Home About us Contact
|
Home About us Contact | ||
|
|
||
Storage Proteins (storage + protein)
Kinds of Storage Proteins Selected AbstractsVegetative Storage Protein with Trypsin Inhibitor Activity Occurs in Sapindus mukorassi, a Sapindaceae Deciduous TreeJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 4 2009Shi-Biao Liu Abstract A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions: nitrogen storage and defense. [source] Azotobacter vinelandii Metal Storage Protein: "Classical" Inorganic Chemistry Involved in Mo/W Uptake and Release ProcessesCHEMBIOCHEM, Issue 4 2008Jörg Schemberg Dr. Abstract The release of Mo (as molybdate) from the Mo storage protein (MoSto), which is unique among all existing metalloproteins, is strongly influenced by temperature and pH value; other factors (incubation time, protein concentration, degree of purity) have minor, though significant effects. A detailed pH titration at 12,°C revealed that three different steps can be distinguished for the Mo-release process. A proportion of ,15,% at pH 6.8,7.0, an additional 25,% at pH 7.2,7.5 and ca. 50,% (up to 90,% in total) at pH 7.6,7.8. This triphasic process supports the assumption of the presence of different types of molybdenum-oxide-based clusters that exhibit different pH lability. The complete release of Mo was achieved by increasing the temperature to 30,°C and the pH value to >7.5. The Mo-release process does not require ATP; on the contrary, ATP prevents, or at least reduces the degree of metal release, depending on the concentration of the nucleotide. From this point of view, the intracellular ATP concentration is suggested to play,in addition to the pH value,an indirect but crucial role in controlling the extent of Mo release in the cell. The binding of molybdenum to the apoprotein (reconstitution process) was confirmed to be directly dependent on the presence of a nucleotide (preferably ATP) and MgCl2. Maximal reincorporation of Mo required 1 mM ATP, which could partly be replaced by GTP. When the storage protein was purified in the presence of ATP and MgCl2 (1 mM each), the final preparation contained 80 Mo atoms per protein molecule. Maximal metal loading (110,115 atoms/MoSto molecule) was only achieved, if Mo was first completely released from the native protein and subsequently (re-) bound under optimal reconstitution conditions: 1 h incubation at pH 6.5 and 12,°C in the presence of ATP, MgCl2 and excess molybdate. A corresponding tungsten-containing storage protein ("WSto") could not only be synthesized in vivo by growing cells, but could also be constructed in vitro by a metalate,ion exchange procedure by using the isolated MoSto protein. The high W content of the isolated cell-made WSto (,110 atoms/protein molecule) and the relatively low amount of tungstate that was released from the protein under optimal "release conditions", demonstrates that the W-oxide-based clusters are more stable inside the protein cavity than the Mo-oxide analogues, as expected from the corresponding findings in polyoxometalate chemistry. The optimized isolation of the W-loaded protein form allowed us to get single crystals, and to determine the crystal X-ray structure. This proved that the protein contains remarkably different types of polyoxotungstates, the formation of which is templated in an unprecedented process by the different protein pockets. (Angew. Chem. Int. Ed.2007, 46, 2408,2413). [source] Fluctuation of Vegetative Storage Proteins in the Seedlings of Swietenia macrophylla, Analogous to the Seasonal Changes of Those in the Shoot of the Adult TreeJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 3 2007Ya-Qin Han Abstract In order to identify appropriate plant materials for studying the gene expression and biological function of vegetative storage proteins (VSPs) in woody plants, the VSPs in the seedlings of Swietenia macrophylla King were investigated by using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blotting. The seed of S. macrophylla was rich in storage proteins that accumulated in the vacuoles of cotyledon parenchyma cells in appearance of compact spherical grains. The growth and development of S. macrophylla seedlings were characterized by an obvious growth rhythm. The storage proteins in seeds disappeared during seedling growth while VSPs appeared in the stem 2 weeks after seedling leaves matured. Thereafter, the VSPs in the seedling stem almost exhausted during new shoot growth, and when the leaves of new shoot just matured, both the stem beneath the new shoot of seedlings and the stem of new shoot started to accumulate VSPs. Nitrogen application dramatically increased the level of VSPs, but had little influence on the dynamics of VSP consumption and accumulation in seedling stem. Together with these data, the fluctuation of VSPs in seedlings was very similar to that in the branches of the adult trees. In addition, seedlings are easy to be treated due to their small size. Our results suggested that S. macrophylla seedlings were suitable for investigating the biological roles of VSPs and the mechanism of nitrogen storage in trees. [source] Iron regulatory protein-independent regulation of ferritin synthesis by nitrogen monoxideFEBS JOURNAL, Issue 16 2006Marc Mikhael The discovery of iron-responsive elements (IREs), along with the identification of iron regulatory proteins (IRP1, IRP2), has provided a molecular basis for our current understanding of the remarkable post-transcriptional regulation of intracellular iron homeostasis. In