to 5 KT lower than one which terminates within the membrane width. The second part of the paper describes the electrical interaction of the charge structure of a membrane channel and the protein helices that surround the pore. The gating charge transfer that is measured when a voltage sensitive ion channel switches, means that the dipole moment of the ion channel changes. This in turn results in a change in the radial forces that act between the pore and the -helices that surround it. A change in these radial forces which tend to open or to close the pore constitutes an electrically silent gating mechanism that must necessarily act subsequent to the gating charge transfer. The gating mechanism could consist of the radial translation of the neighbouring proteins or in their axial rotation under the influence of the torque that would act on a pair of approximately equidistant but oppositely directed -helices. An attempt to calculate the interaction energy of a typical pore and a single -helix spanning the membrane results in an energy of many times KT.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae

Recombinant barley -amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified -amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k _cat/K _m was 2.7 × 10² mM^–1.s^–1, consistent with those of -amylases from plants and other sources.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Role of interferon γ and tumour necrosis factor α in monocyte-mediated cytostasis and cytotoxicity against a human histiocytic lymphoma cell line

Summary In view of cellular adoptive immunotherapy we have studied monocyte-mediated cytostasis and cytotoxicity against U 937 cells, a human histiocytic lymphoma cell line. Highly purified human monocytes and monocytederived macrophages were activated with interferon (IFN) or tumour necrosis factor (TNF) to antileukemic immune effector cells. Antileukemic activity of human monocytes was dependent on monocyte differentiation into macrophages and on a dose- and time-dependent activation with IFN or TNF. Maximum cytostasis of 97.0±0.7% (mean ± SEM) (conventional [³H]dT uptake assay) and 81.9±5.3% cytotoxicity (modified MTT assay) of U 937 cells was obtained by monocytes activated with 100 U/ml IFN for at least 24 h at an effector-to-target-cell ratio of 10. U 937 cells premodified with IFN showed an increase in susceptibility to monocyte-mediated cytotoxicity. U 937 cells premodified with TNF were almost resistant to monocyte-mediated cytotoxicity while activated monocytes maintained their cytotoxic potential. These data show that IFN and TNF are potent activators of monocyte-mediated cytotoxicity. Furthermore, IFN and TNF might be involved in the regulation of the susceptibility of leukemic cells to lysis by interactions with monocytes or macrophages.     

Process optimization for the production of sugar for the bioethanol industry from Tapioca,a non-conventional source of starch

A commercial preparation of -amylase, Biotempase, obtained from Biocon India Pvt. Ltd., and crude glucoamylase produced from Aspergillus sp. NA21 were used to hydrolyse tapioca powder, a non-conventional starchy substrate. Among various concentrations of starch (15–35%, dry weight/volume) tried for maximum liquefaction; slurry made with 25% substrate concentration proved optimal. An economical process of liquefaction was carried out using steam under pressure (0.2–0.3 bar, 104–105 °C) to liquefy a 25% slurry in just 45 min, contrary to a slower process carried out at 95 °C in a water bath. For liquefaction of starch a pH of 5.0 proved to be optimum. The dose of Biotempase as prescribed by the supplier could be reduced by 33% achieving the same degree of liquefaction, by addition of CaCl₂ to the starch slurry at the concentration of 120 mg/l. The conditions for the saccharification of liquefied starch were optimized to be 60 °C and pH 5.0, producing 90% saccharification in 24 h. Supplementation of divalent ions Ca²⁺, Mg²⁺ and Zn²⁺ in the process of saccharification showed no effect. Finally glucose was found to be the main hydrolysis product in the saccharification of tapioca starch.     

Synthesis of α-galacto-oligosaccharides by a cloned α-galactosidase from Bifidobacterium adolescentis

A genomic library of Bifidobacterium adolescentis was constructed in Escherichia coli and a gene encoding an -galactosidase was isolated. The identified open reading frame showed high similarity and identity with bacterial -galactosidases, which belong to Family 36 of the glycosyl hydrolases. For the purification of the enzyme from the medium a single chromatography step was sufficient. The yield of the recombinant enzyme was 100 times higher than from B. adolescentis itself. In addition to hydrolytic activity the -galactosidase showed transglycosylation activity and can be used for the production of -galacto-oligosaccharides.     

