Non-Specific Binding of Thiocholine Ester of Cinnamic Acid to Fibrin

Thiocholine esters inhibit the enzymatic crosslinking of fibrin (1). Thiocholine ester of 14C-labelled cinnamic acid (2-diethylbenzylaminoethyl thioltranscinnamate bromide) was prepared and incubated with human fibrinogen in the presence of factor XIII and thrombin. Polyacrylamide gel electrophoresis with sodium dodecyl sulphate, followed by liquid scintillation counting of the separated protein bands, showed that all three chains of fibrinogen had bound the crosslinking inhibitor(2 moles/mol α chain, 1.5 moles/mol β chain, 2.2 moles/mol γ chain). Cyanogen bromide cleavage of the isolated α chain resulted in 6 major fragments all of whichwere radioactive. These results indicate that the binding of the thiocholine ester of cinnamic acid was not restricted to the specific donor crosslinking sites of fibrin(ogen). This thiocholine ester differs in this respect from dansyl-cadaverine which binds exclusively to the acceptor sites involved in fibrin(ogen) crosslinking.

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Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells

Biochemical Journal ◽

10.1042/bj2360665 ◽

1986 ◽

Vol 236 (3) ◽

pp. 665-670 ◽

Cited By ~ 28

Author(s):

W P Gati ◽

J A Belt ◽

E S Jakobs ◽

J D Young ◽

S M Jarvis ◽

...

Keyword(s):

Gel Electrophoresis ◽

Plasma Membranes ◽

Specific Binding ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Hepatoma Cells ◽

Nucleoside Transport ◽

Facilitated Diffusion ◽

Animal Cells ◽

Novikoff Hepatoma

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

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Quantificação de [32P] fosfoproteínas em gel de pollacrilamida pela Radiação Cerenkov: vantagens sobre outros procedimentos

Ciência e Natura ◽

10.5902/2179460x26714 ◽

1997 ◽

Vol 19 (19) ◽

pp. 21

Author(s):

Carlos A. Gonçalves ◽

Guido Lenz ◽

Christianne Salbego ◽

Carmem Gottfried

Keyword(s):

Liquid Scintillation Counting ◽

Gel Electrophoresis ◽

Liquid Scintillation ◽

Polyacrylamide Gel Electrophoresis ◽

Scintillation Counting ◽

Polyacrylamide Gel ◽

Cerenkov Counting ◽

Phosphorylation Of Proteins ◽

Phosphate Incorporation

The phosphorylation of proteins has been recognized as an important mechanism for controlling cellular activities. There are many ways to measure 32P-labellingof phosphoproteins resolved by polyacrylamide gel electrophoresis, including densitometry of autoradiographs, liquid scintillation counting and Cerenkov counting. This report compares such different procedures and indicates the advantages of Cerenkov counting to determine radioactive phosphate incorporation into proteins.

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The αEC domain of human fibrinogen-420 is a stable and early plasmin cleavage product

Blood ◽

10.1182/blood.v95.7.2297 ◽

2000 ◽

Vol 95 (7) ◽

pp. 2297-2303 ◽

Cited By ~ 6

Author(s):

Dianne Applegate ◽

Lara Stoike Steben ◽

Kathe M. Hertzberg ◽

Gerd Grieninger

Keyword(s):

Gel Electrophoresis ◽

Degradation Products ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cleavage Product ◽

The Other ◽

Cross Linking ◽

Human Fibrinogen ◽

Western Blots ◽

Sds Page

Abstract Human fibrinogen-420, (Eβγ)2, was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (βγ)2, were found to be similar, suggesting little impact of the unique EC domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the EC domain. One was a stable fragment (ECX) comigrating with a 34-kd yeast recombinant EC domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

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Turnover of acyl-CoA-binding protein in four different cell lines measured by using two-dimensional polyacrylamide-gel electrophoresis

Biochemical Journal ◽

10.1042/bj2970555 ◽

1994 ◽

Vol 297 (3) ◽

pp. 555-560 ◽

Cited By ~ 5

Author(s):

C L Buus ◽

K Kristiansen ◽

J Knudsen

Keyword(s):