iron-depleted conditions, IRPs bind to IREs present in the 5,-UTR of ferritin mRNA and the 3,-UTR of transferrin receptor (TfR) mRNA. Such binding blocks the translation of ferritin, the iron storage protein, and stabilizes TfR mRNA, whereas the opposite scenario develops when iron in the intracellular transit pool is plentiful. Nitrogen monoxide (commonly designated nitric oxide; NO), a gaseous molecule involved in numerous functions, is known to affect cellular iron metabolism via the IRP/IRE system. We previously demonstrated that the oxidized form of NO, NO+, causes IRP2 degradation that is associated with an increase in ferritin synthesis [Kim, S & Ponka, P (2002) Proc Natl Acad Sci USA99, 12214,12219]. Here we report that sodium nitroprusside (SNP), an NO+ donor, causes a dramatic and rapid increase in ferritin synthesis that initially occurs without changes in the RNA-binding activities of IRPs. Moreover, we demonstrate that the translational efficiency of ferritin mRNA is significantly higher in cells treated with SNP compared with those incubated with ferric ammonium citrate, an iron donor. Importantly, we also provide definitive evidence that the iron moiety of SNP is not responsible for such changes. These results indicate that SNP-mediated increase in ferritin synthesis is, in part, due to an IRP-independent and NO+ -dependent post-transcriptional, regulatory mechanism. [source] Identification of a 250 kDa putative microtubule-associated protein as bovine ferritinFEBS JOURNAL, Issue 3 2005Evidence for a ferritin, microtubule interaction We reported previously on the purification and partial characterization of a putative microtubule-associated protein (MAP) from bovine adrenal cortex with an approximate molecular mass of 250 kDa. The protein was expressed ubiquitously in mammalian tissues, and bound to microtubules in vitro and in vivo, but failed to promote tubulin polymerization into microtubules. In the present study, partial amino acid sequencing revealed that the protein shares an identical primary structure with the widely distributed iron storage protein, ferritin. We also found that the putative MAP and ferritin are indistinguishable from each other by electrophoretic mobility, immunological properties and morphological appearance. Moreover, the putative MAP conserves the iron storage and incorporation properties of ferritin, confirming that the two are structurally and functionally the same protein. This fact led us to investigate the interaction of ferritin with microtubules by direct electron microscopic observations. Ferritin was bound to microtubules either singly or in the form of large intermolecular aggregates. We suggest that the formation of intermolecular aggregates contributes to the intracellular stability of ferritin. The interactions between ferritin and microtubules observed in this study, in conjunction with the previous report that the administration of microtubule depolymerizing drugs increases the serum release of ferritin in rats [Ramm GA, Powell LW & Halliday JW (1996) J Gastroenterol Hepatol11, 1072,1078], support the probable role of microtubules in regulating the intracellular concentration and release of ferritin under different physiological circumstances. [source] Molecular characterization of artemin and ferritin from Artemia franciscanaFEBS JOURNAL, Issue 1 2003Tao Chen Embryos of the brine shrimp, Artemia franciscana, exhibit remarkable resistance to physiological stress, which is temporally correlated with the presence of two proteins, one a small heat shock/,-crystallin protein termed p26 and the other called artemin, of unknown function. Artemin was sequenced previously by Edman degradation, and its relationship to ferritin, an iron storage protein, established. The isolation from an Artemia expressed sequence tag library of artemin and ferritin cDNAs extends this work. Artemin cDNA was found to contain an ORF of 693 nucleotides, and its deduced amino-acid sequence, except for the initiator methionine, was identical with that determined previously. Ferritin cDNA is 725 bp in length with an ORF of 516 nucleotides. Artemin amino-acid residues 32,185 are most similar to ferritin, but artemin is enriched in cysteines. The abundance of cysteines and their intramolecular spatial distribution suggest that artemin protects embryos against oxidative damage and/or that its function is redox regulated. The conserved regions in artemin and ferritin monomers are structurally similar to one another and both proteins assemble into oligomers. However, modeling of the quaternary structure indicated that artemin multimers lack the central space used for metal storage that characterizes ferritin oligomers, implying different roles for this protein. Probing of Northern blots revealed two artemin transcripts, one of 3.5 kb and another of 2.2 kb. These transcripts decreased in parallel and had almost disappeared by 16 h of development. The ferritin transcript of 0.8 kb increased slightly during reinitiation of development, then declined, and was almost completely gone by 16 h. Clearly, the loss of artemin and ferritin during embryo development is due to transcriptional regulation and proteolytic degradation of the proteins. [source] Seasonal nitrogen storage and remobilization in the forb Rumex acetosaFUNCTIONAL ECOLOGY, Issue 3 2001U. Bausenwein Summary 1,The contribution of N storage and remobilization to the vegetative and reproductive growth of the forb Rumex acetosa was quantified using 15N labelling techniques with plants derived from semi-natural grasslands in Scotland. 