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K. D. Schwenke - One of the best experts on this subject based on the ideXlab platform.
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Physico-chemical characterization of Legumin-T from faba bean (Vicia faba L.)
International journal of peptide and protein research, 2009Co-Authors: K. D. Schwenke, Ralf Mothes, Antonia Staatz, Arndt Seifert, Herbert DautzenbergAbstract:Legumin-T, the high-molecular mass product of limited tryptic hydrolysis of faba bean Legumin, was investigated using hydrodynamic methods, static light scattering, fluorescence and ultraviolet spectroscopy. The following physico-chemical parameters were determined in a high-ionic strength buffer system: molecular mass, 2.4 × 105 g/mol; sedimentation coefficient, s310= 10.8 × 10−13si; diffusion coefficient, D020= 4.1 × 10−7 cm2 s−1; intrinsic viscosity, [n] = 3.51 mL/g; partial specific volume, v∣⃝= O.719 mL/g; frictional ratio, f/fo= 1.22; shape factor, β= 2.17 × 106. Conformational changes during the formation of Legumin-T can be deduced from the fluorescence emission and UV spectra. © Munksgaard 1996
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Comparative studies on thermodynamic characteristics of pea Legumin and Legumin-T thermal denaturation.
International Journal of Biological Macromolecules, 2001Co-Authors: G.o. Kozhevnikov, E. E. Braudo, A. N. Danilenko, K. D. SchwenkeAbstract:Characteristics of thermal denaturation of pea Legumin and a product of its limited proteolysis with trypsin - Legumin-T, in a wide range of NaCl concentrations have bean measured by means of differential scanning microcalorimetry. By the increase of NaCl concentration, the number of cooperative units (domains) increases from 1 per one polypeptide chain to 2 for Legumin and 1.8 for Legumin-T. Deconvolution of denaturation peaks have revealed up to three peaks, which were ascribed to the dissociation of protein macromolecules to subunits and the unfolding of alpha- and beta-polypeptide chains. The analysis of experimental data based on some assumptions showed that the splitting of C-termini of alpha-chains, which are not constituents of cooperative domains, in the course of limited trypsinolysis results in destabilization of the quaternary structure of Legumin and loosening of alpha-chains, as well as decrease of the temperatures of their maximum stability.
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Limited tryptic hydrolysis of pea Legumin: molecular mass and conformational stability of Legumin-T.
International journal of biological macromolecules, 2001Co-Authors: K. D. Schwenke, Steffi Dudek, Thorsten Henning, Herbert Dautzenberg, Anatolij N Danilenko, Georgij O Kozhevnikov, Evgenij E BraudoAbstract:The investigation of hydrodynamic and thermodynamic properties and the determination of the molecular mass of Legumin-T, the product of limited tryptic hydrolysis of the 11-S-globulin from pea seeds, was carried out to ascertain the structural relationship to globulin-T's from other Legumin-like proteins. The obtained Legumin-T preparation has a molecular mass M(W)=260+/-10 kDa and M(S,D)=270+/-20 kDa. The secondary structure of Legumin-T is characterised by a high percentage of beta-sheet conformation, comparable to that of native Legumin and a reduced percentage of helical conformation. The conformational stability of Legumin-T evaluated by equilibrium unfolding in the presence of guanidinium chloride was only slightly reduced in comparison to the native Legumin, whereas the calorimetrically determined denaturation enthalpy and Gibbs energy of denaturation were found to be increased for Legumin-T. These physicochemical properties are very similar to those of faba bean Legumin-T.