Syntheses of 1-O-benzyl-α-glucoside and 1-O-benzyl-α-maltoside by transglycosylation of α- amylase from soluble starch in aqueous solution

Glycosides, 1-O-benzyl--glucoside (BG) and 1- O-benzyl--maltoside (BM), were synthesized from soluble starch and benzyl alcohol by transglycosylation with an -amylase in a water system. BG was mostly obtained in a reaction mixture of pH 5.0, while BM was synthesized in pH 8.0. The synthesized glycosides had -configuration linkage between sugar and benzyl alcohol. The BG was rapidly hydrolyzed to benzyl alcohol and glucose by -glucosidase. The BM was hydrolyzed to BG and glucose below pH 5.0 by the -amylase used for its synthesis but it was not hydrolyzed above pH 8.0.     

Purification and characterization of GDP L -Fuc: N-acetyl β D -glucosaminide α1→6fucosyltransferase from human blood platelets

-6 L -Fucosyltransferase (1,6FucT; EC 2.4.1.68) from human platelets, the enzyme that is released into serum during coagulation of blood, was purified 100,000-fold. The purification required three sequential chromatographic steps: chromatofocusing, affinity column chromatography on GnGn-Gp(asialo-aglacto-transferrin glycopeptide)-CH-Sepharose, and gel filtration of Sephadex G-200. The final preparation contained a protein that migrated as a single discrete band Mr of 58,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, and as a single enzymatically active peak Mr of 58,000 in gel filtration. Although the purified enzyme utilized the biantennary GnGn-Gp as substrate, it was twice as active with the triantennary oligosaccharide when the Man 1,3 antenna was substituted with GlcNac1,4. On the other hand the tetraantennary oligosaccharide was not a preferred substrate. The Km values for the substrate asialo-agalactotransferrin-glycopeptide, and GDP L -fucose were 29 and 28 M, respectively. The optimum pH of the enzyme was 6.0. The activity of 1,6FucT was abolished in the presence of -mercaptoethanol. Divalent cations such as Mg2+ and Ca2+ activated, but Cu2+, Zn2+ and Ni2+ strongly inhibited the activity.     

Crystal structure of the pig pancreatic alpha-amylase complexed with malto-oligosaccharides

The structural X-ray map of a pig pancreatic -amylase crystal soaked (and flash-frozen) with a maltopentaose substrate showed a pattern of electron density corresponding to the binding of oligosaccharides at the active site and at three surface binding sites. The electron density region observed at the active site, filling subsites –3 through –1, was interpreted in terms of the process of enzyme-catalyzed hydrolysis undergone by maltopentaose. Because the expected conformational changes in the flexible loop that constitutes the surface edge of the active site were not observed, the movement of the loop may depend on aglycone site being filled. The crystal structure was refined at 2.01 å resolution to an R factor of 17.0% (R _free factor of 19.8%). The final model consists of 3910 protein atoms, one calcium ion, two chloride ions, 103 oligosaccharide atoms, 761 atoms of water molecules, and 23 ethylene glycol atoms.     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

High maltose-producing amylolytic system of a Streptomyces sp.

The -amylases of Streptomyces sp. IMD 2679 produced yields of 79% (w/w) maltose from starch by reactions other than simple hydrolysis. The enzymes also had a low affinity (Km 8.0–8.2 mm) for maltotriose and each possessed a temperature maximum in the range 60–65°C.     

Resistance of α-globulin fromSesamum indicum L. to proteases in relationship to its structure

-Globulin, the high-molecular-weight protein fraction fromSesamum indicum L., was hydrolyzed to low-molecular-weight protein and peptides by pepsin, while its resistance to hydrolysis by group-specific enzymes, trypsin or -chymotrypsin, was very high. The protein showed definite structural changes after proteolysis, especially after peptic hydrolysis, as evidenced from various biophysical data. The sedimentation velocity pattern of -globulin hydrolyzed by trypsin or -chymotrypsin indicated reduction in the percentage of 11S component, while the pepsinhydrolyzed sample was devoid of any 11S component, indicating the absence of a native protein molecule. The fluorescence emission spectra of the various hydrolyzed -globulin showed a red shift in the fluorescence emission maximum. The red shift was maximum with -globulin hydrolyzed by pepsin and minimum with the trypsin-hydrolyzed sample. The far-ultraviolet-circular dichroic measurements indicated that most of the ordered structure of -globulin was absent after pepsin hydrolysis, while after trypsin and chymotrypsin hydrolysis conformational changes were less.     