2,The contribution of remobilized N to the total N in the new above-ground tissues was highest at the beginning of the growing season at 58%. New leaves and reproductive organs contained equal amounts of remobilized N. 3,During early vegetative growth, the taproot was the main source of remobilized N, whereas during reproductive growth, N was additionally remobilized from fine roots and leaves. 4,Free amino acids (mainly arginine and glutamine) and proteins were identified as the main storage compounds in the taproots. The protein pool did not show any seasonal variations that indicated the existence of a vegetative storage protein, indicating that such proteins are not a necessary component of N storage/remobilization in all species. 5,The ability to store and remobilize N provides a mechanism for growth in the spring when the availability of soil N is low, and means that growth depends upon environmental conditions during more than one year. [source] The use of muscle protein for egg production in the Zebra Finch Taeniopygia guttataIBIS, Issue 2 2002Mat Cottam Pectoral muscle can be an important source of protein for birds. During egg formation Zebra Finches Taeniopygia guttata are able to compensate for nutritional inadequacies in their diet by utilization of the protein in their flight muscles. This analysis of flight muscle sarcoplasm supported earlier observations of protein depletion during egg production. However, SDS gel electrophoresis of the sarcoplasm produced no evidence to support a previous suggestion of the existence of a high molecular weight storage protein, and it is thought that the original observation may have arisen as an artefact of experimental methodology. During laying, protein removal from the sarcoplasm occurred over a range of different proteins and was not confined to any one specific protein band. Additionally, the protein band most reduced over the course of laying did not contain elevated levels of the amino acids most limiting to egg production. These results indicate that during laying, flight muscle sarcoplasm contributes towards the nutrient requirements of egg production from general protein reserves, rather than from a specific storage protein containing elevated levels of limiting amino acids. [source] cDNA of an arylphorin-type storage protein from Pieris rapae with parasitism inducible expression by the endoparasitoid wasp Pteromalus puparumINSECT SCIENCE, Issue 3 2009Jia-Ying Zhu Abstract, This report presents the cDNA cloning of a storage protein, PraAry, from Pieris rapae and investigates its expression regulated by parasitism of an endoparasitoid wasp Pteromalus puparum. The full-length cDNA of PraAry is 2 270 nucleotides and contains a 2 121 nucleotide open reading frame encoding 707 amino acids with calculated molecular weights of approximately 83 kDa. Analysis of the primary protein sequence revealed that it possesses a signal peptide of 16 amino acids at the N-terminus and contains two highly conserved storage protein signature motifs. According to both phylogenetic analysis and the criteria for amino acid composition, PraAry belongs to the subfamily of arylphorin-type storage protein (1.42% methionine and 18.82% aromatic amino acids). Reverse transcription , polymerase chain reaction analysis indicated that the transcriptional level of PraAry mRNA in P. rapae pupae fat body is inducible in response to parasitism by P. puparum. [source] Vegetative Storage Protein with Trypsin Inhibitor Activity Occurs in Sapindus mukorassi, a Sapindaceae Deciduous TreeJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 4 2009Shi-Biao Liu Abstract A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions: nitrogen storage and defense. [source] Ancient conserved domain protein-1 binds copper and modifies its retention in cellsJOURNAL OF NEUROCHEMISTRY, Issue 1 2007Alexandra Alderton Abstract The ancient conserved domain protein (ACDP) family are a recently identified group of homologous mammalian proteins. Some family members have been suggested to have roles in the metabolism of metals. We investigated the capacity of ACDP-1 to bind metals. Using immobilised metal affinity chromatography and isothermal titration calorimetry we determined that ACDP-1 is a high affinity copper binding protein able to bind copper at nanomolar concentrations. In addition the promoter of ACDP-1 contains metal response elements and the cellular expression of ACDP-1 alters cellular retention of copper. However, cellular expression of ACDP-1 does not alter cellular resistance to the toxicity of copper or other metals. As our findings place the subcellular localisation of ACDP-1 in the cytoplasm it is possible that ACDP-1 represent a novel copper chaperone or storage protein. [source] Divergent modulation of iron regulatory proteins and ferritin biosynthesis by hypoxia/reoxygenation in neurones and glial cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2005Carlo Irace Abstract Ferritin, the main iron storage protein, exerts a cytoprotective effect against the iron-catalyzed production of reactive oxygen species, but its role in brain injury caused by hypoxia/reoxygenation is unclear. Ferritin expression is regulated mainly at post-transcriptional level by iron regulatory proteins (IRP1 and IRP2) that bind specific RNA sequences (IREs) in the 5,untranslated region of ferritin mRNA. Here, we show that hypoxia decreases IRP1 binding activity in glial