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Acetylation of faba bean Legumin: conformational changes and aggregation
Journal of the Science of Food and Agriculture, 2000Co-Authors: K. D. Schwenke, Constanze Knopfe, Arndt Seifert, Eckhard Görnitz, Dietrich ZirwerAbstract:The effect of progressive acetylation upon the conformation of the 11S globulin Legumin from faba bean has been studied using chemical analysis, UV, fluorescence and CD spectroscopy, viscometry and analytical ultracentrifugation. The modification did not induce complete dissociation of the oligomeric protein. Only 30% of the protein was found to be a dissociated 3S subunit after excessive acetylation, whereas 70% was a dimeric Legumin aggregate with a molecular mass of about 700 kDa. The aggregation of the highly modified Legumin in high-ionic-strength buffer solution leads to soluble higher Legumin oligomers. The acetylation resulted in a moderate molecular expansion of Legumin due to a changed tertiary structure, whereas the far-UV circular dichroism spectra did not provide definitive evidence of a decrease in domain-stabilizing β-sheet conformations in their secondary structure. © 2000 Society of Chemical Industry
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Interfacial behaviour of succinylated faba bean Legumin at low ionic strength
Nahrung Food, 1999Co-Authors: J.-p. Krause, R. Wüstneck, K. D. SchwenkeAbstract:Changes in the tensio-active properties of the main storage protein from faba beans (Legumin) after succinylation were studied at a low salt concentration. Surface tension, surface dilatational properties of monolayers and emulsifying activity were measured at a ionic strength of = = 0.02. The results were compared with those at a high ionic strength of = = 0.3. Parameters of the Gibbs' adsorption isotherm indicate that the most surface-active derivatives are Legumins with a moderate degree of succinylation (34% and 65%). The equilibrium surface pressure, Π e , inreased from 18.47 (native Legumin) to 20.72 mN/m (65% succinylation). The critical association concentration, CAC, i.e., the subphase concentration at which the plateau of Π e was reached, decreased from 15.9. 10 -6 to 7.12. 10 -6 g/ml after 34% succinylation. The film forming properties differed from the adsorption behaviour. Only monolayers of the 65% succinylated Legumin exhibited viscoelastic behaviour. By contrast, the emulsifying activity, EAI, reached the highest values for the 65% and 95% succinylated Legumins. Low salt concentrations favour the adsorption of the native Legumin and reduce the surface activity of succinylated Legumin. Monolayer formation and especially the ability to form elastic networks seems to be diminished by the repulsive interaction of like-charged molecules. The emulsifying properties of the higher succinylated Legumins are not influenced by the ionic strength whereas those of the native and low succinylated Legumin are distinctly lower at 1 = 0.02. This result points to different adsorption and stabilizing processes during emulsion formation.
Helmut Bäumlein - One of the best experts on this subject based on the ideXlab platform.
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Sequence peculiarity of gnetalean Legumin-like seed storage proteins
Journal of Molecular Evolution, 1998Co-Authors: Andrei D. Shutov, H. Braun, Yu.v. Chesnokov, Ch. Horstmann, I. A. Kakhovskaya, Helmut BäumleinAbstract:The development of seeds as a specialized organ for the nutrition, protection, and dispersal of the next generation was an important step in the evolution of land plants. Seed maturation is accompanied by massive synthesis of storage compounds such as proteins, starch, and lipids. To study the processes of seed storage protein evolution we have partially sequenced storage proteins from maturing seeds of representatives from the gymnosperm genera Gnetum, Ephedra, and Welwitschia—morphologically diverse and unusual taxa that are grouped in most formal systems into the common order Gnetales. Based on partial N-terminal amino acid sequences, oligonucleotide primers were derived and used for PCR amplification and cloning of the corresponding cDNAs. We also describe the structure of the nuclear gene for Legumin of Welwitschia mirabilis. This first gnetalean nuclear gene structure contains introns in only two of the four conserved positions previously characterized in other spermatophyte Legumin genes. The distinct phylogenetic status of the gnetalean taxa is also reflected in a sequence peculiarity of their Legumin genes. A comparative analysis of exon/intron sequences leads to the hypothesis that Legumin genes from Gnetales belong to a monophyletic evolutionary branch clearly distinct from that of Legumin genes of extant Ginkgoales and Coniferales as well as from all angiosperms.
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Biphasic expression of a Vicia faba Legumin B gene in developing seeds of transgenic tobacco
Journal of Plant Physiology, 1997Co-Authors: Reinhard Panitz, Helmut Bäumlein, Renate Manteuffel, Ulrich WobusAbstract:Summary We analysed the spatial and temporal expression of the Vicia faba Legumin gene LeB4 in developing seeds of transgenic tobacco plants by means of in situ hybridization and immunohistostaining. The results indicate that the expression of the LeB4 gene is regulated in embryo and endosperm in a biphasic manner. Legumin accumulation starts during early seed development at low amounts in single cells of the integument. Accumulation proceeds successively in the proembryo, suspensor and endosperm reaching maximum levels in all cells of these organs around 10 to 12 DAP. With the beginning of the heart stage (around 13 DAP) Legumin reserves disappear completely from all parts of the seed. The described early phase of Legumin accumulation occurs in embryo and endosperm still before the onset of the cell expansion phase in a period of continuous mitotic activity. A second expression phase starts in embryo and endosperm around 18 DAP, as the embryo enters late torpedo stage. Within the embryo the accumulation is restricted to the parenchyma cells of cotyledons and hypocotyl, while the embryonic root and provascular tissue do not accumulate LeB4 protein. In addition, a low percentage ( V. faba for the Legumin and vicilin genes during early embryogenesis (Panitz et al., 1995), but differs from that of tobacco 12S globulin genes, which are expressed in the embryo not before the late heart stage.
