MicroELISA method for the determination of thymosin β9 discriminating between thymosin β9 and the structurally closely related thymosin β4

Mihelić, M.; Kalbacher, Hubert; Hannappel, Ewald; Voelter, Wolfgang

doi:10.1016/0022-1759(89)90328-1

Cited by 17 publications

(11 citation statements)

References 11 publications

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“…The conjugate obtained by crosslinking with MBS was further characterized by Western blotting. Using antiserum against T/I4 exhibiting 35% cross-reactivity against T/?9 [15] in an ELISA, we were able to detect T/39 in the covalently AK AKj39 AK linked state (data not shown). Based on this possibility, and using the same antiserum in combination with antibodies against actin, we could show that the 47 kDa band seen in SDS-PAGE contains both actin and T/?9 ( Fig.…”

Section: Resultsmentioning

confidence: 95%

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Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

et al. 1993

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By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB-actin decreased significantly on addition of thymosinp4 (T/94), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation, Based on this effect, we determined the Kn value of the thymosin @l complex as 0.8 PM, a value that is in agreement with previous determinations. In addition to the main compound thymosin/?4, bovine tissue contains a related peptide, thymosin p (Tp9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that T/$?, similar to Tp4, forms a 1:l complex with monomeric actin, and hereby inhibits actin polymerization. With a K, value of 1.1 FM the affinity of TjT9 is in the same range as that of Tp4, suggesting that T@9, like T/?4, contributes to maintaining the pool of monomeric. actin in bovine non-muscle cells. Further proof of the interaction of T/I9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslink~g experiments. Using different crosslinking reagents, like water-soluble ~~iimide (EDC), ~-maleimido~~oyl-~-hydroxys~nimi~te (MBS), and disu~inimidylsu~rate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tfi9 could be identified by imm~oblot~ng. When, in the MBS crosslinking experiments, native actin was replaced with (374-NEM)-actin, the 47 kDa band was not seen, indicating that Cys-374 takes part in the thiol-specific crosslinking reaction. This suggests that part of the binding site of T/39 must be located close to the carboxy-terminus.

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Section: Resultsmentioning

confidence: 95%

“…K Tp9 was isolated together with Tp4 from bovine lung, following a procedure described for the isolation of these peptides from bovine tissue [15]. Separation of T/I9 from Tp4 was achieved using preparative isoelectric focussing based on different pI values of the two peptides, which are 4.6 and 5.1, respectively.…”

Section: Methodsmentioning

confidence: 99%

Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

et al. 1993

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“…The endogenous Nterminal tetrapeptide of thymosin b 4 was found to inhibit bone-marrow haematopoietic cell and hepatocyte proliferation [19,20]. Some members of the bthymosin family as well as their fragments have been synthesized using different approaches [21±25] and used for immunization and development of specific antibodies [24,26,27]. In order to extend the phylogenetic distribution of this peptide family, regulating fundamental cell processes, for the first time a b-thymosin from an invertebrate, the sea urchin, has been isolated and its sequence determined.…”

Section: Introductionmentioning

confidence: 99%

A novel β-thymosin from the sea urchin: extending the phylogenetic distribution of β-thymosins from mammals to echinoderms

1997

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The study of the phylogenetic distribution of the beta-thymosin family is important to elucidate its biological function further. A new thymosin, designated as thymosin beta 14, consisting of 40 amino acid residues and with a molecular weight of 4537 Da as determined by ion spray mass spectrometry, was isolated from the sea urchin. The N-terminus of this polypeptide is blocked by an acetyl group as found by matrix-assisted laser desorption mass spectrometric and amino acid analysis. The primary structure was elucidate by Edman degradation of the HPLC-purified thymosin beta 14 fragments produced by digestion with endoproteinase Asp-N and trypsin. Sequence comparison reveals that thymosin beta 14 is 73% homologous to thymosin beta 4, obtained from calf thymus. By isolating and characterising the structure of thymosin beta 14 from the sea urchin, an invertebrate, substantial knowledge about the phylogenetic distribution and evolution of beta-thymosins is gained.

