scholarly journals Homodimerization and hetero-oligomerization of the single-domain trefoil protein pNR-2/pS2 through cysteine 58

1997 ◽  
Vol 327 (1) ◽  
pp. 117-123 ◽  
Author(s):  
Mark P. CHADWICK ◽  
Bruce R. WESTLEY ◽  
Felicity E. B. MAY
Keyword(s):  
Gel Filtration ◽  
Biological Action ◽  
Single Domain ◽  
Disulphide Bond ◽  
Intestinal Trefoil Factor ◽  
Pvdf Membrane ◽  
C Terminus ◽  
Form Part ◽  
Equilibrium Ultracentrifugation ◽  
Protein Treatment

The single-domain human trefoil proteins [pNR-2/pS2 and human intestinal trefoil factor (hITF)] have seven cysteine residues, of which six are involved in maintaining the structure of the trefoil domain. The seventh does not form part of the trefoil domain and is located three residues from the C-terminus. The ability of the pNR-2/pS2 single trefoil domain protein to dimerize was examined by using recombinant protein with either a cysteine or a serine residue at this position by equilibrium ultracentrifugation, laser-assisted desorption MS, gel filtration and PAGE. pNR-2/pS2 Cys58 formed dimers, whereas pNR-2/pS2 Ser58 did not. Experiments in which the dimer was treated with thiol agents demonstrated that the dimer was linked via a disulphide bond and that the intermolecular disulphide bond was more susceptible to reduction than the intramolecular disulphide bonds. To examine whether dimeric pNR-2/pS2 was secreted by oestrogen-responsive breast cancer cells, which are known to express pNR-2/pS2 mRNA, conditioned medium was separated on non-denaturing polyacrylamide gels, transferred to PVDF membrane and reacted with antiserum against pNR-2/pS2. Monomeric and dimeric pNR-2/pS2 were detected but the majority of the protein reactivity was associated with a larger protein. Treatment of this protein with thiol agents suggested that it is an oligomer containing pNR-2/pS2 linked to another protein by a disulphide bond. These studies suggest that the biological action of pNR-2/pS2 single-domain trefoil protein might involve the formation of homodimers or oligomers with other proteins.

scholarly journals Production and comparison of mature single-domain ‘trefoil’ peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58

1995 ◽  
Vol 308 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
M P Chadwick ◽  
F E B May ◽  
B R Westley
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Gel Filtration ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Exchange Chromatography ◽  
Single Domain ◽  
Trefoil Peptides ◽  
Trefoil Peptide

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the ‘trefoil’ family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.

scholarly journals A novel peptide-processing activity of insect peptidyl-dipeptidase A (angiotensin I-converting enzyme): the hydrolysis of lysyl-arginine and arginyl-arginine from the C-terminus of an insect prohormone peptide

1998 ◽  
Vol 330 (1) ◽  
pp. 61-65 ◽  
Author(s):  
R. Elwyn ISAAC ◽  
Liliane SCHOOFS ◽  
A. Tracy WILLIAMS ◽  
Dirk VEELAERT ◽  
Mohammed SAJID ◽  
...  
Keyword(s):  
Neurosecretory Cells ◽  
Single Domain ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Recognition Sequence ◽  
Angiotensin I Converting Enzyme ◽  
C Terminus ◽  
Processing Activity ◽  
Hydrolysis Of

Insect peptidyl-dipeptidase A [angiotensin I-converting enzyme (ACE)] is a soluble single-domain peptidyl-dipeptidase that has many properties in common with the C-domain of mammalian somatic ACE and with the single-domain mammalian germinal ACE. Mammalian somatic ACE is important in blood homoeostasis, but the role of ACE in insects is not known. Immunocytochemistry has been used to localize ACE in the neuroendocrine system of the locust, Locusta migratoria. Staining was observed in five groups of neurosecretory cells in the brain and suboesophageal ganglion, in the nervi corpori cardiaci, the storage part of the corpora cardiaca and in the nervi corpori allati. In three groups of neurosecretory cells, ACE co-localized with locustamyotropins, suggesting a possible role for the enzyme in the metabolism of these neuropeptides. We demonstrate in vitro a novel activity of ACE that removes pairs of basic amino acid residues from a locustamyotropin peptide extended at the C-terminus with either Gly-Lys-Arg or Gly-Arg-Arg, corresponding to a consensus recognition sequence for endoproteolysis of prohormone proteins by prohormone convertases. The low Km and high kcat values (Km 7.3 and 5.0 μM, kcat 226 and 207 s-1 for the hydrolysis of Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg, respectively) obtained for the hydrolysis of these two peptides by insect ACE means that these peptides, along with mammalian bradykinin, are the most favoured in vitro ACE substrates so far identified. The discovery of this in vitro prohormone-processing activity of insect ACE provides a possible explanation for the intracellular co-localization of the enzyme with locustamyotropin peptides, and provides evidence for a new role for ACE in the biosynthesis of peptide hormones and transmitters.