cells and enhances it in cortical neurons. These effects were reversed by reoxygenation in both cell types. In glial cells there was an early increase of ferritin synthesis during hypoxia and reoxygenation. Conversely, in cortical neurons, ferritin synthesis increased during the late phase of reoxygenation. Steady-state analysis of ferritin mRNA levels suggested that ferritin synthesis is regulated mainly post-transcriptionally by IRPs in glioma cells, both transcriptionally and post-transcriptionally in type-1 astrocytes, and mainly at transcriptional level in an IRP-independent way in neurons. The different regulation of ferritin expression may account for the different vulnerability of neurons and glial cells to the injury elicited by oxygen and glucose deprivation (OGD)/reoxygenation. The greater vulnerability of cortical neurons to hypoxia-reoxygenation was strongly attenuated by the exogenous administration of ferritin during OGD/reoxygenation, suggesting the possible cytoprotective role exerted by this iron-segregating protein. [source] Differential proteomic analysis of the endoplasmic reticulum from developing and germinating seeds of castor (Ricinus communis) identifies seed protein precursors as significant components of the endoplasmic reticulumPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2007Daniel J. Maltman Abstract The endoplasmic reticulum is a major compartment of storage protein and lipid biosynthesis. Maximal synthesis of these storage compounds occurs during seed development with breakdown occurring during germination. In this study, we have isolated four independent preparations of ER from both developing and germinating seeds of castor bean (Ricinus communis) and used 2-D DIGE, and a combination of PMF and MS/MS sequencing, to quantify and identify differences in protein complement at both stages. Ninety protein spots in the developing seeds are up-regulated and 19 individual proteins were identified, the majority of these are intermediates of seed storage synthesis and protein folding. The detection of these transitory storage proteins in the ER is discussed in terms of protein trafficking and processing. In germinating seed ER 15 spots are elevated, 5 of which were identified, amongst them was malate synthetase which is a component of the glyoxysome which is believed to originate from the ER. Notably no proteins involved in complex lipid biosynthesis were identified in the urea soluble ER fraction indicating that they are probably all integral membrane proteins. [source] NRAMP genes function in Arabidopsis thaliana resistance to Erwinia chrysanthemi infectionTHE PLANT JOURNAL, Issue 2 2009Diego Segond Summary AtNRAMP3 and AtNRAMP4 are two Arabidopsis metal transporters sharing about 50% sequence identity with mouse NRAMP1. The NRAMP1/Slc11A1 metal ion transporter plays a crucial role in the innate immunity of animal macrophages targeted by intracellular bacterial pathogens. AtNRAMP3 and AtNRAMP4 localize to the vacuolar membrane. We found that AtNRAMP3 is upregulated in leaves challenged with the bacterial pathogens Pseudomonas syringae and Erwinia chrysanthemi, whereas AtNRAMP4 expression is not modified. Using single and double nramp3 and nramp4 mutants, as well as lines ectopically expressing either of these genes, we show that AtNRAMP3 and, to a lesser extent, AtNRAMP4 are involved in Arabidopsis thaliana resistance against the bacterial pathogen E. chrysanthemi. The susceptibility of the double nramp3 nramp4 mutant is associated with the reduced accumulation of reactive oxygen species and ferritin (AtFER1), an iron storage protein known to participate in A. thaliana defense. Interestingly, roots from infected plants accumulated transcripts of AtNRAMP3 as well as the iron-deficiency markers IRT1 and FRO2. This finding suggests the existence of a shoot-to-root signal reminiscent of an iron-deficiency signal activated by pathogen infection. Our data indicate that the functions of NRAMP proteins in innate immunity have been conserved between animals and plants. [source] FUSCA3 from barley unveils a common transcriptional regulation of seed-specific genes between cereals and ArabidopsisTHE PLANT JOURNAL, Issue 6 2008Miguel Ángel Moreno-Risueno Summary Accumulation of storage compounds in the embryo and endosperm of developing seeds is a highly regulated process that allows seedling growth upon germination until photosynthetic capacity is acquired. A critical regulatory element in the promoters of seed storage protein (SSP) genes from dicotyledonous species is the RY box, a target of B3-type transcription factors. However, the functionality of this motif in the transcriptional regulation of SSP genes from cereals has not been fully established. We report here the identification and molecular characterization of barley FUSCA3, a B3-type transcription factor as yet uncharacterized in monocotyledonous plants. Our results show that both the barley and Arabidopsis FUS3 genes maintain a conserved functionality for the regulation of SSP genes and anthocyanin biosynthesis in these two distantly related phylogenetic groups. Complementation of the loss-of-function mutant fus3 in Arabidopsis by the barley HvFus3 gene resulted in restored transcription from the At2S3 gene promoter and normal accumulation of anthocyanins in the seed. In barley, HvFUS3 participates in transcriptional activation of the endosperm-specific genes Hor2 and Itr1. HvFUS3, which specifically binds to RY boxes in EMSA experiments, trans -activates