Mechanism Of The Inactivation Of Trypsin By Antithrombin III

1981 ◽  
Author(s):  
A Danielsson ◽  
I Björk
Keyword(s):  
Disulfide Bonds ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
General Mechanism ◽  
Reducing Conditions ◽  
Sds Page ◽  
Activity Measurements ◽  
General Aspects ◽  
Modified Forms

The reaction between bovine antithrombin III (AT) and bovine trypsin was'studied and compared to the inactivation of thrombin by the inhibitor in order to elucidate general aspects of the mechanism of AT action. AT and trypsin formed inactive 1:1 complexes, as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/ PAGE) and trypsin activity measurements. In the absence of heparin, the reaction was about 100-fold faster than the AT-thrombin reaction. The reaction rate increased when AT was preincubated with heparin before trypsin was added. Purified AT-trypsin complex dissociated at pH 10 into free enzyme and two proteolytically modified forms of AT, while no intact AT appeared. In SDS/PAGE, the two modified inhibitors gave bands corresponding to apparent molecular weights of 52000 and 48000 under reducing conditions, while both forms co-migrated with intact AT (mol wt 56000) under non-reducing conditions. This indicates that each of the two modified forms of AT had been cleaved into two or more chains held together by disulfide bonds. Under reducing conditions, the larger of the modified AT chains co-migrated grated with the large chain of thrombin-modified AT, i.e. the form of AT which dissociates from the AT-thrombin complex and which is cleaved at the reactive Arg-Ser bond of the inhibitor. Control experiments showed that the smaller of the two chains was formed by tryptic cleavage of the larger chain. Antisera specific for thrombin-modified AT reacted with purified AT-trypsin complex, demonstrating that the inhibitor was present in the complex in a form immunologically identical to thrombin-modified AT. An analogous finding has been reported earlier for the anti thrombin- thrombin complex. Together, these results suggest the same general mechanism for inhibition of trypsin and thrombin by AT.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

scholarly journals Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1206-1212 ◽  
Author(s):  
RJ Olds ◽  
DA Lane ◽  
R Caso ◽  
M Panico ◽  
HR Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Sds Page ◽  
Additional Support ◽  
Variant Protein

Abstract Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin- Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

scholarly journals Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1206-1212 ◽  
Author(s):  
RJ Olds ◽  
DA Lane ◽  
R Caso ◽  
M Panico ◽  
HR Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Sds Page ◽  
Additional Support ◽  
Variant Protein

Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin- Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

scholarly journals Bovine Herpesvirus 1 Glycoprotein M Forms a Disulfide-Linked Heterodimer with the UL49.5 Protein

1998 ◽  
Vol 72 (4) ◽  
pp. 3029-3036 ◽  
Author(s):  
S. X. Wu ◽  
X. P. Zhu ◽  
G. J. Letchworth
Keyword(s):  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Herpesvirus ◽  
In Vitro Translation ◽  
Bovine Herpesvirus 1 ◽  
Western Blots ◽  
Sds Page ◽  
Herpesvirus 1

ABSTRACT Nine glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, and gL) have been identified in bovine herpesvirus 1 (BHV-1). gM has been identified in many other alpha-, beta-, and gammaherpesviruses, in which it appears to play a role in membrane penetration and cell-to-cell fusion. We sought to express BHV-1 open reading frame UL10, which encodes gM, and specifically identify the glycoprotein. We corrected a frameshift error in the published sequence and used the corrected sequence to design coterminal peptides from the C terminus. These were expressed as glutathione S-transferase fusion proteins inEscherichia coli. The fusion protein containing the 63 C-terminal amino acids from the corrected gM sequence engendered antibodies that immunoprecipitated a 30-kDa protein from in vitro translation reactions programmed with the UL10 gene. Proteins immunoprecipitated by this antibody from virus-infected cells ran at 36 and 43 kDa in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 and 48 kDa in nonreducing SDS-PAGE. Only the larger of the pair was present in virions. A 7-kDa protein was released from gM by reducing agents. The 7-kDa protein was not recognized in Western blots probed with the anti-gM antibody but reacted specifically with antibodies prepared against BHV-1 UL49.5, previously reported to be a 9-kDa protein associated with an unidentified 39-kDa protein (X. Liang, B. Chow, C. Raggo, and L. A. Babiuk, J. Virol. 70:1448–1454, 1996). This is the first report of a small protein covalently bound to any herpesvirus gM. Similar patterns of hydrophobic domains and cysteines in all known gM and UL49.5 homologs suggest that these two proteins may be linked by disulfide bonds in all herpesviruses.

scholarly journals Characterization of Immunochemically Nonreactive Urinary Albumin