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Evolution of seed storage protein genes: Legumin genes of Ginkgo biloba
Journal of molecular evolution, 1995Co-Authors: K P Häger, H. Braun, B. Müller, A. Czihal, Helmut BäumleinAbstract:Legumin-like seed storage proteins have been intensively studied in crop plants. However, little is known about the molecular evolution of these proteins and their genes and it was assumed that they originated from an ancestral gene that already existed at the beginning of angiosperm evolution. We have evidence for the ubiquitous occurrence of homologous proteins in gymnosperms as well. We have characterized the major seed storage globulin from Ginkgo biloba by amino acid sequencing, which reveals clear homology to Legumin-like proteins from angiosperms. The Ginkgo Legumin is encoded by a gene family; we describe two of its members. The promoter regions contain sequence motifs which are known to function as regulatory elements involved in seed-specific expression of angiosperm Legumins, although the tissues concerned are different in gymnosperms and angiosperms. The Ginkgo Legumin gene structure is divergent from that of angiosperms and suggests that the evolution of Legumin genes implicated loss of introns. From our data and from functional approaches recently described it becomes obvious that the posttranslational processing site of Legumin precursors is less conserved than hitherto assumed. Finally, we present a phylogenetic analysis of Legumin encoding sequences and discuss their utility as molecular markers for the reconstruction of seed plant evolution.
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Legumin-like and vicilin-like seed storage proteins: Evidence for a common single-domain ancestral gene
Journal of molecular evolution, 1995Co-Authors: Andrei D. Shutov, H. Braun, I. A. Kakhovskaya, Helmut Bäumlein, Klaus MüntzAbstract:Legumin-like 11S and vicilin-like 7S globulins are the main storage proteins of most angiosperms and gymnosperms. The subunits of the hexameric Legumin are synthesized as a precursor comprising a N-terminal acidic α- and a C-terminal basic β-chain. The trimeric vicilin molecule consists of subunits composed of two symmetrical N- and C-terminal structural domains.
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The Legumin gene family: a reconstructed Vicia faba Legumin gene encoding a high-molecular-weight subunit is related to type B genes.
Plant molecular biology, 1994Co-Authors: Ute Heim, Helmut Bäumlein, Ulrich WobusAbstract:Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large Legumin subunit, designated LelB3. The length difference to the well-characterized major Legumin subunits is caused by an extended glutamin/glutamic acid-rich region encoded by the C-terminal part of the α chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type Legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342–350) and limited own data.
Yu. N. Polikarpov - One of the best experts on this subject based on the ideXlab platform.
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calorimetric investigation of the thermodynamic basis of the effect of maltodextrins on the foaming ability of Legumin in the presence of small molecule surfactant
Food Hydrocolloids, 2005Co-Authors: M. S. Anokhina, Maria G Semenova, Larisa E Belyakova, Michael M Ilin, Yu. N. PolikarpovAbstract:Abstract In this investigation, using a combination of mixing and differential scanning calorimetry, the relationships have been clarified between thermodynamics of the intermolecular interactions, the conformational stability of the protein globule and foaming ability of the protein in the ternary mixtures: neutral polysaccharide (maltodextrins with different dextrose equivalent (DE=2, 6, 10))+globular protein-Legumin (11 S globulin from broad beans)+water-dispersible ionic small-molecule surfactant that represents generally the mixture of the esters of stearic and palmitic acids with a citric acid (CITREM). In addition, it was also shown that the found peculiarities of the intermolecular interactions in the ternary mixtures are also determined by the aggregation states (monomolecular or micellar) of the small-molecule surfactant in aqueous medium. The combined calorimetric data suggest that the role of the unadsorbed maltodextrin in the foaming ability of its mixture with Legumin in the presence of CITREM is principally governed, on the one hand, by the marked addition of hydrophilicity from both maltodextrins and CITREM to the protein as a result of the formation of the ternary complex, maltodextrin+CITREM+Legumin, and, on the other hand, by the partial protein unfolding of the protein in the ternary (maltodextrin+CITREM+Legumin) complex.
