scholarly journals Functional characterization of thrombin Salakta: an abnormal thrombin derived from a human prothrombin variant

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 556-561 ◽  
Author(s):  
A Bezeaud ◽  
J Elion ◽  
MC Guillin
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Functional Characterization ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Specific Pattern ◽  
Factor V ◽  
Functional Abnormality ◽  
V Protein ◽  
Fibrinogen Binding

Abstract The genetic variant prothrombin Salakta has been described in a patient presenting with a normal level of prothrombin antigen but reduced prothrombin activity. Initial studies indicated that factor Xa- catalyzed cleavages proceed normally but lead to the production of a thrombin molecule with an altered enzymatic activity. To characterize the functional abnormality of thrombin Salakta more precisely, it was purified by chromatography on heparin-Sepharose and diethylaminoethyl- Sephadex. The purified variant does not differ from normal thrombin by size, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and is 93.1% +/- 7.6% active by titration with p- nitrophenyl-p'-guanidinobenzoate. Its activity, however, is altered to various extents toward the following substrates: H-D-phenylalanyl-L- pipecolyl-L-arginine paranitroanilide (S 2238), fibrinogen, factor V, protein C, and antithrombin III. The Michaelis constant (Km) of thrombin Salakta for S 2238 is higher (12.2 +/- 3.3 mumol/L) than normal (2.8 +/- 0.7 mumol/L), whereas the turnover number (Kcat) is normal (84.4 +/- 6.6 s-1 v 85.9 +/- 14.0 s-1 for normal thrombin). The interaction of thrombin Salakta with benzamidine is also altered as evidenced by an increased inhibition constant (Ki = 3.5 mmol/L v 0.28 mmol/L for normal thrombin). The inability of fibrinogen to act as a competitor in the inactivation of thrombin Salakta by diisopropylfluorophosphate clearly indicates that fibrinogen binding to the fibrinopeptide groove is drastically impaired. In contrast, interactions involving sites remote from the active site such as those with fibrin and thrombomodulin are only slightly impaired. These results indicate that thrombin Salakta exhibits a specific pattern of functional alterations different from those reported for other variants. The structural defect seems to affect essentially the primary substrate binding site and to a lesser extent recognition site(s) remote from the catalytic site such as those for fibrin and thrombomodulin.

scholarly journals Functional characterization of thrombin Salakta: an abnormal thrombin derived from a human prothrombin variant

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 556-561
Author(s):  
A Bezeaud ◽  
J Elion ◽  
MC Guillin
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Functional Characterization ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Specific Pattern ◽  
Factor V ◽  
Functional Abnormality ◽  
V Protein ◽  
Fibrinogen Binding

The genetic variant prothrombin Salakta has been described in a patient presenting with a normal level of prothrombin antigen but reduced prothrombin activity. Initial studies indicated that factor Xa- catalyzed cleavages proceed normally but lead to the production of a thrombin molecule with an altered enzymatic activity. To characterize the functional abnormality of thrombin Salakta more precisely, it was purified by chromatography on heparin-Sepharose and diethylaminoethyl- Sephadex. The purified variant does not differ from normal thrombin by size, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and is 93.1% +/- 7.6% active by titration with p- nitrophenyl-p'-guanidinobenzoate. Its activity, however, is altered to various extents toward the following substrates: H-D-phenylalanyl-L- pipecolyl-L-arginine paranitroanilide (S 2238), fibrinogen, factor V, protein C, and antithrombin III. The Michaelis constant (Km) of thrombin Salakta for S 2238 is higher (12.2 +/- 3.3 mumol/L) than normal (2.8 +/- 0.7 mumol/L), whereas the turnover number (Kcat) is normal (84.4 +/- 6.6 s-1 v 85.9 +/- 14.0 s-1 for normal thrombin). The interaction of thrombin Salakta with benzamidine is also altered as evidenced by an increased inhibition constant (Ki = 3.5 mmol/L v 0.28 mmol/L for normal thrombin). The inability of fibrinogen to act as a competitor in the inactivation of thrombin Salakta by diisopropylfluorophosphate clearly indicates that fibrinogen binding to the fibrinopeptide groove is drastically impaired. In contrast, interactions involving sites remote from the active site such as those with fibrin and thrombomodulin are only slightly impaired. These results indicate that thrombin Salakta exhibits a specific pattern of functional alterations different from those reported for other variants. The structural defect seems to affect essentially the primary substrate binding site and to a lesser extent recognition site(s) remote from the catalytic site such as those for fibrin and thrombomodulin.

scholarly journals Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 565-569
Author(s):  
T Inomoto ◽  
A Shirakami ◽  
S Kawauchi ◽  
T Shigekiyo ◽  
S Saito ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Molecular Defect ◽  
Sds Page

A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of “prothrombin” by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of “prothrombin.” Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of “thrombin.” Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of “thrombin” when Boc-Val- Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p′-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as “thrombin” in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than “thrombin” when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as “thrombin” in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.

scholarly journals Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 565-569 ◽  
Author(s):  
T Inomoto ◽  
A Shirakami ◽  
S Kawauchi ◽  
T Shigekiyo ◽  
S Saito ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Molecular Defect ◽  
Sds Page

Abstract A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of “prothrombin” by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of “prothrombin.” Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of “thrombin.” Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of “thrombin” when Boc-Val- Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p′-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as “thrombin” in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than “thrombin” when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as “thrombin” in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

scholarly journals Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Preeti Anand ◽  
Jay Prakash Pandey ◽  
Dev Mani Pandey
Keyword(s):  
San Francisco ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Evolutionary Genetics ◽  
Imaging Analysis ◽  
Silk Cocoon ◽  
Natural Color ◽  
Sds Page

Abstract Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

scholarly journals Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 508-514 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Time Dependent ◽  
Binding Studies ◽  
Fibrinogen Binding ◽  
Human Thrombin ◽  
Membrane Bound ◽  
Independent Association ◽  
Triton X

Abstract Previous studies indicated a correlation between the formation of EDTA- resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA- resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA- resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet- fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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