Über die Struktur der Pilzkörper des Insektenhirns

Zusammenfassung Die Organisation der -Loben der Pilzkörper im Gehirn von Acheta domesticus L. wird nach licht- und elektronenmikroskopischen Befunden beschrieben. Der säulenartige Faserkomplex des -Lobus besteht aus Fortsätzen von Pilzkörperzellfasern (intrinsischen Fasern, IF) und pilzkörperfremden Fasern (extrinsischen Fasern, EF), die in den Lobus eindringen. Die feinen IF durchziehen den Lobus hauptsächlich parallel zu seiner Längsachse, während die EF zumeist senkrecht zur Längsachse angeordnet sind. Der Lobus erscheint von seiner Peripherie bis zu seinem Zentrum durch IF-Zonen gegliedert. Die Verteilung der EF weist auf eine zusätzliche Ordnung von der Basis zur Spitze des Lobus hin. Zahlreiche polarisierte Synapsen verbinden IF mit EF. Die IF zeigen Vesikelanhäufungen und präsynaptische Apparate besonders in Erweiterungen, die auch in Golgi-Präparaten lichtmikroskopisch zu sehen sind. Es werden zwei EF-Typen unterschieden: 1. Postsynaptische EF (zahlreich) und 2. EF mit prä- und postsynaptischen Kontakten, die nur in einigen Regionen des -Lobus gefunden wurden. Präsynaptische IF konvergieren auf dendritische EF, die Verbindungen mit anderen Teilen des Hirns und des Nervensystems herstellen. Funktionelle Gesichtspunkte werden diskutiert.

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On the structure of the mushroom-bodies in the brain of insectsII. Synaptic connexions in the alpha-lobe of the cricket Acheta domesticus L.

Summary The organization of the -lobes of the corpora pedunculata in the brain of the cricket Acheta domesticus L. has been investigated in the light and electron microscopes. The cylindrical fibre complex is composed of branches of mushroom-body fibres (intrinsic fibres) and extrinsic fibres, which penetrate the -lobe. Intrinsic fibres (IF) run through the -lobe in the same direction, but not strictly parallel to each other or to the axis of the -lobe. Extrinsic fibres (EF) and their fine branches are often arranged perpendicular to the axis of the -lobe. There is some evidence that different IF zones occur in the -lobe when passing from its periphery to its centre. The distribution of EF may reflect a structural order when passing from the base of the lobe to its top. Numerous polarized synapses connect the IF with the EF. The IF show clusters of vesicles and presynaptic figures especially in their blebs, which can be seen in Golgi preparations for light microscopy. Two types of EF are distinguished on the basis of their synaptic junctions: (1) postsynaptic EF (abundant) and (2) EF with pre- and postsynaptic sites (perhaps restricted to some regions of the -lobe). Presynaptic IF converge on EF, which may transfer excitation from the -lobe to different parts of the brain and nervous system.

     

Chemical modification of the α-amylase ofBacillus caldovelox with diethyl pyrocarbonate: Evidence for an essential histidine at the active site

Summary The -amylase ofBacillus caldovelox is inactivated by diethyl pyrocarbonate at pH 6.6 and 20°C by a monomolecular reaction with a second-order rate constant of 41.7 M^–1·min^–1. The rate of inactivation increases with decreasing pH, suggesting participation of an amino acid residue with a pK a of 6.6. The increase in absorbance at 240 nm, unchanged absorbance at 280 nm and reactivation in the presence of hydroxylamine suggest the participation of a histidine residue. Statistical analyses of inactivation suggest that only one histidine residue is essential for activity. Substrate afforded complete protection against inactivation, indicating the involvement of the histidine residue at the active site of the enzyme.     

Lysosomal α-D-mannosidase

-mannosidosis in the human is an autosomal recessive lysosomal storage disease caused by a deficiency of lysosomal -D-mannosidasea actvity. Lysosomal -D-mannosidase is involved in the catabolism of N-linked glycoproteins through the sequential degradation of high-mannose, hybrid and complex oligosaccharides. This review is focused on human, mouse, bovine and feline genes coding for lysosomal -D-mannosidase. In particular the exon-intron structure of the genes, their promoters, and the identification of mutations causing the disease have been examined. The construction, by homologous recombination, of a mouse model of -mannosidosis is reported.