Biochemical and genetic characterisation of a D glutenin subunit encoded at the Glu-B3 locus

Genome ◽  
1998 ◽  
Vol 41 (2) ◽  
pp. 215-220 ◽  
Author(s):  
M T Nieto-Taladriz ◽  
M Rodríguez-Quijano ◽  
J M Carrillo
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Wheat Gluten ◽  
Gene Clusters ◽  
Glutenin Subunits ◽  
Key Words Wheat ◽  
Sds Page ◽  
A Subunit ◽  
Omega Gliadins ◽  
Prolamin Loci

The SDS-PAGE pattern of reduced and alkylated glutenins from the bread wheat cultivar Prinqual presents a subunit (named d4) in the mobility zone of the omega -gliadins that only appears under reduced conditions. This subunit was isolated and characterised at the biochemical and genetic levels. Subunit d4 was shown to form disulphide aggregates with glutenins and had an acidic pI. These characteristics correspond to those of the D glutenin subunits. The N-terminal amino acid sequence of subunit d4 was coincident with the SRL sequence type characteristic of omega -gliadins encoded by genes on the 1B chromosome, and confirms the similarity between D glutenin subunits and omega -gliadins. The genetic study of subunit d4 was performed in the F2 progeny from the 'Prinqual' x 'Ablaca' cross, based on four prolamin loci: Glu-B1, Glu-B3, Gli-B1, and Gli-B5. The recombinants found between Glu-B3 and Gli-B1 demonstrated that subunit d4 was encoded at the Glu-B3 locus, and reinforces the hypothesis of the duplication of prolamin gene clusters in wheat. A preliminary study of the effect of subunit d4 on gluten strength showed that lines with the Glu-B3 allele from 'Prinqual', which includes subunit d4, had a significantly higher sedimentation volume than those with the allele from 'Ablaca'.Key words: wheat gluten proteins, D glutenin subunits, amino acid sequence, linkage mapping, complex loci duplication.

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

1989 ◽  
Vol 61 (03) ◽  
pp. 437-441 ◽  
Author(s):  
Cindra Condra ◽  
Elka Nutt ◽  
Christopher J Petroski ◽  
Ellen Simpson ◽  
P A Friedman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Salivary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Potent Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Sds Page ◽  
Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Identification of TMEM45B as a protein clearly showing thermal aggregation in SDS–PAGE gels and dissection of its amino acid sequence responsible for this aggregation

2011 ◽  
Vol 77 (1) ◽  
pp. 118-123 ◽  
Author(s):  
Naoto Okada ◽  
Takenori Yamamoto ◽  
Masahiro Watanabe ◽  
Yuuya Yoshimura ◽  
Eriko Obana ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Thermal Aggregation ◽  
Sds Page

scholarly journals Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-d-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene

2004 ◽  
Vol 380 (1) ◽  
pp. 211-218 ◽  
Author(s):  
Chi-Wah TSEUNG ◽  
Laura G. McMAHON ◽  
Jorge VÁZQUEZ ◽  
Jan POHL ◽  
Jesse F. GREGORY
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Northern Blot ◽  
Sequence Data ◽  
Cdna Probe ◽  
Protein Mass ◽  
Sds Page ◽  
Mrna Analysis ◽  
Hydrolysis Of

We have previously identified and purified a novel β-glucosidase, designated PNGH (pyridoxine-5´-β-d-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5´-β-d-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase–phlorizin hydrolase), the β-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568–784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.

Characterization of cytoplasmic fibril structures found in gliding cells of Saprospira sp.

2005 ◽  
Vol 51 (10) ◽  
pp. 875-880 ◽  
Author(s):  
Gou Furusawa ◽  
Takeshi Yoshikawa ◽  
Yoshitaka Takano ◽  
Kazuyuki Mise ◽  
Iwao Furusawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Band ◽  
Amino Acid Sequences ◽  
Gliding Motility ◽  
Pcr Product ◽  
Nutrient Agar Medium ◽  
Sds Page ◽  
Tail Sheath ◽  
A Subunit ◽  
Triton X

