ANTITHROMBIN III NORTHWICK PARK: CHARACTERIZATION OF AN INACTIVE HIGH MW COMPLEX WITH INCREASED AFFINITY FOR HEPARIN

1987 ◽  
Author(s):  
H Erdjument ◽  
D A Lane ◽  
A M Flynn ◽  
H Ireland ◽  
M Panico ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Exchange Chromatography ◽  
Terminal Sequence ◽  
Structural Investigations ◽  
Crossed Immunoelectrophoresis ◽  
Sds Page ◽  
Affected Family Member ◽  
At Iii ◽  
Salt Gradient

It has been shown previously that antithrombin III Northwick Park (AT III NWP) has reduced ability to inactivate thrombin and is characterised by an additional anodal component on crossed immunoelectrophoresis. We have applied plasma from an affected family member to heparin-Sepharose and eluted the AT III with a salt gradient. Evidence will be presented that the anodal component has* higher affinity for heparin than normal AT III. Furthermore, this variant component is present in plasma as a MW >120,000 inactive complex whose tryptic peptide FAB map contains numerous signals not characteristic of normal AT . 111 . This complex can be reduced with dithiothreitol to two non identical bands on SDS PAGE with MW ~60,000, only one of which reacts with anti-AT III. Using ion-exchange chromatography and HPLC these two components have been isolated and separated. The N-terminal sequence of the protein that does not react with anti-AT III is believed to be Asp-Ala-His-Ile-Ser-Glu. Structural investigations on the variant AT III are underway.

Isolation and Partial Characterization of Equine Antithrombin III.

1979 ◽  
Author(s):  
D.W. Estry ◽  
T.G. Bell ◽  
G.H. Tishkoff ◽  
J.C. Mattson ◽  
S.C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

Antithrombin III Avranches, a New Variant with Defective Serine-Protease Inhibition - Comparison with Antithrombin III Charleville

1988 ◽  
Vol 60 (01) ◽  
pp. 094-096 ◽  
Author(s):  
M Aiach ◽  
M Roncato ◽  
G Chadeuf ◽  
P Dezellus ◽  
L Capron ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Protease Inhibition ◽  
Sds Page ◽  
Recurrent Venous Thromboembolism ◽  
Factor Xa Inhibition ◽  
New Variant ◽  
At Iii

SummaryA decreased plasma antithrombin activity in presence or in absence of heparin was discovered in a 47-year-old patient presenting with recurrent venous thromboembolism. The immunoreactive material (AT ΠΙ-IR) was normal. The same biological abnormalities were found in two relatives of the patient, leading to the diagnosis of hereditary qualitative AT III deficiency.The propositus’ AT III was coeluted with normal AT III from an heparin-sepharose column. An additional step of ion-exchange chromatography on a Mono Q column using a FPLC system (Pharmacia, St-Quentin en Yvelines, France) allowed the purification of a protein which was homogenous in SDS-10% polyacrylamide electrophoresis gel (PAGE). AT III purified from propositus’ plasma, normal plasma and the plasma of the patient known to have an AT III variant with defective protease binding (AT III Charleville) were compared. The specific activities measured as heparin cofactor anti thrombin or factor Xa inhibition in absence of heparin were decreased to half the normal value.Kinetic studies confirmed a decreased rate of thrombin inhibi-tion for both abnormal AT III preparations. SDS-PAGE experi-ments performed in purified system and immunoblots obtained from plasma showed that the two variants have different behaviour: in the case of AT III Charleville thrombin induced an apparent 5 Δ increase in molecular mass, probably due to a conformational change. AT III Avranches did not form stoechiometric complexes with thrombin, but was unmodified by the protease.

Isolation and Partial Characterization of Equine Antithrombin III

1979 ◽  
Author(s):  
D. W. Estry ◽  
T. G. Bell ◽  
G. H. Tishkoff ◽  
J. C. Mattson ◽  
S. C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. in order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichiometry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

Characterization Of Rabbit Antithrombin III

1981 ◽  
Author(s):  
D Estry ◽  
J C Mattson ◽  
T G Bell ◽  
G H Tishkoff
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Cross Reactivity ◽  
Antigenic Determinants ◽  
Heparin Binding ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Immunologic Assays

The rabbit is a well established model for studying the disseminated intravascular coagulation (DIC) associated with endotoxic syndromes. In order to establish the role of antithrombin III (AT III) in the modulation of DIC in the rabbit, characterization of rabbit AT III was undertaken. Rabbit antithrombin III, isolated according to modifications of the method of Thaler and Schmer, has a molecular weight comparable to that of human AT III (62,000 daltons) as measured by mobility on SDS-PAGE gels. Mixtures of rabbit and human AT III co-migrate as a single band on 7.5% SDS-PAGE gels. Rabbit AT III possesses both progressive and heparin activated (immediate) antithrombin activity in assays using human thrombin. Antisera raised against rabbit AT III demonstrates no cross reactivity with human AT III suggesting that despite physiologic and molecular weight similarities, antigenic differences are present. Incubation of rabbit antithrombin III with specific antisera, either prior to or after addition of heparin, did not alter the ability of antithrombin III to inhibit thrombin in either the immediate or progressive assays indicating that the antigenic determinants are not found in either the heparin binding or active thrombin binding sites. Crossed immunoelectrophoresis (IEP) demonstrates that antisera to rabbit AT III reacts with both free rabbit antithrombin III and AT III-thrombin complexes and can therefore be used in immunologic assays to quantitate total rabbit AT III (bound and free) and in crossed IEP to demonstrate the mobility of both free and complexed AT III.