Hor2 and Itr1 promoters containing intact RY boxes in transient expression assays in developing endosperms. Mutations in the RY boxes abolished the HvFUS3-mediated trans -activation. HvFus3 transcripts accumulate in the endosperm and in the embryo of developing seeds, peaking at mid maturation phase. Remarkably, HvFUS3 interacts with the Opaque2-like bZIP factor BLZ2 in yeast, and this interaction is essential for full trans -activation of the seed-specific genes in planta. [source] The effect of calorie restriction on growth and development in silkworm, Bombyx moriARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Yijia Li Abstract Caloric restriction (CR) is known to extend the life span in different species from yeast to mammals. In this report, a simple organism silkworm (Bombyx mori) was used to study the effect of moderate CR on the growth and development processes of insects. Here we show that an extension of life span upon moderate CR was observed in the silkworm. The total protein level in the 5th instar larvae hemolymph appeared to decline significantly under CR. SDS-PAGE analysis showed that the influence of CR was sex-dependent. The CR effects on female animals were much more significant than on the males. The MALDI-TOF MS study identified 16 proteins that expressed differentially among six groups of the male or female larvae fed at different time frequencies. Four of them, storage protein 1 (SP1), arylphorin (SP2), imaginal disk growth factor (IDGF), and 30-kDa lipoprotein, showed significant differences. It was demonstrated that these four proteins were up-regulated when the larvae were over-fed and down-regulated when the larvae were less-fed. © 2009 Wiley Periodicals, Inc. [source] Expression, purification and preliminary crystallization of amaranth 11S proglobulin seed storage protein from Amaranthus hypochondriacus L.ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Mary Rose Tandang-Silvas 11S globulin is one of the major seed storage proteins in amaranth. Recombinant protein was produced as up to ,80% of the total bacterial protein using Escherichia coli Rosetta-gami (DE3) containing pET21d with amaranth 11S globulin cDNA. The best expression condition was at 302,K for 20,h using LB medium containing 0.5,M NaCl. The recombinant protein was easily separated from most of the Escherichia coli proteins by precipitation with 0,40% ammonium sulfate solution. It formed aggregates at low temperature and at low salt concentrations. This behaviour may imply that it has a more hydrophobic nature than other 11S seed globulins. The crystals diffracted to 6,Å resolution and belonged to space group P63, with unit-cell parameters a = b = 97.6, c = 74.8,Å, , = 120.0°. One subunit of a trimer was estimated to be present in the asymmetric unit, assuming a Vsol of 41%. To obtain the complete structure solution, experiments to improve crystallization and flash-cooling conditions are in progress. [source] Crystallization and initial crystallographic characterization of a vicilin-type seed storage protein from Pinus koraiensisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2007Tengchuan Jin The cupin superfamily of proteins includes the 7S and 11S seed storage proteins. Many members of this family of proteins are known allergens. In this study, the Korean pine (Pinus koraiensis) vicilin-type 7S seed storage protein was isolated from defatted pine-nut extract and purified by sequential gel-filtration and anion-exchange chromatography. Well diffracting single crystals were obtained by the vapor-diffusion method in hanging drops. The crystals belong to the primitive cubic space group P213, with unit-cell parameters a = b = c = 148.174,Å. Two vicilin molecules were present in the asymmetric unit and the Matthews coefficient was determined to be 2.90,Å3,Da,1, with a corresponding solvent content of ,58%. A molecular-replacement structural solution has been obtained using the program Phaser. Refinement of the structure is currently under way. [source] Azotobacter vinelandii Metal Storage Protein: "Classical" Inorganic Chemistry Involved in Mo/W Uptake and Release ProcessesCHEMBIOCHEM, Issue 4 2008Jörg Schemberg Dr. Abstract The release of Mo (as molybdate) from the Mo storage protein (MoSto), which is unique among all existing metalloproteins, is strongly influenced by temperature and pH value; other factors (incubation time, protein concentration, degree of purity) have minor, though significant effects. A detailed pH titration at 12,°C revealed that three different steps can be distinguished for the Mo-release process. A proportion of ,15,% at pH 6.8,7.0, an additional 25,% at pH 7.2,7.5 and ca. 50,% (up to 90,% in total) at pH 7.6,7.8. This triphasic process supports the assumption of the presence of different types of molybdenum-oxide-based clusters that exhibit different pH lability. The complete release of Mo was achieved by increasing the temperature to 30,°C and the pH value to >7.5. The Mo-release process does not require ATP; on the contrary, ATP prevents, or at least reduces the degree of metal release, depending on the concentration of the nucleotide. From this point of view, the intracellular ATP concentration is suggested to play,in addition to the pH value,an indirect but crucial role in controlling the extent of Mo release in the cell. The binding of molybdenum to the apoprotein (reconstitution process) was confirmed to be directly dependent on the presence of a nucleotide (preferably ATP) and MgCl2. Maximal reincorporation