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thermodynamic and functional properties of Legumin 11s globulin from vicia faba in the presence of small molecule surfactants effect of temperature and ph
Journal of Colloid and Interface Science, 2004Co-Authors: Michael M Ilin, Maria G Semenova, Larisa E Belyakova, Anna S Antipova, Yu. N. PolikarpovAbstract:We report on the effect of a set of water-dispersible small-molecule surfactants (the main and the longest-hydrocarbon components of which are a citric acid ester of monostearate, a sodium salt of stearol-lactoyl lactic acid, and a polyglycerol ester of stearic acid) on molecular, thermodynamic, and functional properties of the major storage protein of broad beans (Vicia faba) Legumin in different molecular states (native, heated, and acid-denatured). The interaction between Legumin and the surfactants has been characterized by a combination of thermodynamic methods, namely, mixing calorimetry and multiangle laser static and dynamic light scattering. It was found that hydrogen bonds, electrostatic interactions, and hydrophobic contacts provided a basis for the interactions between the surfactants and both the native and the denatured protein in aqueous medium. Intensive association of the protein molecules in a bulk aqueous medium in the presence of the surfactants was revealed by static and dynamic laser light scattering. In consequence of this, both the surface activity and the gel-forming ability of Legumin increased markedly, which has been shown by tensiometry, estimation of protein foaming capacity, and steady-state viscometry. A likely molecular mechanism underlying the effects of small-molecule surfactants on Legumin structure-forming properties at the interface and in a bulk aqueous medium is discussed.
Jacques Guéguen - One of the best experts on this subject based on the ideXlab platform.
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Adaptive immune responses of Legumin nanoparticles.
Journal of drug targeting, 2002Co-Authors: T. Mirshahi, Jacques Guéguen, Juan M. Irache, C. Nicolas, M. Mirshahi, Jean-pierre Faure, C. Hecquet, A. M. OrecchioniAbstract:Legumin is one of the main storage proteins in the pea seeds (Pisum sativum L.) and the molecules of this protein have the capacity of binding together to form nanoparticles after aggregation and chemical cross-linkage with glutaraldehyde. The aim of this work was to study the adaptive immune response of Legumin nanoparticles in rats. Following intradermal immunisation with the native protein Legumin and Legumin nanoparticles of about 250 nm, the humoral and cell-mediated immune responses were analysed in rats. The humoral responses against Legumin and Legumin nanoparticles were examined by western blot and ELISA analysis. Both techniques clearly showed that sera from rats immunised with Legumin strongly expressed antibodies against this protein. On the contrary, serum samples from rats inoculated with Legumin nanoparticles did not contain detectable amounts of antibodies. These results may be explained by a reduction on the antigenic epitopes of the protein induced by the glutaraldehyde used during the c...
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Functional properties of the α-chain of a major Legumin subunit of Pisum sativum Legumin
Nahrung-food, 1998Co-Authors: Steffi Dudek, Jacques Guéguen, J.‐p. Krause, K. D. SchwenkeAbstract:The constituting polypeptides of a heavy pea Legumin subunit were separated. The interfacial and emulsifying properties of the α-chains were studied and compared with those of the respective subunit and the native Legumin molecules. The α-polypeptides show modified interface behaviour as compared to the Legumin. Faster diffusion and higher adsorption barriers during the phase of rearrangement, higher interface coverage in the equilibrium state and improved emulsifying properties were measured.
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Optimization and in vitro stability of Legumin nanoparticles obtained by a coacervation method
International Journal of Pharmaceutics, 1995Co-Authors: Juan M. Irache, Jacques Guéguen, Laurence Bergougnoux, Isabel Ezpeleta, A. M. OrecchioniAbstract:Legumin (a storage protein from Pisum sativum L.) nanoparticles of about 250 nm were prepared by means of a pH-coacervation method and chemical cross-linking with glutaraldehyde. Non-stabilized nanoparticles or coacervates were obtained by mixing an aqueous solution of Legumin with a buffer. The influence of some experimental parameters (pH, surfactant content and ionic strength) on the size and yield of coacervates was studied. These systems were then treated with glutaraldehyde. After 2 h, a concentration of at least 0.05 mg glutaraldehyde/mg Legumin was necessary to stabilize these colloidal systems. No significant differences in size and percentage of yield were obtained between Legumin nanoparticles cross-linked with different glutaraldehyde concentrations. Legumin nanoparticles were quite stable in pH conditions close to neutrality. On the other hand, nanoparticles stored under acidic conditions (pH 5.5; 37°C) showed a rapid degradation and this fact may be of interest for pharmaceutical applications like cutaneous or transdermal administration of drugs.
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Selected physico-chemical properties of succinylated Legumin from pea (Pisum sativum L.).