2004 ◽  
Vol 50 (12) ◽  
pp. 2286-2291 ◽  
Author(s):  
Tanya M Osicka ◽  
Wayne D Comper
Keyword(s):  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Clinical Importance ◽  
Urinary Albumin ◽  
Albumin Concentration ◽  
Urinary Proteins ◽  
Native Page ◽  
Sds Page ◽  
Urinary Albumin Concentration

Abstract Background: Conventional immunoassays underestimate the urinary albumin concentration because intact albumin in urine exists in two forms, immunoreactive and immunochemically nonreactive. Methods: Urinary albumin concentration measured by HPLC (which measures total albumin, i.e., the sum of immunoreactive albumin + immunochemically nonreactive albumin) or RIA was compared with densitometric analysis of albumin bands in diabetic urine samples separated by either native polyacrylamide gel electrophoresis (PAGE) or reducing sodium dodecyl sulfate (SDS)-PAGE. Immunochemically nonreactive albumin was also isolated from diabetic urine (relative amount detected, 70–80% of the expected) and was tested for contamination by common urinary proteins by native PAGE, ELISA, and capillary electrophoresis. Results: Urinary albumin concentrations measured by native PAGE and HPLC were better correlated (r2 = 0.83) than concentrations measured by native PAGE and RIA (r2 = 0.62) because under native conditions both native PAGE and HPLC detect total albumin and not only the immunoreactive albumin alone that is measured by RIA. Urinary albumin concentrations measured by reducing SDS-PAGE and RIA were better correlated (r2 = 0.84) than concentrations measured by reducing SDS-PAGE and HPLC (r2 = 0.65) because under reducing conditions immunochemically nonreactive albumin is unstable and fragments into many smaller peptides. The partially purified preparation was found to contain <1% contamination by common urinary proteins and is stable to freezing and frequent freeze/thaw cycles. Conclusions: The results are consistent with the interpretation that immunochemically nonreactive albumin has a limited number of polypeptide chain scissions and is held together by noncovalent intrachain bonding and disulfide bonds. Detection of this molecule is likely to be of clinical importance in diagnosing kidney disease as well as cardiovascular disease.

scholarly journals PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF A T/TN SPECIFIC LECTIN FROM LEPECHINIA BULLATA SEEDS (LAMIACEAE)

2017 ◽  
Vol 9 (10) ◽  
pp. 165
Author(s):  
Wilches Torres A. ◽  
Rojas Caraballo J. ◽  
Sanabria E. ◽  
Reyes MontaÑo E ◽  
FernÁndez Alonso Jl ◽  
...  
Keyword(s):  
Isoelectric Focusing ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reducing Conditions ◽  
Sds Page ◽  
Unique Band

Objective: This study focused on purifying and characterizing a lectin from Lepechinia bullata (L. bullata) seeds, and determining its specificity towards tumour-associated carbohydrate-antigens.Methods: Pigments were removed by washing the seeds with NH4OH 0.1 M pH 9.4 and treating the crude extracts with Pectinex®. The purification procedure consisted of anion exchange chromatography on diethylaminoethyl (DEAE)-Sephadex followed by affinity chromatography. For the characterization, the phase was used polyacrylamide gel electrophoresis-sodium dodecyl sulphate (SDS-PAGE), isoelectric focusing, hemagglutination assays, enzyme-linked lectinosorbent assay (ELLA) and thermal shift assay (TSA).Results: 6.2 mg of lectin were obtained from 100 g of seeds. It was able to agglutinate enzymatically treated erythrocytes with a minimal required lectin concentration of 7 μg. ml-1. Strong binding to asialo bovine submaxillary mucine (aBSM) was determined, corroborating Tn recognition.The isoelectric focusing showed a unique band at pH 8.5. Lectin pure shown bands at 28, 48 and 93 kDa by SDS-PAGE, with an incomplete dissociation of the last species despite trying several reduction conditions. By preparative electrophoresis under different conditions, three species were observed too, in all fractions one band at 28 kDa on Tricine-PAGE in reducing and no reducing conditions were found.Amino acid composition, carbohydrate content, thermal stability and Ca2+and Mn2+requirements were determined. N-acetylgalactosamine (GalNAc) and desialylated mucins inhibited the agglutinant activity on human cells. Fetuin inhibited hemagglutination of rabbit erythrocytes.Conclusion: A new lectin was isolated and characterized from L. bullata seeds, it recognizes T/Tn antigen and shows some similarities with other Lamiaceae lectins.

scholarly journals Iodine-mediated inactivation of lipid- and nonlipid-enveloped viruses in human antithrombin III concentrate

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 791-796 ◽  
Author(s):  
F Highsmith ◽  
H Xue ◽  
X Chen ◽  
L Benade ◽  
J Owens ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Albumin ◽  
Enveloped Viruses ◽  
Complete Inactivation ◽  
Ensure Patient Safety ◽  
Sds Page ◽  
At Iii

