scholarly journals Localization of Ca2+-dependent conformational changes of calretinin by limited tryptic proteolysis

1995 ◽  
Vol 308 (2) ◽  
pp. 607-612 ◽  
Author(s):  
J Kuźnicki ◽  
T L Wang ◽  
B M Martin ◽  
L Winsky ◽  
D M Jacobowitz
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Conformational Changes ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Amino Acid Sequence Analysis ◽  
Sds Page ◽  
Ef Hand ◽  
Digestion Pattern

Calretinin is an EF-hand Ca(2+)-binding protein expressed predominantly in some neurons. We have found that the tryptic digestion pattern of rat recombinant calretinin depends on Ca2+ concentration as determined by SDS/PAGE, amino-acid-sequence analysis and electrospray-ionization MS. Ca(2+)-saturated calretinin was cleaved between amino acids 60 and 61 to yield two fragments, which accumulated during cleavage. Small amounts of the larger fragment (amino acid residues 61-271) were further cleaved from the C-terminal end. Ca(2+)-free calretinin was also cleaved between residues 60 and 61; however, under the latter conditions the fragment 61-271 was further cleaved from the N-terminal end. Native rat calretinin was cleaved by trypsin in a similar Ca(2+)-dependent fashion. All identified fragments of recombinant calretinin bound 45Ca2+ on nitrocellulose filters, although to a different extent. The 61-271 fragment was released by EGTA from an octyl-agarose column in a manner similar to intact calretinin, while fragment 61-233 was not eluted by EGTA. These observations show that there are trypsin cleavage sites in calretinin that are available regardless of Ca2+ binding, other sites that are completely protected against trypsin on Ca(2+)-binding and sites which become partially available on Ca(2+)-binding. Together these data show that calretinin changes its conformation on Ca2+ binding and identify the regions which are exposed in apo and Ca(2+)-bound form.

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.

scholarly journals Ornithine decarboxylase stability in HMOA and DH23b cells is not due to post-translational truncation of a C-terminal recognition site

1996 ◽  
Vol 318 (3) ◽  
pp. 879-882
Author(s):  
John L. A. MITCHELL ◽  
Chung-youl CHOE ◽  
Gary G JUDD
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ornithine Decarboxylase ◽  
Cdna Sequence ◽  
Carboxypeptidase Y ◽  
Amino Acid Residues ◽  
Wild Type ◽  
C Terminus ◽  
Variant Cell ◽  
Sds Page

The normally labile ornithine decarboxylase (ODC) becomes unusually stable when Cys-441 is replaced with Trp in the variant cell lines HMOA and DH23b. This stable ODC is also observed to have higher mobility on SDS/PAGE. Because previous studies have shown that ODC stability can be achieved when as few as five amino acid residues are removed from its C-terminus, it was suggested that the amino acid substitution in the variant ODC might alter its conformation sufficiently to promote a similar proteolytic loss of a C-terminal degradation signal, resulting in a stable yet active ODC. To examine this mechanism, amino acids in the C-terminal regions of both wild-type and stable (Trp-441) ODC proteins were released, by means of carboxypeptidase-Y digestion, and identified by HPLC. The C-terminal ends were found to be the same, and are as predicted from the cDNA sequence. This study proves that stability of the Trp-441 form of ODC is not simply due to proteolytic removal of a C-terminal proteasome-targeting sequence, thereby implying that the stabilization of this mutant ODC form must result directly from a conformational change associated with the loss of Cys-441.

scholarly journals Molecular Characterization of a Secreted Enzyme with Phospholipase B Activity from Moraxella bovis

2001 ◽  
Vol 183 (22) ◽  
pp. 6717-6720 ◽  
Author(s):  
Jacinta L. Farn ◽  
Richard A. Strugnell ◽  
Peter A. Hoyne ◽  
Wojtek P. Michalski ◽  
Jan M. Tennent
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Amino Acid Sequence Analysis ◽  
Lipolytic Enzymes ◽  
Infectious Bovine Keratoconjunctivitis ◽  
Phospholipase B ◽  
Moraxella Bovis

ABSTRACT A candidate for a vaccine against infectious bovine keratoconjunctivitis (IBK) has been cloned and characterized fromMoraxella bovis. The plb gene encodes a protein of 616 amino acids (molecular mass of ∼65.8 kDa) that expresses phospholipase B activity. Amino acid sequence analysis revealed that PLB is a new member of the GDSL (Gly-Asp-Ser-Leu) family of lipolytic enzymes.