β-Carotene and protein complex found in microsomal membranes of bovine corpora lutea

1996 ◽  
Vol 1996 ◽  
pp. 69-69
Author(s):  
AJ Holt ◽  
RG Rodway ◽  
JBC Findlay ◽  
HS Sands ◽  
DN Batchelder
Keyword(s):  
Corpus Luteum ◽  
High Performance ◽  
Gel Filtration ◽  
Biological Action ◽  
Corpora Lutea ◽  
Luteal Cells ◽  
Bovine Corpus Luteum ◽  
Microsomal Membranes ◽  
Ruminant Animals ◽  
Β Carotene

The role of β-carotene in the fertility of ruminant animals has long been acknowledged (Friesecke, 1978). Initially this was thought to be due to its action as a vitamin A precursor but recently β-carotene itself has been considered responsible for improving reproductive performance (Hurley & Doane, 1989). The mechanism by which β-carotcne acts is unclear, but as its concentration in the corpus luteum was typically found to be 70μg per gram of tissue, its biological action is probably exerted in this area.β-Carotene has been studied in the bovine corpus luteum using Raman spectroscopy, high performance liquid chromatography (HPLC) and gel filtration chromatography. The structure of β-carotene produces a characteristic Raman spectrum and by utilising an imaging technique, photographs of isolated luteal cells were obtained indicating the regions of β-carotene within them. Differential centrifugation was used to obtain pure subcellular fractions of luteal cells.

Purification and characterization of a thermostable mycelial lectin from basidiomycete Lentinus squarrosulus

Biologia ◽  
2013 ◽  
Vol 68 (6) ◽  
Author(s):  
Ram Singh ◽  
Hemant Kaur ◽  
Pradeep Kumar ◽  
Harpreet Kaur
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Specific Binding ◽  
Action Spectrum ◽  
Gel Filtration ◽  
Biological Action ◽  
Exchange Chromatography ◽  
Blood Type ◽  
Carbohydrate Binding ◽  
Lectin Activity ◽  
Neuraminidase Treatment

AbstractLectins are non-immune carbohydrate-binding proteins or glycoproteins with specific binding sites for certain glycoconjugates. Fungal lectins have been documented for their antitumour, antiproliferative, immunomodulatory, hypotensive and insecticidal effects. In the present study, a mycelial lectin having molecular mass 55 kDa has been purified and characterized from Lentinus squarrosulus. Biological action spectrum of the lectin revealed agglutination of all human blood types (A, B, O, AB), goat, sheep, rabbit and pig erythrocytes. Neuraminidase treatment of blood type O erythrocytes considerably augmented hemagglutination titre. Carbohydrate inhibition studies showed its high affinity to mucin and asialofetuin. Lectin was purified by a combination of ammonium sulphate precipitation, dialysis, ion exchange chromatography and gel filtration chromatography. Optimum pH for lectin activity was observed to be 6.5–8.0 and optimum temperature was 25–30°C. Lectin showed poor pH stability and was stable within pH 7.0–7.5. It was highly thermostable and could withstand temperature upto 70°C. Lectin activity was sensitive to ethylenediaminetetraacetic acid and denaturants.

scholarly journals Influence of the peptide-chain length on disulphide-bond formation in neurohypophysial hormones and analogues

1978 ◽  
Vol 173 (2) ◽  
pp. 403-409 ◽  
Author(s):  
G Moore
Keyword(s):  
Acetic Acid ◽  
Arginine Vasopressin ◽  
Solid Phase ◽  
Bond Formation ◽  
Gel Filtration ◽  
Arginine Vasotocin ◽  
Disulphide Bond ◽  
Peptide Chain ◽  
Phase Method ◽  
Disulphide Bond Formation

(8-Arginine)vasopressin, (8-arginine)vasotocin, oxytocin and oxypressin, the ‘ring’ derivatives pressinamide and tocinamide, and the extended-chain analogues Pro-Arg-Val-(8-arginine)vasopressin and (8-arginine)vasopressinoyl-Ala-Met-Ala-NH(2), were synthesized by the solid-phase method and purified by sequential gel filtration on Sephadex G-15 in 50% acetic acid and 0.2M-acetic acid. Controlled oxidation of the thiol groups of the reduced peptides obtained after deprotection with sodium in liquid ammonia gave rise to products that depended on the length of the peptide chain: (i) nonapeptides gave monomer and dimer species, (ii) hexapeptides produced mixtures containing higher polymers, and (iii) dodecapeptides gave predominantly monomer with some dimerized material. The evidence suggests that the presence of the acyclic tail tripeptide in the nonapeptide hormones induces a conformation in the preceding hexapeptide that favours the formation of an intramolecular disulphide bond. For (8-arginine)vasopressin, intramolecular disulphide-bond formation is enhanced by extension of the peptide chain from either the N- or the C-terminus. The possible significance of these studies to neurohypophysial hormone-prohormone relationships is discussed.