of Mo required 1 mM ATP, which could partly be replaced by GTP. When the storage protein was purified in the presence of ATP and MgCl2 (1 mM each), the final preparation contained 80 Mo atoms per protein molecule. Maximal metal loading (110,115 atoms/MoSto molecule) was only achieved, if Mo was first completely released from the native protein and subsequently (re-) bound under optimal reconstitution conditions: 1 h incubation at pH 6.5 and 12,°C in the presence of ATP, MgCl2 and excess molybdate. A corresponding tungsten-containing storage protein ("WSto") could not only be synthesized in vivo by growing cells, but could also be constructed in vitro by a metalate,ion exchange procedure by using the isolated MoSto protein. The high W content of the isolated cell-made WSto (,110 atoms/protein molecule) and the relatively low amount of tungstate that was released from the protein under optimal "release conditions", demonstrates that the W-oxide-based clusters are more stable inside the protein cavity than the Mo-oxide analogues, as expected from the corresponding findings in polyoxometalate chemistry. The optimized isolation of the W-loaded protein form allowed us to get single crystals, and to determine the crystal X-ray structure. This proved that the protein contains remarkably different types of polyoxotungstates, the formation of which is templated in an unprecedented process by the different protein pockets. (Angew. Chem. Int. Ed.2007, 46, 2408,2413). [source] A putative hexamerin from a Campodea sp. suggests an independent origin of haemocyanin-related storage proteins in HexapodaINSECT MOLECULAR BIOLOGY, Issue 5 2009C. Pick Abstract Haemocyanins are copper-containing respiratory proteins in the arthropod haemolymph. In hexapods, haemocyanins gave rise to hexamerins, which have lost the ability to bind copper and thus oxygen. Hexamerins are thought to act mainly as storage proteins in nonfeeding periods. So far, hexamerins have only been identified in ectognathan hexapods, but not in Entognatha. Here we report the identification of a putative hexamerin from Campodea sp. (Diplura). The full-length cDNA of Campodea sp. hexamerin 1 (CspHex1) measures 2188 bp and translates into a native polypeptide of 667 amino acids. As in other hexamerins, the six copper-coordinating histidines are not conserved. However, sequence comparison and phylogenetic analyses demonstrated that CspHex1 is not closely related to other hexapod hexamerins, which derive from hexapod type 1 haemocyanin subunits in the ectognathan lineage, but rather resembles a derivative of hexapod type 2 haemocyanin subunits. Hence, haemocyanin-related storage proteins emerged at least two times independently in Hexapoda. [source] Functional dissection of the hexamerin receptor and its ligand arylphorin in the blowfly Calliphora vicinaINSECT MOLECULAR BIOLOGY, Issue 5 2003I. A. Hansen Abstract The process of receptor-mediated uptake of hexamerin storage proteins from insect haemolymph by fat body cells is a unique feature of the class Insecta. We identified the binding domains of the hexamerin receptor and the hexamerin ligand arylphorin in the blowfly, by means of the yeast-two-hybrid-system. The receptor-binding domain of arylphorin was located within domain 3 of the arylphorin monomer. The ligand-binding domain of the hexamerin receptor was mapped to the extreme N-terminus of the receptor. The binding domains identified exhibit no similarity to any functional protein domains known to date. Additionally, we identified two previously unknown protein-interactors of the hexamerin receptor. The results of this study provide further insights regarding the mechanism of the receptor-mediated endocytosis of storage proteins in insects. [source] Alleviation of the adverse effect of cooking on sorghum protein digestibility through fermentation in traditional African porridgesINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 2 2002Janet Taylor Cooking sorghum is well known to reduce its protein digestibility. In southern Africa fermented sorghum porridges are commonly consumed. Knowledge is lacking as to how their preparation affects sorghum protein digestibility. Five sorghum varieties of varied origin were fermented using traditional semi-solid state fermentation. In vitro protein digestibility and a new index, in vitro insoluble protein digestibility, were measured. Both increased during fermentation, generally within the first day, coinciding with a strong decrease in pH. The increase in insoluble protein digestibility suggests fermentation causes structural changes in the sorghum storage proteins (prolamins and glutelins), making them more accessible to pepsin attack. Wet cooking during porridge-making greatly reduced protein digestibility. Combining fermentation with cooking, either fermenting then cooking or cooking then fermenting, significantly improved protein digestibility over wet cooking alone. Thus natural fermentation, as applied in traditional African porridge preparation is an effective method of improving the protein digestibility of cooked sorghum. [source] Dietary vitamin E reduces labile iron in rat tissuesJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2005Wissam Ibrahim Abstract Previous studies have shown that dietary vitamin E reduced generation and/or levels of superoxide. As superoxide has potential to release iron from its transport and storage proteins, and labile or available form of iron is capable of catalyzing the formation of reactive hydroxyl