Cloning and Sequence Analysis of Two cDNA Encoding Invertase Inhibitors from Cassava (Manihot esculenta Crantz)

2013 ◽  
Vol 726-731 ◽  
pp. 4326-4329 ◽  
Author(s):  
Meng Ting Geng ◽  
Yuan Yao ◽  
Xiao Hui Wu ◽  
Yi Min ◽  
Shao Ping Fu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Manihot Esculenta ◽  
Disulfide Bridges ◽  
Amino Acid Sequence Analysis ◽  
Manihot Esculenta Crantz ◽  
Typical Structure ◽  
Invertase Inhibitor

In this study, two encoding invertase inhibitors (INH) named as MeINH1 and MeINH2 were isolated from cassava (Manihot esculenta Crantz). TheMeINH1 contains a 583 bp fragment in length and codes a region of 172 amino acids; and the MeINH2 contains a 538 bp fragment in length and codes a region of 169 amino acids. The amino acid sequence analysis showed that both MeINH1 and MeINH2 have typical structure of invertase inhibitor proteins with a N-terminal signal peptide, four conserved cysteine residues and two disulfide bridges, respectively; whereas, they showed low homology to other organisms. This research provides a foundation for further study of the cassava INH.

Identification of the gene encoding 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase in Burkholderia sp. HME13

2020 ◽  
Vol 85 (3) ◽  
pp. 626-629
Author(s):  
Hisashi Muramatsu ◽  
Hiroki Maguchi ◽  
Taisuke Harada ◽  
Takehiro Kashiwagi ◽  
Chul-Sa Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Propionic Acid ◽  
Unknown Function ◽  
Protein Family ◽  
Amino Acid Sequence Analysis ◽  
Gene Encoding

ABSTRACT Here, we report the identification of the gene encoding a novel enzyme, 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase, in Burkholderia sp. HME13. The enzyme converts 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid and H2O to 3-(2,5-dioxoimidazolidin-4-yl) propionic acid and H2S. Amino acid sequence analysis of the enzyme indicates that it belongs to the DUF917 protein family, which consists of proteins of unknown function.

scholarly journals Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein.

1988 ◽  
Vol 263 (25) ◽  
pp. 12559-12563
Author(s):  
T L Wasmoen ◽  
M P Bell ◽  
D A Loegering ◽  
G J Gleich ◽  
F G Prendergast ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Basic Protein ◽  
Major Basic Protein ◽  
Amino Acid Sequence Analysis ◽  
Human Eosinophil ◽  
Eosinophil Granule

Developing structure-based models to predict substrate specificity of D-group (Type II) molybdenum enzymes: application to a molybdo-enzyme of unknown function from Archaeoglobus fulgidus

2006 ◽  
Vol 34 (1) ◽  
pp. 118-121 ◽  
Author(s):  
E.J. Dridge ◽  
D.J. Richardson ◽  
R.J. Lewis ◽  
C.S. Butler
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Archaeoglobus Fulgidus ◽  
Substrate Selectivity ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Sequence Alignments ◽  
X Ray ◽  
Group Type

The AF0174–AF0176 gene cluster in Archaeoglobus fulgidus encodes a putative oxyanion reductase of the D-type (Type II) family of molybdo-enzymes. Sequence analysis reveals that the catalytic subunit AF0176 shares low identity (31–32%) and similarity (41–42%) to both NarG and SerA, the catalytic components of the respiratory nitrate and selenate reductases respectively. Consequently, predicting the oxyanion substrate selectivity of AF0176 has proved difficult based solely on sequence alignments. In the present study, we have modelled both AF0176 and SerA on the recently determined X-ray structure of the NAR (nitrate reductase) from Escherichia coli and have identified a number of key amino acid residues, conserved in all known NAR sequences, including AF0176, that we speculate may enhance selectivity towards trigonal planar (NO3−) rather than tetrahedral (SeO42− and ClO4−) substrates.

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