Cell Lines ◽

Total Protein ◽

Gel Electrophoresis ◽

Liquid Scintillation ◽

Binding Protein ◽

Biological Activities ◽

Polyacrylamide Gel Electrophoresis ◽

Growth Conditions ◽

Scintillation Counting ◽

Two Dimensional

Acyl-CoA-binding protein (ACBP), also named diazepam-binding inhibitor or endozepine, is a 10 kDa protein for which a surprisingly large number of biological activities has been suggested. Some of these would seem to require a rapid intracellular turnover of the protein. In this paper we report on the turnover of ACBP in cell lines derived from mouse, rat and man. ACBP was identified in two-dimensional gels by using specific antibodies. Cells were labelled with [35S]methionine and chased for various periods of time. Total protein was extracted, subjected to two-dimensional PAGE, and radioactivity in the spot containing ACBP was determined by liquid-scintillation counting. ACBP half-life was determined, and varied from 25 to 53 h depending on the cell line and the growth conditions. In all cases, radioactivity in ACBP was lost slightly faster than radioactivity in total protein. These results are discussed in relation to the possible function suggested for ACBP.

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The αEC domain of human fibrinogen-420 is a stable and early plasmin cleavage product

Blood ◽

10.1182/blood.v95.7.2297.007k39_2297_2303 ◽

2000 ◽

Vol 95 (7) ◽

pp. 2297-2303 ◽

Cited By ~ 1

Author(s):

Dianne Applegate ◽

Lara Stoike Steben ◽

Kathe M. Hertzberg ◽

Gerd Grieninger

Keyword(s):

Gel Electrophoresis ◽

Degradation Products ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cleavage Product ◽

The Other ◽

Cross Linking ◽

Human Fibrinogen ◽

Western Blots ◽

Sds Page

Human fibrinogen-420, (Eβγ)2, was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (βγ)2, were found to be similar, suggesting little impact of the unique EC domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the EC domain. One was a stable fragment (ECX) comigrating with a 34-kd yeast recombinant EC domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

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Isolation and renaturation of α2-macroglobulin receptor from diploid human fibroblasts

Biochemical Journal ◽

10.1042/bj2140629 ◽

1983 ◽

Vol 214 (2) ◽

pp. 629-631 ◽

Cited By ~ 14

Author(s):

J Frey ◽

E G Afting

Keyword(s):

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Specific Binding ◽

Human Fibroblasts ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Single Step ◽

Polyacrylamide Gel ◽

Human Diploid Fibroblasts ◽

Α2 Macroglobulin

alpha 2-Macroglobulin receptor was extracted from human diploid fibroblasts and purified by affinity chromatography in a single step. The receptor had mol.wt. 125 000 after sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. The isolated receptor was separated by SDS/polyacrylamide-gel electrophoresis, transferred on to nitrocellulose sheets and subsequently renatured, as shown by a specific binding test, by incubation with Nonidet P40.

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Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645062 ◽

1990 ◽

Vol 63 (03) ◽

pp. 439-444 ◽

Cited By ~ 19

Author(s):

C Kuyas ◽

A Haeberli ◽

P Walder ◽

P W Straub

Keyword(s):

Gel Electrophoresis ◽

Affinity Purification ◽

Polyacrylamide Gel Electrophoresis ◽

Ease Of Use ◽

Terminal Sequence ◽

Purification Method ◽

Human Fibrinogen ◽

Isolation Methods ◽

One Step ◽

Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

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Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646313 ◽

1992 ◽

Vol 68 (05) ◽

pp. 534-538 ◽

Cited By ~ 5

Author(s):

Nobuhiko Yoshida ◽

Shingi Imaoka ◽

Hajime Hirata ◽

Michio Matsuda ◽

Shinji Asakura

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl Sulfate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Tetraacetic Acid ◽

Fibrin Monomer ◽

Sds Page ◽

Thrombotic Tendency ◽

Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

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Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657115 ◽

1982 ◽

Vol 47 (01) ◽

pp. 014-018 ◽

Cited By ~ 13

Author(s):

H Sumi ◽

N Toki ◽

S Takasugi ◽

S Maehara ◽

M Maruyama ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Chromatography ◽

Urinary Trypsin Inhibitor ◽

Plasmin Activity ◽

Molecular Weights ◽

Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

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Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

YARSI Medical Journal ◽

10.33476/jky.v26i2.394 ◽

2018 ◽

Vol 26 (2) ◽

pp. 058

Author(s):

Anna P. Roswiem ◽

Triayu Septiani

Keyword(s):

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Sds Page

Bahan baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.

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