scholarly journals A novel tetrameric lectin from Lycoris aurea with four mannose binding sites per monomer.

2007 ◽  
Vol 54 (1) ◽  
pp. 159-166 ◽  
Author(s):  
Jiwei Liu ◽  
Xiaochao Xu ◽  
Jinzhi Liu ◽  
Jan Balzarini ◽  
Yongtin Luo ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Binding Sites ◽  
Tertiary Structure ◽  
Gel Filtration ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Hydrophobic Cluster Analysis ◽  
Mannose Binding ◽  
Narcissus Pseudonarcissus ◽  
Lycoris Aurea

The mannose-binding agglutinin from bulbs of Lycoris aurea (LAA) agglutinates rabbit but not human erythrocytes. The molecular mass of the monomer in SDS/PAGE is 12 kDa while the apparent molecular mass in gel filtration is 48 kDa, indicating that LAA is a homotetramer. The full-length cDNA of LAA contains 683 bp with an open reading frame encoding a protomer of 162 amino-acid residues. Hydrophobic Cluster Analysis and molecular modeling of the 109-residue mature polypeptide suggested a similar secondary and tertiary structure to those of Narcissus pseudonarcissus agglutinin (NPA). Molecular docking revealed that, besides the three mannose-binding sites common among Amaryllidaceae lectins, LAA also contains a fourth unique mannose-binding site formed by a tryptophan cluster. The existence of four mannose-binding sites in each monomer of LAA is very unusual and has only been reported for NPA earlier.

Features of the cellodextrinase gene from Fibrobacter succinogenes S85

1994 ◽  
Vol 40 (7) ◽  
pp. 592-596 ◽  
Author(s):  
Abiye H. Iyo ◽  
Cecil W. Forsberg
Keyword(s):  
Gel Filtration ◽  
Fibrobacter Succinogenes ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Base Pairs ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Promoter Sequences ◽  
Putative Ribosome Binding Site ◽  
Coli Promoter

The nucleotide sequence of a 2.3-kb DNA fragment containing a cellodextrinase gene (cedA) from the ruminal anaerobe Fibrobacter succinogenes S85 was determined. Activity was expressed from this fragment when it was cloned in both orientations in pBluescript KS+ and SK−, indicating a functional F. succinogenes promoter in Escherichia coli. Promoter sequences (TTGAACA and AATAA) were identified upstream of the ATG initiation codon preceded by a putative ribosome binding site. The cedA open reading frame of 1071 base pairs encoded a protein of 357 amino acid residues with a calculated molecular mass of 41.9 kDa, similar to the40-kDa size of the native protein as determined by gel filtration chromatography. CedA is proposed to belong to family 5 (family A) of the glycosyl hydrolases. The primary structure of the cellodextrinase showed over 40% similarity with endoglucanase 3 from F. succinogenes S85. Short regions of similarity were also demonstrated with endoglucanase C from Clostridium thermocellum, CelA from Ruminococcus flavefaciens, and two exoglucanases from yeast.Key words: Fibrobacter succinogenes, cedA, cellodextrinase, sequence, rumen, gene.

Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

1999 ◽  
Vol 65 (7) ◽  
pp. 3001-3007 ◽  
Author(s):  
Frederic Chavagnat ◽  
Michael G. Casey ◽  
Jacques Meyer
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Streptococcus Thermophilus ◽  
Maximal Activity ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
T7 Promoter ◽  
Reading Frame ◽  
Endopeptidase Activity

ABSTRACT The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of thepepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.

Cloning and Characterization of an Agglutinin Gene from Arisaema lobatum

2005 ◽  
Vol 25 (5-6) ◽  
pp. 345-362 ◽  
Author(s):  
Juan Lin ◽  
Xuanwei Zhou ◽  
Yongzhen Pang ◽  
Han Gao ◽  
Jiong Fei ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Regulation Mechanism ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Mannose Binding ◽  
Pcr Technology ◽  
Plant Families ◽  
Race Pcr