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“…According to the results received from the antigenic index plots (Figures 1, 2), the troponin-T segment 34 -62 should be indeed a peptide fragment, suitable for the development of specific antibodies, recognizing the selected segment as well as the parent protein, as we could demonstrate in the past in connection with the development of specific immunoassays for quantitative determinations of thymosin β 9 [36], thymosin β 4 [37,38], transmembrane glycoprotein gp41 env [39], human relaxin [40] or immunostaining experiments of β-thymosins [41,25]. In our laboratory, solution [42], as well as solid phase [43 -45, 26] peptide syntheses are well established for many years, and improved, highly efficient strategies are available.…”

Section: Synthesis Of Troponin-t Fragment 34 -62mentioning

confidence: 98%

Analytical Tools for Rapid, Sensitive, Quantitative Identification of Potential Meat Quality Markers

Voelter¹,

Stoeva

Echner

et al. 2000

J. prakt. Chem.

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Conversion of muscle tissue to meat, including tenderization, is a complex process. As the consumer considers meat tenderness to be the most important criterion for meat quality, this conversion should be elucidated on a biomolecular basis. With such a biochemical background, specific sensitive analytical tools could be developed which would correlate physicochemical parameters with the degree of tenderness.Various events take place at the level of the myofibrils which have been correlated with meat tenderness. These include Z-disk weakening [1], degradation of desmin or titin, leading to fragmentation or weakening of myofibrils, the disappearance of troponin-T and simultaneous appearance of 28 -32 kDa proteins or detection of a 110 kDa protein fragment, as monitored thoughout the conditioning period by electron microscopic studies and SDS polyacrylamide gel electroAbstract. Myofibrillar extracts from bovine Musculus longissimus dorsi (MLD) were subjected to SDS-PAGE, electroblotted and fragments of the 30 kDa band determined by internal and N-terminal Edman sequencing, giving unequivocal proof, troponin-T (TNT) to be the origin of this band. Based on the N-terminal primary sequence of the 30 kDa band, a peptide with high antigenic sites was synthesized, conjugated to keyhole limpet hemocyanin (KLH), antibodies were generated and an enzyme-linked immunosorbent assay (ELI-SA) was developed for the determination of TNT concentrations in meat samples. For routine determinations of meat quality markers it seems more convenient to analyse soluble meat extracts, produced by trichloroacetic acid (TCA) or HCl treatment. In the supernatants of TCA-treated MLDs, prominent peptide fragments from glyceraldehyde-3-phosphate Keywords: Capillary electrophoresis, Immunoassays, Peptides, Proteins, Meat quality markers phoresis, respectively [2 -7]. In particular, the disappearance of troponin-T and the concomitant appearance of 28 -32 kDa proteins seem to be good indicators of post-mortem proteolysis. However, the origin of the 30 kDa fragments was speculative and not determined on a structural basis [8 -10]. Therefore, we recently applied the technique of internal microsequencing to provide for the first time unequivocal proof for the origin of the 30 kDa protein [11]. The 30 kDa protein transferred to ProBlott and Immobilon membranes was digested with trypsin, the peptide mixture separated with HPLC, selected peptides isolated and sequenced. Eight peptides, varying from 5 to 13 amino acid residues, displayed between 50 and 100% homology to rabbit troponin-T, according to our protein homology search. As SDS-PAGE operations are tedious, inconvenient and time-consuming, we suggest in this communication could be separated by HPLC and identified by Edman degradation. Both fragments were found to increase with ageing and might become useful indicators of meat quality. After HPLC separation and structure elucidation of MLD HCl extracts, fructose-biphosphate aldolase, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase and myogl...

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MicroELISA method for the determination of thymosin β9 discriminating between thymosin β9 and the structurally closely related thymosin β4

Cited by 17 publications

References 11 publications

Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

A novel β-thymosin from the sea urchin: extending the phylogenetic distribution of β-thymosins from mammals to echinoderms

Analytical Tools for Rapid, Sensitive, Quantitative Identification of Potential Meat Quality Markers

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