radicals, the effect of dietary vitamin E on labile iron pool was studied in rats. One-month-old Sprague-Dawley male and female rats were fed a basal vitamin E-deficient diet supplemented with 0, 20, 200, or 2,000 IU vitamin E/kg diet for 90 days. The levels of labile iron were measured in the liver, kidney, spleen, heart and skeletal muscle. Additionally, the levels of lipid peroxidation products were measured. The results showed that, except for labile iron in the heart of male rats, dietary vitamin E dose dependently reduced the levels of labile iron and lipid peroxidation products in all tissues of male and female rats. The findings suggest that dietary vitamin E may protect against oxidative tissue damage by reducing the generation and/or level of superoxide, which in turn attenuates the release of iron from its protein complexes. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:298,303, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20094 [source] Effects of NaCl Concentration on Salting-in and Dilution During Salting-out on Soy Protein FractionationJOURNAL OF FOOD SCIENCE, Issue 4 2006N. A. Deak ABSTRACT:, Glycinin and ,-conglycinin are the main storage proteins in soybeans that can be fractionated by using alkali extraction, SO2, salting-in with NaCl, salting-out by dilution and pH adjustment to produce a glycinin-rich fraction, a ,-conglycinin,rich fraction, and an intermediate fraction, which is a mixture of the two proteins. Two different strategies were employed to optimize the procedure to achieve high efficiency in recovering the ,-conglycinin,rich fraction. The first strategy was to optimize salting-in effects of NaCl, and the effects of NaCl concentration on the yields and purities of the protein fractions were investigated. The maximum protein yield of the ,-conglycinin,rich fraction was obtained at 500 mM NaCl, but at the expense of purity. The optimum NaCl concentration was 250 mM, at which good protein yield (18.5%) and purity (84.5%) were achieved. At higher NaCl concentrations, the protein yields of the intermediate fractions were significantly lower, and the protein loss in the whey fraction increased. The second strategy was to improve the salting-out step for the ,-conglycinin,rich fraction. At 0- and 0.5-fold dilution, the purities and yields of the ,-conglycinin,rich fractions were significantly lower than at 1.0- and 2.0-fold dilution. There were no differences in protein yields or purities when using 1.0- or 2.0-fold dilution. According to these results, the recommended NaCl concentration for the salting-in step is 250 mM and the dilution factor for salting-out is 1.0. [source] Fluctuation of Vegetative Storage Proteins in the Seedlings of Swietenia macrophylla, Analogous to the Seasonal Changes of Those in the Shoot of the Adult TreeJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 3 2007Ya-Qin Han Abstract In order to identify appropriate plant materials for studying the gene expression and biological function of vegetative storage proteins (VSPs) in woody plants, the VSPs in the seedlings of Swietenia macrophylla King were investigated by using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blotting. The seed of S. macrophylla was rich in storage proteins that accumulated in the vacuoles of cotyledon parenchyma cells in appearance of compact spherical grains. The growth and development of S. macrophylla seedlings were characterized by an obvious growth rhythm. The storage proteins in seeds disappeared during seedling growth while VSPs appeared in the stem 2 weeks after seedling leaves matured. Thereafter, the VSPs in the seedling stem almost exhausted during new shoot growth, and when the leaves of new shoot just matured, both the stem beneath the new shoot of seedlings and the stem of new shoot started to accumulate VSPs. Nitrogen application dramatically increased the level of VSPs, but had little influence on the dynamics of VSP consumption and accumulation in seedling stem. Together with these data, the fluctuation of VSPs in seedlings was very similar to that in the branches of the adult trees. In addition, seedlings are easy to be treated due to their small size. Our results suggested that S. macrophylla seedlings were suitable for investigating the biological roles of VSPs and the mechanism of nitrogen storage in trees. [source] LC-MSMS identification of Arabidopsis thaliana heat-stable seed proteins: Enriching for LEA-type proteins by acid treatmentJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2007E. Oliveira Abstract Protein identification in systems containing very highly abundant proteins is not always efficient and usually requires previous enrichment or fractionation steps in order to uncover minor proteins. In plant seeds, identification of late embryogenesis abundant (LEA) proteins is often masked by the presence of the large family of storage proteins. LEA-proteins are predicted to play a role in plant stress tolerance. They are highly hydrophilic proteins, generally heat-stable, and correlate with dehydration in seeds or vegetative tissues. In the present work, we analyze the protein composition of heat-stable Arabidopsis thaliana seed extracts after treatment with trichloroacetic acid (TCA). The composition of the proteins that precipitate and those that remain in solution in 3% TCA was analyzed by two different approaches: 1D SDS-PAGE coupled to LC-ESI-MSMS analysis and a gel-free protocol associated with LC-MALDI-MSMS. Our results indicate that treating total