A novel agglutinin gene was cloned from Arisaema lobatum using SMART RACE-PCR technology. The full-length cDNA of Arisaema lobatum agglutinin (ala) was 1078 bp and contained a 774 bp open reading frame encoding a lectin precursor (proproprotein) of 258 amino acid residues with a 23 aa signal peptide. ALA contained three mannose-binding sites (QXDXNXVXY) with two-conserved domains of 45% identity, ALA-DOM1 and ALA-DOM2. The three-dimensional structure of ALA was very similar to that of GNA (Galanthus nivalis agglutinin). ALA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families, such as Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. Genomic sequence of ala was also cloned using genomic walker technology, and it was found to contain three putative TATA boxes and eight possible CAAT boxes in the 5′-flanking region. No intron was found within the region of genomic sequence. Southern blot analysis indicated that the ala belonged to a multi-copy gene family. Expression pattern analysis revealed that the ala preferentially expressed in the tissues with the higher expression being found in spadix, bud, leaf, spathe and tuber. The cloning of the ala gene not only provides a basis for further investigation of its structure, expression and regulation mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.

Isolation and characterization of a mannose-binding lectin from leaves of the Chinese daffodil Narcissus tazetta

1998 ◽  
Vol 76 (4) ◽  
pp. 601-608 ◽  
Author(s):  
Linda SM Ooi ◽  
Hexiang Wang ◽  
T B Ng ◽  
Vincent EC Ooi
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Mannose Binding Lectin ◽  
Narcissus Tazetta ◽  
Isolation And Characterization ◽  
Mannose Binding ◽  
Narcissus Pseudonarcissus ◽  
Considerable Homology ◽  
Binding Lectin

A mannose-binding lectin was isolated from leaves of the Chinese daffodil Narcissus tazetta (family Amaryllidaceae) using a procedure that comprised extraction with aqueous buffer, ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel Blue gel and mannose-agarose, and FPLC-gel filtration on Superose 12. The lectin was adsorbed on mannose-agarose and unadsorbed on DEAE-cellulose and Affi-gel Blue gel. It was an unglycosylated homodimer with a molecular mass of 26 kDa. Analysis of the N-terminal sequence of the N. tazetta lectin revealed considerable homology to lectins from the daffodil Narcissus pseudonarcissus, the snowdrop Galanthus nivalis (family Amaryllidaceae), the tulip Tulipa, and Kidachi aloe Aloe arborescens (family Liliaceae), and the orchid lectins (family Orchidaceae). The most striking likeness exists among the Amaryllidaceae lectins. The N. tazetta lectin exhibits hemagglutinating activity toward rabbit erythrocytes.Key words: daffodil, mannose-binding lectin, agglutinin.

Characterization, expression and localization of S-adenosylhomocysteine hydrolase from amphioxus Branchiostoma belcheri tsingtaunese

2008 ◽  
Vol 28 (3) ◽  
pp. 135-144 ◽  
Author(s):  
Yuan Wang ◽  
Bosheng Zhao ◽  
Shicui Zhang ◽  
Xiaojuan Qu
Keyword(s):  
Molecular Mass ◽  
Sea Urchin ◽  
Amino Acid Residues ◽  
Adenosylhomocysteine Hydrolase ◽  
Reading Frame ◽  
Catalytic Activities ◽  
Native Molecular Mass ◽  
Immunohistochemistry Analysis ◽  
Branchiostoma Belcheri ◽  
Genomic Dna Sequence

A cDNA clone encoding AmphiSAHH [amphioxus SAHH (S-adenosylhomocysteine hydrolase)] protein was isolated from a cDNA library from the gut of Branchiostoma belcheri tsingtaunese. It contained a 1305 bp open reading frame corresponding to a deduced protein of 434 amino acid residues, with a predicted molecular mass of approx. 47.8 kDa. Phylogenetic analysis showed that AmphiSAHH and sea-urchin SAHH joined together and positioned at the base of the vertebrate SAHH clade, suggesting that both AmphiSAHH and sea-urchin SAHH might share some characteristics of the archetype of vertebrate SAHH proteins. The genomic DNA sequence of AmphiSAHH contained eight exons and seven introns, which was similar to B. floridae and sea-urchin SAHH exon/intron organization. Sequence comparison suggested the evolutionary appearance of the ten exon/nine intron organization of SAHH genes after the split of invertebrates and vertebrates, after which it has been highly conserved. AmphiSAHH has been successfully expressed in Escherichia coli and purified. Western blotting confirmed that the enzyme has a native molecular mass of approx. 48 kDa, and the catalytic activities and NAD+/NADH binding affinity of recombinant AmphiSAHH were measured. Immunohistochemistry analysis showed that SAHH was strongly expressed in hepatic caecum, gill, spermary and ovary of amphioxus.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

scholarly journals Properties of subunits of the multicatalytic proteinase complex revealed by the use of subunit-specific antibodies