Die Nahrung, 1993Co-Authors: K. D. Schwenke, Ralf Mothes, Barbara Raab, H. Rawel, Jacques GuéguenAbstract:Selected physico-chemical properties of pea Legumin before and after succinylation have been investigated using isoelectric focusing, PAGE, SDS-PAGE, hydrophobicity measurements, SE-HPLC and RP-HPLC. Exhaustive succinylation shifted the I.P. of Legumin from 4.75 to 3.5. The stepwise dissociation of Legumin by increasing succinylation has been confirmed both by means of PAGE in a nondenaturing system, and by SE-HPLC. The results of SDS-PAGE provided evidence for the exposure of α-polypeptide chains in the native Legumin. High succinylation resulted in a decrease of the surface hydrophobicity (S0) measured by both fluorescence probes (cis-parinaric acid and anilino-naphtalene sulfonic acid). RP-HPLC gave a response both to conformational changes and the introduced succinyl residues. Ausgewahlte physiko-chemische Eigenschaften von succinyliertem Legumin der Erbse (Pisum sativum L.) Ausgewahlte physiko-chemische Eigenschaften des ErbsenLegumins vor und nach Succinylierung wurden mit Hilfe von isoelektrischer Fokussierung, PAGE, SDS-PAGE, Hydrophobizitats-Messung, SE-HPLC und RP-HPLC untersucht. Durch erschopfende Succinylierung wird der isoelektrische Punkt des Legumins von 4.75 nach 3.5 verschoben. Die bereits fruher nachgewiesene stufenweise Dissoziation des modifizierten Legumins wird durch Untersuchungen mittels PAGE und SE-HPLC bestatigt, wahrend die Ergebnisse der SDS PAGE die Exponierung der α-Ketten im Leguminmolekul belegen. Die mittels Fluoreszenzsonden-Techniken unter Einsatz von cis-Parinarsaure und 8-Anilinonaphtalen-1-sulfonsaure gemessenen relativen Hydrophobizitatswerte (S0) nehmen bei hohen Succinylierungsgraden signifikant ab. Die Ergebnisse der RP-HPLC-Analyse spiegeln sowohl durch die Modifizierung eingetretene Konformationsanderungen des Legumins als auch den Einflus der eingefuhrten Succinylreste wider.
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Action of transglutaminase on an 11 S seed protein (pea Legumin): influence of the substrate conformation
Journal of Agricultural and Food Chemistry, 1992Co-Authors: Colette Larré, Z. Marek. Kedzior, Michel. Chenu, Gerard. Viroben, Jacques GuéguenAbstract:The reactivity of globular seed protein (pea Legumin) as substrate for transglutaminase is studied as a function of its conformation. Citraconylation of Legumin induced, depending on the level of modification, a total or partial dissociation of the protein into subunits. The acylated Legumins were used as substrates for guinea pig liver transglutaminase, and the transfer and hydrolytic activities were investigated
Ulrich Wobus - One of the best experts on this subject based on the ideXlab platform.
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Biphasic expression of a Vicia faba Legumin B gene in developing seeds of transgenic tobacco
Journal of Plant Physiology, 1997Co-Authors: Reinhard Panitz, Helmut Bäumlein, Renate Manteuffel, Ulrich WobusAbstract:Summary We analysed the spatial and temporal expression of the Vicia faba Legumin gene LeB4 in developing seeds of transgenic tobacco plants by means of in situ hybridization and immunohistostaining. The results indicate that the expression of the LeB4 gene is regulated in embryo and endosperm in a biphasic manner. Legumin accumulation starts during early seed development at low amounts in single cells of the integument. Accumulation proceeds successively in the proembryo, suspensor and endosperm reaching maximum levels in all cells of these organs around 10 to 12 DAP. With the beginning of the heart stage (around 13 DAP) Legumin reserves disappear completely from all parts of the seed. The described early phase of Legumin accumulation occurs in embryo and endosperm still before the onset of the cell expansion phase in a period of continuous mitotic activity. A second expression phase starts in embryo and endosperm around 18 DAP, as the embryo enters late torpedo stage. Within the embryo the accumulation is restricted to the parenchyma cells of cotyledons and hypocotyl, while the embryonic root and provascular tissue do not accumulate LeB4 protein. In addition, a low percentage ( V. faba for the Legumin and vicilin genes during early embryogenesis (Panitz et al., 1995), but differs from that of tobacco 12S globulin genes, which are expressed in the embryo not before the late heart stage.
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The Legumin gene family: a reconstructed Vicia faba Legumin gene encoding a high-molecular-weight subunit is related to type B genes.
Plant molecular biology, 1994Co-Authors: Ute Heim, Helmut Bäumlein, Ulrich WobusAbstract:Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large Legumin subunit, designated LelB3. The length difference to the well-characterized major Legumin subunits is caused by an extended glutamin/glutamic acid-rich region encoded by the C-terminal part of the α chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type Legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342–350) and limited own data.