heat-soluble extracts with 3% TCA is an effective procedure to remove storage proteins by selective precipitation and this fractionation step provides a soluble fraction highly enriched in Lea-type proteins. The analysis and determination of protein identities in this acid-soluble fraction by MS technology is a suitable system for large-scale identification of Lea-proteins present in seeds. Copyright © 2007 John Wiley & Sons, Ltd. [source] The content and distribution of condensed tannins in different species of the genus sorghum (Sorghum Moench) and their effect on seed protein electrophoresis,JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 9 2009Min-Xuan Liu Abstract BACKGROUND: The interaction between condensed tannins (CTs) and seed protein in varieties of sorghum interferes with protein extraction and the separation by electrophoresis, so electrophoresis can not be used widely for determining seed purity and identifying a variety. The objective of this research was to classify the effect of CTs on the extraction of seed storage proteins and on their analysis by SDS,PAGE, and to search for a promising solution to reduce the negative effect of CTs in sorghum. RESULTS: The vanillin,HCl test confirmed that CTs were localised mainly in the glumes of grain sorghum, but distributed in every fraction of sudangrass. Samples with high CT content did not produce any bands in the gel after electrophoresis. Removal of the glumes and pericarp/testa prevented the influence of CTs on electrophoresis for grain sorghum but had little effect for sudangrass. Adding tannin/catechin to the protein extraction of sorghum kernel decreased the number of bands in the gel. Adding polyvinylpyrrolidine to the protein extraction of sudangrass increased the bands. CONCLUSION: Tannin,protein interactions are responsible for the absence of bands in varieties with high CT content. For grain sorghum, decortication can prevent the influence. Adding polyvinylpyrrolidine during the extraction of seed protein could solve the problem of tannin,protein interactions for varieties of sudangrass. Copyright © 2009 Society of Chemical Industry [source] High-lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulationPLANT BIOTECHNOLOGY JOURNAL, Issue 6 2005Shihshieh Huang Summary Corn is one of the major crops in the world, but its low lysine content is often problematic for animal consumption. While exogenous lysine supplementation is still the most common solution for today's feed corn, high-lysine corn has been developed through genetic research and biotechnology. Reducing the lysine-poor seed storage proteins, zeins, or expressing a deregulated lysine biosynthetic enzyme, CordapA, has shown increased total lysine or free lysine content in the grains of modified corn plants, respectively. Here, by combining these two approaches through genetic crosses, the total lysine content has more than doubled in F1 progeny. We also observe a synergy between the transgenic zein reduction and the enhanced lysine biosynthesis by CordapA expression. The zein reduction plants are found to accumulate higher levels of aspartate, asparagine and glutamate, and therefore, provide excess precursors for the enhanced lysine biosynthesis. [source] Gene expression associated with N-induced shifts in resource allocation in poplarPLANT CELL & ENVIRONMENT, Issue 5 2003J. E. K. COOKE ABSTRACT Surprisingly little is known about molecular mechanisms by which nitrogen (N) availability acts to modulate the growth of forest trees. To address this issue, differential display was used in conjunction with filter-based arrays to identify 52 partial cDNA clones that were significantly regulated within days in response to limiting or luxuriant levels of NH4NO3 fertilization in Populus trichocarpa Torr. & Gray × deltoides Bartr. ex Marsh. A subset of these cDNAs also demonstrated shifts in expression patterns in stem-girdled trees, a manipulative physiology technique that disrupts phloem transport. Stem girdling also induced changes in glutamine and asparagine pools which were correlated with the observed changes in expression profiles for these genes. The identity of these genes provides insight into biochemical processes that are altered by N availability in poplar. Carbon,nitrogen interactions appear to figure prominently in the N-response. The gene expression data suggest that N availability modulates the partitioning of C and N resources into metabolic fates that have the potential to alter both wood quality and quantity, including synthesis of vegetative storage proteins, cell wall components, and terpenoids. [source] Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean ,-amylase inhibitor genePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2009Hancai Chen Abstract Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for ,-amylase inhibitor-1 (,AI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean ,AI1 protein and the corresponding ,AI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for ,AI1. Proteomic analysis showed that in addition to the presence of ,AI1, 33 other proteins were differentially accumulated in the ,AI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of ,AI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium -mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the ,AI1-expressing GM lines and one new protein was present in both the ,AI1-expressing GM lines and their ,AI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested. [source] | ||||||||||||||||||||||||||||