1991 ◽  
Vol 278 (1) ◽  
pp. 171-177 ◽  
Author(s):  
A J Rivett ◽  
S T Sweeney
Keyword(s):  
Molecular Mass ◽  
Rat Liver ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Rat Tissues ◽  
Specific Antibodies ◽  
Multicatalytic Proteinase ◽  
Cell Extracts ◽  
Liver And Kidney ◽  
Lysosomal Proteinase

The multicatalytic proteinase (MCP) is a high-molecular-mass non-lysosomal proteinase that gives rise to a characteristic pattern of bands of molecular mass 22-34 kDa on SDS/PAGE gels. Isoelectric-focusing gels of the enzyme purified from rat liver show 16 bands with isoelectric points in the range of pH 5-8.5. Two-dimensional PAGE gels reveal that there are more than the previously reported 13 polypeptides associated with the MCP from rat liver and show a pattern of 15-20 major spots and several minor ones, similar to that of MCP isolated from some other sources. Possible relationships between the different polypeptides were investigated by immunoblot analysis of electrophoretically purified proteinase subunits with affinity-purified subunit-specific antibodies as well as antibodies raised against individual denatured subunits of the complex. The results demonstrate that many of the major polypeptide components of the MCP complex are antigenically distinct. Moreover comparison of immunoreactive material in crude cell extracts with that in purified MCP preparations has shown that the polypeptides are not derived from a smaller number of higher-molecular-mass subunits. Also, individual subunits have the same apparent molecular mass in a variety of rat tissues, suggesting close similarity between MCPs of different tissues. The highest concentrations of MCP subunits occur in liver and kidney. Gel-filtration analysis of crude extracts has demonstrated that MCP polypeptides are also associated with a higher-molecular-mass complex, which may be the 26 S proteinase that has been implicated in the degradation of ubiquitin-protein conjugates.

scholarly journals Identification of NAD+ Synthetase from Streptococcus sobrinus as a B-Cell-Stimulatory Protein

2004 ◽  
Vol 186 (2) ◽  
pp. 419-426 ◽  
Author(s):  
Isabel Veiga-Malta ◽  
Margarida Duarte ◽  
Márcia Dinis ◽  
Pedro Madureira ◽  
Paula Ferreira ◽  
...  
Keyword(s):  
B Cell ◽  
Active Form ◽  
Lymphocyte Activation ◽  
Amino Acid Residues ◽  
Streptococcus Sobrinus ◽  
Reading Frame ◽  
High Sequence Homology ◽  
Culture Supernatants ◽  
Nad Synthetase

ABSTRACT Streptococcus sobrinus, one agent of dental caries, secretes a protein that induces lymphocyte polyclonal activation of the host as a mechanism of immune evasion. We have isolated from culture supernatants of this bacterium a protein with murine B-cell-stimulatory properties and subsequently cloned the relevant gene. It contains an open reading frame of 825 bp encoding a polypeptide with 275 amino acid residues and a molecular mass of 30 kDa. The protein displays high sequence homology with NAD+ synthetases from several organisms, including a conserved fingerprint sequence (SGGXD) characteristic of ATP pyrophosphatases. The polypeptide was expressed in Escherichia coli as a hexahistidine-tagged protein and purified in an enzymatically active form. The recombinant NAD+ synthetase stimulates murine B cells after in vitro treatment of spleen cell cultures, as demonstrated by its ability to induce up-regulation of the expression of CD69, an early marker of lymphocyte activation. Stimulation with the recombinant NAD+ synthetase was also observed with other B-cell markers, such as CD19+, B220+, and CD21+. Cell proliferation follows the activation induced by the recombinant NAD+ synthetase.

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