scholarly journals Bovine acetylcholinesterase: cloning, expression and characterization

1998 ◽  
Vol 334 (1) ◽  
pp. 251-259 ◽  
Author(s):  
Itai MENDELSON ◽  
Chanoch KRONMAN ◽  
Naomi ARIEL ◽  
Avigdor SHAFFERMAN ◽  
Baruch VELAN
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Hek 293 ◽  
Human Sequence ◽  
Tetramer Formation ◽  
Glycosylation Sites ◽  
293 Cells ◽  
Sds Page ◽  
High Conservation ◽  
Fetal Bovine

The bovine acetylcholinesterase (BoAChE) gene was cloned from genomic DNA and its structure was determined. Five exons coding for the AChE T-subunit and the alternative H-subunit were identified and their organization suggests high conservation of structure in mammalian AChE genes. The deduced amino acid sequence of the bovine T-subunit is highly similar to the human sequence, showing differences at 34 positions only. However, the cloned BoAChE sequence differs from the published amino acid sequence of AChE isolated from fetal bovine serum (FBS) by: (1) 13 amino acids, 12 of which are conserved between BoAChE and human AChE, and (2) the presence of four rather than five potential N-glycosylation sites. The full coding sequence of the mature BoAChE T-subunit was expressed in human embryonal kidney 293 cells (HEK-293). The catalytic properties of recombinant BoAChE and its reactivity towards various inhibitors were similar to those of the native bovine enzyme. Soluble recombinant BoAChE is composed of monomers, dimers and tetramers, yet in contrast to FBS-AChE, tetramer formation is not efficient. Comparative SDS/PAGE analysis reveals that all four potential N-glycosylation sites identified by DNA sequencing appear to be utilized, and that recombinant BoAChE comigrates with FBS-AChE. A major difference between the recombinant enzyme and the native enzyme was observed when clearance from circulation was examined. The HEK-293-derived enzyme was cleared from the circulation at a much faster rate than FBS-AChE. This difference in behaviour, together with previous studies on the effect of post-translation modification on human AChE clearance [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959–967] suggests that cell-dependent glycosylation plays a key role in AChE circulatory residence.

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.

scholarly journals Primary structure of bovine complement activation fragment C4a, the third anaphylatoxin. Purification and complete amino acid sequence

1982 ◽  
Vol 207 (2) ◽  
pp. 253-260 ◽  
Author(s):  
M A Smith ◽  
L M Gerrie ◽  
B Dunbar ◽  
J E Fothergill
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Primary Structure ◽  
Gel Filtration ◽  
Complete Amino Acid Sequence ◽  
The Third ◽  
Human Sequence ◽  
Bovine Plasma ◽  
Bovine Sequence ◽  
C 50

Purification of C4a from heat-activated bovine plasma by elution from CM-Sephadex C-50 at pH 7.4 and gel filtration on Sephadex G-50 gives a 20% yield of pure C4a. The complete amino acid sequence of bovine C4a has been determined by automatic sequencer degradation of CNBr and enzymic fragments, and by carboxypeptidase digestion. The 77-residue bovine sequence shows 12 differences from the human sequence with five of these differences occurring in the C-terminal 11 residues. The sequence of C4a confirms earlier suggestions of homology with C3a and C5a: the three sequences show an almost equal number of identities with each other. The six cysteine residues of the ‘disulphide knot’ are conserved as well as seven other residues including the C-terminal arginine.

Plasma membrane delivery of the gastric H,K-ATPase: the role of β-subunit glycosylation

2003 ◽  
Vol 285 (4) ◽  
pp. C968-C976 ◽  
Author(s):  
O. Vagin ◽  
S. Denevich ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Glycosylation Site ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Hek 293 ◽  
Glycosylation Sites ◽  
293 Cells

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase β-subunit (YFP-β) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase α-subunit (CFP-α) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-α, wild-type YFP-β, and YFP-β mutants lacking one or two of the seven β-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-β resulted in the plasma membrane localization of the protein, whereas the expressed CFP-α was retained intracellularly. When coexpressed, both CFP-α and YFP-β were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-β prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the β-subunit are essential for the plasma membrane delivery of the β-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the β-subunits from different species, is not critical for plasma delivery of the protein.

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.

scholarly journals The proteolytic processing site of the precursor of lysyl oxidase

1995 ◽  
Vol 306 (1) ◽  
pp. 279-284 ◽  
Author(s):  
A D Cronshaw ◽  
L A Fothergill-Gilmore ◽  
D J S Hulmes
Keyword(s):  
Cleavage Site ◽  
Lysyl Oxidase ◽  
Peptide Fragmentation ◽  
Proteolytic Processing ◽  
Amino Acid Sequencing ◽  
Human Sequence ◽  
Glycosylation Sites ◽  
Sds Page ◽  
Processing Site ◽  
Partial Chemical

The precise cleavage site of the N-terminal propeptide region of the precursor of lysyl oxidase has not yet been established, due to N-terminal blocking of the mature protein. Using a combination of peptide fragmentation, amino acid sequencing, time-of-flight m.s. and partial chemical unblocking procedures, it is shown that the mature form of lysyl oxidase begins at residue Asp-169 of the precursor protein (numbered according to the human sequence). The cleavage site is 28 residues to the C-terminal side of the site previously suggested on the basis of apparant molecular mass by SDS/PAGE, with the consequence that the two putative, N-linked glycosylation sites and the position of the Arg/Gln sequence polymorphism are now all in the precursor region.

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.

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.

Cloning of a bovine renal epithelial Na+ channel subunit

1997 ◽  
Vol 272 (6) ◽  
pp. 1-1
Author(s):  
C. M. Fuller ◽  
M. S. Awayda ◽  
M. P. Arrate ◽  
A. L. Bradford ◽  
R. G. Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Na Channel ◽  
Terminal Portion ◽  
Human Sequence ◽  
Relative Molecular Weight ◽  
Epithelial Na Channel

Pages C641-C654: C. M. Fuller, M. S. Awayda, M. P. Arrate, A. L. Bradford, R. G. Morris, C. M. Canessa, B. C. Rossier, and D. J. Benos. “Cloning of a bovine renal epithelial Na+ channel subunit.” Page C642: Fig. 1 contains two errors in the published sequence of the cDNA agr-bENaC clone presented. A severe COOH compression at nucleotide positions 1750 and 1760 resulted in a double frameshift in the COOH-terminal portion of the sequence. Correction of the nucleotide sequence causes the termination codon to fall at position 1951 (as opposed to position 2092 as previously published), predicting a translated polypeptide of 650 amino acids as opposed to 697 residues as previously reported. This shortened protein has a calculated molecuar mass of 73.4 kDa, although it is observed to migrate with a relative molecular weight of ap80,000 on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The overall homology of the nucleotide sequence with the rat and human agr-ENaC clones is slightly increased by this sequence change to 80 and 84% identities, respectively. In the COOH-terminal region, the homology increases to 53% identity from 43% identity for the rat clone and to 64% identity from 51% identity for the human sequence. A revised nucleotide and amino acid sequence is given in the revised Fig. 1. The sites of the COOH insertion are underlined and the altered amino acid sequence is given in bold. However, this sequence revision does not affect the conclusions of this or subsequent papers from our laboratory concerning this cDNA clone. The amended sequence has been deposited with GenBank (accession no. U14944). The authors apologize for any inconvenience caused by this error. (See PDF)

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