The cytoplasmic fibril structures of Saprospira sp. strain SS98-5 grown on a low-nutrient agar medium were purified from cell lysates treated with Triton X-100 and were observed by electron microscopy to be about 7 nm in width and 200–300 nm in length. SDS–PAGE of the fibril structures exhibited a single protein band with a molecular mass of 61 kDa. A Saprospira cytoplasmic fibril protein (SCFP), which is a subunit of the fibril structures, was digested with trypsin to oligopeptides and analyzed for amino acid sequences. A partial nucleotide sequence of the SCFP gene was determined after PCR using primers designated from the amino acid sequences of the oligopeptides. SCFP gene including DNA fragments were detected by Southern hybridization using the PCR product for an SCFP gene as a probe and were cloned to determine whole nucleotide sequences. The SCFP gene indicated relatively higher similarity to conserved hypothetical phage tail sheath proteins. A Western immunoblotting analysis showed that SCFP was significantly expressed in gliding cells as compared with nongliding cells. The above findings with the previously reported results suggest that the cytoplasmic fibril structures are possibly related to the gliding motility of Saprospira sp. strain SS98-5.Key words: Saprospira, gliding motility, Saprospira cytoplasmic fibril protein (SCFP).

scholarly journals Neurotoxin Gene Clusters in Clostridium botulinum Type Ab Strains

2009 ◽  
Vol 75 (19) ◽  
pp. 6094-6101 ◽  
Author(s):  
Carolina Lúquez ◽  
Brian H. Raphael ◽  
Susan E. Maslanka
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Clostridium Botulinum ◽  
Gene Diversity ◽  
Gene Clusters ◽  
Toxin Gene ◽  
Predicted Amino Acid Sequence ◽  
Botulinum Type ◽  
Type Ab

ABSTRACT There is limited knowledge of the neurotoxin gene diversity among Clostridium botulinum type Ab strains. Only the sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene in C. botulinum type Ab strain CDC588 have been reported. In this study, we sequenced the entire bont/A- and bont/B-associated neurotoxin gene clusters of C. botulinum type Ab strain CDC41370 and the bont/A gene of strain CDC588. In addition, we analyzed the organization of the neurotoxin gene clusters in strains CDC588 and CDC1436. The bont/A nucleotide sequence of strain CDC41370 differed from those of the known bont/A subtypes A1 to A4 by 2 to 7%, and the predicted amino acid sequence differed by 4% to 14%. The bont/B nucleotide sequence in strain CDC41370 showed 99.7% identity to the sequence of subtype B1. The bont/A nucleotide sequence of strain CDC588 was 99.9% identical to that of subtype A1. Although all of the C. botulinum type Ab strains analyzed contained the two sets of neurotoxin clusters, similar to what has been found in other bivalent strains, the intergenic spacing of p21-orfX1 and orfX2-orfX3 varied among these strains. The type Ab strains examined in this study had differences in their toxin gene cluster compositions and bont/A and bont /B nucleotide sequences, suggesting that they may have arisen from separate recombination events.

scholarly journals Amino Acid Sequence of the Smaller Subunit of Conglutin ?, a Storage Globulin of Lupinus angustifolius

1977 ◽  
Vol 30 (2) ◽  
pp. 33 ◽  
Author(s):  
TC Elleman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Storage Protein ◽  
Storage Proteins ◽  
Lupinus Angustifolius ◽  
Amino Acid Residues ◽  
A Subunit

The amino acid sequence of the smaller subunit of conglutin y, the simplest of the three globulins from the seeds of Lupinus angusti/olius cv. Uniwhite, has been determined. The subunit was homogeneous and contained 154 amino acid residues, including five sulphur-containing amino acids-a considerably higher content than is found in most other legume storage proteins. There was no indication of the complexity experienced in studies of many other legume storage proteins. This is perhaps the first sequence of a subunit of a legume storage protein to be determined.

FIBRINOGENS KYOTO AND TOCHIGI, EACH WITH AN APPARENT ABNORMAL MOL. WT. γ CHAIN, ARE CHARACTERIZED BY REPLACEMENT OF γ ASN-308 BY LYS AND γ ARG-275 BY CYS, RESPECTIVELY

1987 ◽  
Author(s):  
N Yoshida ◽  
S Terukina ◽  
M Matsuda ◽  
M Moroi ◽  
M Okuma ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Cross Linking ◽  
Short Peptides ◽  
Amino Acid Sequence Analysis ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Fibrin Monomers