Human plasma-derived protein concentrates intended for clinical use must be treated for viral inactivation to ensure patient safety. This study explored the use of liquid iodine for inactivation of several lipid- and nonlipid-enveloped viruses in an antithrombin III (AT-III) concentrate. Iodine at levels of 0.01% to 0.02% caused between 43% and 94% loss of AT-III activity, as well as degradation of AT-III as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. However, addition of up to 0.1% human albumin protected the AT-III against both inactivation and fragmentation. At albumin levels sufficient to retain greater than 75% of AT-III activity, greater than 6 logs of sindbis, encephalomyocarditis, and vesicular stomatitis viruses, greater than 4 logs of pseudorabies, and greater than 3 logs of human immunodeficiency virus were inactivated. Except with sindbis virus, this represented complete inactivation of all the viruses spiked into the AT-III concentrate.

In Vitro Characterization of High Purity Factor IX Concentrates for the Treatment of Hemophilia B

1995 ◽  
Vol 73 (04) ◽  
pp. 584-591 ◽  
Author(s):  
Steven A Limentani ◽  
Kerry P Gowell ◽  
Steven R Deitcher
Keyword(s):  
High Purity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Ix ◽  
Factor Vii ◽  
Dominant Factor ◽  
Factor X ◽  
Reducing Conditions ◽  
Sds Page ◽  
Activation Products

SummaryThis study employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting to assess the purity of seven high purity factor IX concentrates: Aimafix (Aima), AlphaNine-SD (Alpha Therapeutic), Factor IX VHP (Biotransfusion), Immunine (Immuno), Mononine (Armour Pharmaceutical), Nanotiv (Kabi Pharmacia), and 9MC (Blood Products Laboratory). The mean specific activity of these products ranged from 68 U factor IX/mg (Aimafix) to 246 U factor IX/mg (Mononine). SDS-PAGE analysis showed that the highest purity product, Mononine, had a single contaminating band under non-reducing conditions. Two additional bands were detected when this product was analyzed under reducing conditions. All other products had multiple contaminating bands that were more apparent under reducing than non-reducing conditions. The immunoblot for factor IX showed a dominant factor IX band for all products. In addition, visible light chain of factor IX was detected for AlphaNine-SD, Factor IX VHP, Immunine, Mononine, Nanotiv, and 9MC, suggesting that the factor IX in these products had undergone partial activation to factor IXa. Another contaminating band was visible at 49,500 for all of the products except 9MC. In addition to this band, high molecular weight contaminants were apparent for some products, most notably AlphaNine-SD. The identity of these bands is unknown. Immunoblotting failed to demonstrate factor VII as a contaminant of any of the high purity products, although factor Vila could be detected in some lots of Immunine, Nanotiv, and 9MC by a clot-based assay. Factor X contaminated Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Nanotiv, and 9MC, but activation products of factor X were not detected. Prothrombin contaminated all of the products except Mononine. Activation products of prothrombin were identified for three of four lots of Immunine and for one lot of Factor IX VHP. These results thus demonstrate that high purity factor IX concentrates differ substantially in the degree to which they are contaminated by potentially thrombogenic materials.

scholarly journals Multimeric Association of Purified Novel Bowman-Birk Inhibitor From the Medicinal Forage Legume Mucuna pruriens (L.) DC.

2021 ◽  
Vol 12 ◽  
Author(s):  
Jafar K. Lone ◽  
Mandapanda A. Lekha ◽  
Rajiv P. Bharadwaj ◽  
Fasil Ali ◽  
M. Arumugam Pillai ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Specific Activity ◽  
Therapeutic Potential ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mucuna Pruriens ◽  
Reducing Conditions ◽  
Functional Efficiency ◽  
Sds Page ◽  
Functional Features

A Bowman-Birk protease, i.e., Mucuna pruriens trypsin inhibitor (MPTI), was purified from the seeds by 55.702-fold and revealed a single trypsin inhibitor on a zymogram with a specific activity of 202.31 TIU/mg of protein. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under non-reducing conditions, the protease trypsin inhibitor fraction [i.e., trypsin inhibitor non-reducing (TINR)] exhibited molecular weights of 74 and 37 kDa, and under reducing conditions [i.e., trypsin inhibitor reducing (TIR)], 37 and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 and 18 kDa protein bands on SDS–PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 56.085, and 74.78 kDa on ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization/quadrupole time-of-flight-mass spectrometry (ESI/QTOF-MS). Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Furthermore, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by the UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of Bowman-Birk inhibitors (BBIs). The multimeric association helps to achieve more stability, thus enhancing their functional efficiency. MPTI was found to be a competitive inhibitor which again suggested that it belongs to the BBI family of inhibitors, displayed an inhibitor constant of 1.3 × 10–6 M, and further demonstrates potent anti-inflammatory activity. The study provided a comprehensive basis for the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in the native state from M. pruriens.

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.

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