p53 domains: structure, oligomerization, and transformation

1994 ◽  
Vol 14 (8) ◽  
pp. 5182-5191
Author(s):  
P Wang ◽  
M Reed ◽  
Y Wang ◽  
G Mayr ◽  
J E Stenger ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Gel Filtration ◽  
Dominant Negative ◽  
Wild Type ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Wild Type P53 ◽  
Negative Phenotype

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

scholarly journals Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

2002 ◽  
Vol 68 (4) ◽  
pp. 1980-1987 ◽  
Author(s):  
Yolanda Sanz ◽  
Fidel Toldrá
Keyword(s):  
Specific Activity ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Sulfhydryl Group ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Lactobacillus Sakei ◽  
C Terminus ◽  
Arginine Aminopeptidase

ABSTRACT An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu2+, Hg2+, and Zn2+) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The Km values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.

scholarly journals Plasmin alters the activity and quaternary structure of human plasma carboxypeptidase N

2005 ◽  
Vol 388 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Mercy O. QUAGRAINE ◽  
Fulong TAN ◽  
Hironori TAMEI ◽  
Ervin G. ERDÖS ◽  
Randal A. SKIDGEL
Keyword(s):  
Quaternary Structure ◽  
Gel Filtration ◽  
Catalytic Subunit ◽  
Surface Proteins ◽  
Covalent Bonds ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
C Terminus ◽  
Carboxypeptidase N ◽  
Structure And Activity

Human CPN (carboxypeptidase N) is a tetrameric plasma enzyme containing two glycosylated 83 kDa non-catalytic/regulatory subunits that carry and protect two active catalytic subunits. Because CPN can regulate the level of plasminogen binding to cell surface proteins, we investigated how plasmin cleaves CPN and the consequences. The products of hydrolysis were analysed by activity assays, Western blotting, gel filtration and sequencing. When incubated with intact CPN tetramer, plasmin rapidly cleaved the 83 kDa subunit at the Arg457–Ser458 bond near the C-terminus to produce fragments of 72 and 13 kDa, thereby releasing an active 142 kDa heterodimer, and also cleaved the active subunit, decreasing its size from 55 kDa to 48 kDa. Further evidence for the heterodimeric form of CPN was obtained by re-complexing the non-catalytic 72 kDa fragment with recombinant catalytic subunit or by immunoprecipitation of the catalytic subunit after plasmin treatment of CPN using an antibody specific for the 83 kDa subunit. Upon longer incubation, plasmin cleaved the catalytic subunit at Arg218–Arg219 to generate fragments of 27 kDa and 21 kDa, held together by non-covalent bonds, that were more active than the native enzyme. These data show that plasmin can alter CPN structure and activity, and that the C-terminal 13 kDa fragment of the CPN 83 kDa subunit is a docking peptide that is necessary to maintain the stable active tetrameric form of human CPN in plasma.

scholarly journals Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence of protein SCMKB-IIIA3

1973 ◽  
Vol 133 (4) ◽  
pp. 641-654 ◽  
Author(s):  
Louis S. Swart ◽  
Thomas Haylett
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Minor Component ◽  
Amino Acid Sequences ◽  
Edman Degradation ◽  
Merino Wool ◽  
C Terminus ◽  
A Minor

The complete amino acid sequences of wool protein SCMKB-IIIA3 (131 residues) and a minor component SCMKB-IIIA3A (130 residues) have been determined. The proteins are mutually homologous and have free threonine as the N-terminal residue and carboxymethylcysteine as the C-terminus. The peptides used for the sequence work were obtained by trypsin, thermolysin, pepsin and chymotrypsin digestions and were fractionated by chromatography on DEAE-cellulose, gel filtration on Sephadex G-25 and G-50, paper chromatography and electrophoresis. The Edman degradation method (employing both the Beckman Sequencer and a non-automatic procedure) was used to obtain the sequences of the peptides.

scholarly journals Bovine lens aldehyde dehydrogenase. Purification and preliminary characterization

1983 ◽  
Vol 215 (2) ◽  
pp. 351-359 ◽  
Author(s):  
H H Ting ◽  
M J C Crabbe
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Bovine Lens ◽  
Systemic Corticosteroids ◽  
Apparent Homogeneity ◽  
Stokes Radius ◽  
Equilibrium Ultracentrifugation

Cytoplasmic aldehyde dehydrogenase from bovine lens was purified to apparent homogeneity by using ion-exchange and affinity chromatography. Sedimentation-equilibrium ultracentrifugation, gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis show that the enzyme is a dimer of Mr 114000, with subunits of Mr 57000. The enzyme does not dissociate into monomers in the presence of Ca2+ or Mg2+. The enzyme has a pI of 5.0, an activation energy of 35.1kJ/mmol and a pK value of 8.6 with acetaldehyde as substrate. The enzyme is a prolate ellipsoid with a Stokes radius of 4nm. Progesterone, deoxycorticosterone and chlorpropamide inhibited enzyme activity, and this inhibition may play a role in cataract formation in patients maintained on systemic corticosteroids and in tablet-dependent diabetics.

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