Congenital inherited abnormal fibrinogens (Fbgs) Kyoto and Tochigi showed prolonged thrombin- and reptilase-time, normal release of fibrinopeptides A and B, normal cross linking ability and impaired polymerization of the fibrin monomer.Purified Fbg analyzed on SDS-PAGE under the reduced condition in the system of Laemmli contained 50 % of an apparent lower mol. wt. γ chain (γ Kyoto)(mol. wt.= 48,000 compared with 50,000 for the normal) in Fbg Kyoto and an apparent higher mol. wt. γ chain (γ Tochigi)(mol. wt.= 50,500) in Fbg Tochigi. Apparent mol. wt. differences were also detected in reduced and carboxymethyl ated Fbg, Fbg fragment D1, and D2, but not in D3. This suggested that the abnormality of γ chains in both Fbgs is in γ 303-356.Amino acid sequence analysis was performed for CNBr- or lysylendopeptidase-digested peptides of the γ chain or D1 peptides after fractionation on HPLC. In Fbg Kyoto, γ Asn-308 was substituted by Lys, and a deletion of short peptides corresponding to the mol. wt. difference of 2,000 could not be detected. In Fbg Tochigi, γ Arg-275 was substituted by Cys, and no abnormality of amino acid sequence was found in γ 303-356.These results suggest that some lesions or conformations containing γ 275 and γ 308 will directly or indirectly affect polymerization of fibrin monomers. Although the reason for apparent mol. wt. differences is not known yet, SDS-PAGE in the system of Laemmli will be useful for the analysis of abnormal Fbgs.Fbg Kyoto could not be separated into two or three populations and may contain hetero-dimer molecules, but Fbg Tochigi had unclottable Fbg with predominant γ Tochigi and may contain abnormal homo-dimer molecules and normal molecules.

An approach for isolating high-molecular-weight glutenin subunit genes using monoclonal antibodies

Genome ◽  
2006 ◽  
Vol 49 (2) ◽  
pp. 181-189 ◽  
Author(s):  
H Q Wang ◽  
X Y Zhang
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Triticum Aestivum ◽  
Amino Acid ◽  
High Molecular Weight ◽  
Storage Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Glutenin Subunits ◽  
Sds Page

High-molecular-weight glutenin subunits (HMW-GSs) play an important role in the breadmaking quality of wheat flour. In China, cultivars such as Triticum aestivum 'Xiaoyan No. 6' carrying the 1Bx14 and 1By15 glutenin subunits usually have attributes that result in high-quality bread and noodles. HMW-GS 1Bx14 and 1By15 were isolated by preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and used as an antigen to immunize BALB/c mice. A resulting monoclonal antibody belonging to the IgG1 subclass was shown to bind to all HMW-GSs of Triticum aestivum cultivars, but did not bind to other storage proteins of wheat seeds in a Western blot analysis. After screening a complementary DNA expression library from immature seeds of 'Xiaoyan No. 6' using the monoclonal antibody, the HMW-GS 1By15 gene was isolated and fully sequenced. The deduced amino acid sequence showed an extra stretch of 15 amino acid repeats consisting of a hexapeptide and a nonapeptide in the repetitive domain of this y-type HMW subunit. Bacterial expression of a modified 1By15 gene, in which the coding sequence for the signal peptide was removed and a BamHI site eliminated, gave rise to a protein with mobility identical to that of HMW-GSs extracted from seeds of 'Xiaoyan No. 6' via SDS-PAGE. This approach for isolating genes using specific monoclonal antibody against HMW-GS genes is a good alternative to the extensively used polymerase chain reaction (PCR) technology based on sequence homology of HMW-GSs in wheat and its relatives.Key words: wheat, HMW-GS, monoclonal antibody, immunoscreen.

scholarly journals Conformational differences between two wheat (Triticum aestivum) ‘high-molecular-weight’ glutenin subunits are due to a short region containing six amino acid differences

1989 ◽  
Vol 263 (3) ◽  
pp. 837-842 ◽  
Author(s):  
A P Goldsbrough ◽  
N J Bulleid ◽  
R B Freedman ◽  
R B Flavell
Keyword(s):  
Molecular Weight ◽  
Triticum Aestivum ◽  
Amino Acid ◽  
High Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Sequences ◽  
Glutenin Subunits ◽  
Sds Page ◽  
Hmw Glutenin

‘High-molecular-weight’ (HMW, high-Mr) glutenin subunits are protein constituents of wheat (Triticum aestivum) seeds and are responsible in part for the viscoelasticity of the dough used to make bread. Two subunits, numbered 10 and 12, are the products of allelic genes. Their amino acid sequences have been derived from the nucleic acid sequences of the respective genes. Subunit 10 has fewer amino acids than subunit 12, but migrates more slowly on SDS/PAGE (polyacrylamide-gel electrophoresis). This anomaly is due to between one and six of the amino acid differences between the subunits, localized towards the C-terminal end of the proteins. This has been established by making chimaeric genes between the genes for subunits 10 and 12, transcribing and translating them in vitro and analysing the products by SDS/PAGE. The postulated conformational differences between subunits 10 and 12 are discussed in relation to current hypotheses for the structure of HMW glutenin subunits.

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