Purification and Partial Characterization of a Hereditary Abnormal Antithrombin III Fraction of a Patient with Recurrent Thrombophlebitis

1980 ◽  
Vol 44 (02) ◽  
pp. 087-091 ◽  
Author(s):  
T H Tran ◽  
H Bounameaux ◽  
C Bondeli ◽  
H Honkanen ◽  
G A Marbet ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Agarose Gel ◽  
Normal Range ◽  
Antigen Concentration ◽  
Superficial Thrombophlebitis ◽  
Antithrombin Activity ◽  
Crossed Immunoelectrophoresis ◽  
At Iii ◽  
Heparin Affinity

SummaryA relatively low heparin cofactor activity (0.60 U/ml) was observed in a patient with recurrent superficial thrombophlebitis of the left leg. However, the antigen concentration was in the normal range (1.04 U/ml) and the progressive antithrombin activity was normal. The crossed immunoelectrophoresis in presence of heparin in agarose gel separated the patient's AT-III antigen in 2 fractions with different mobilities. The patient's AT-III was purified for further characterization. The last step of the purification procedure, a heparin-agarose chromatography, led to a separation and a purification of 2 AT-III fractions with different heparin affinities: an abnormal AT-III with reduced heparin affinity and a normal AT-III with a heparin affinity similar to that of AT-III isolated from normal plasmas. Abnormal and normal AT-III share several identical properties as molecular weight, ability to form complexes with thrombin and progressive antithrombin activity.

Antithrombin III Alger: A New Homozygous AT III Variant

1986 ◽  
Vol 55 (02) ◽  
pp. 218-221 ◽  
Author(s):  
A M Fischer ◽  
P Cornu ◽  
C Sternberg ◽  
F Mériane ◽  
M D Dautzenberg ◽  
...  
Keyword(s):  
Family History ◽  
Antithrombin Iii ◽  
Antigen Concentration ◽  
Crossed Immunoelectrophoresis ◽  
The Family ◽  
At Iii ◽  
Molecular Abnormality ◽  
Slow Moving ◽  
Cofactor Activity ◽  
Plasma Heparin

SummaryA qualitative abnormality of antithrombin III (AT III) was found in the plasma of a 41-year old patient. The plasmatic AT III antigen concentration was 130% and the progressive anti-F IIa and anti-F Xa activities were normal (105% and 137%). The plasma heparin cofactor activity was less than 10%, when measured by F Ila or F Xa inhibition. Crossed immunoelectrophoresis of AT III in the presence of heparin revealed in the plasma an abnormal slow-moving peak. When tested by affinity chromatography on heparin Sepharose, this abnormal AT III did not bind to heparin. Among the investigated relatives, 5 subjects had normal AT III levels, whatever the test used, the nine others having reduced levels of antithrombin heparin cofactor activity (45-61%) but normal levels of immunoreactive AT III (97-122%). Consanguinity was found in the family history. We therefore considered our patient as homozygous for an AT III molecular abnormality affecting the binding site for heparin.

Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

1986 ◽  
Vol 56 (03) ◽  
pp. 349-352 ◽  
Author(s):  
A Tripodi ◽  
A Krachmalnicoff ◽  
P M Mannucci
Keyword(s):  
Venous Thromboembolism ◽  
Active Site ◽  
Inhibitory Activity ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Molecular Defect ◽  
Affinity Adsorption ◽  
Italian Family ◽  
Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

scholarly journals PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
Author(s):  
Dzun Noraini Jimat ◽  
Intan Baizura Firda Mohamed ◽  
Azlin Suhaida Azmi ◽  
Parveen Jamal
Keyword(s):  
Specific Activity ◽  
Hot Spring ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Bacillus Sp ◽  
Sds Page ◽  
Fast Flow ◽  
Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60°C.

The Thrombin Protecting Function of a Complement Inhibitor in Human Plasma

1979 ◽  
Author(s):  
E.R. Podack ◽  
J.G. Curd ◽  
J.H. Griffin ◽  
H.J. Müller-Eberherd
Keyword(s):  
Complex Formation ◽  
Antithrombin Iii ◽  
Membrane Attack Complex ◽  
Protein S ◽  
Thrombin Time ◽  
Two Dimensional ◽  
Complement Inhibitor ◽  
Functional Studies ◽  
Sds Page ◽  
At Iii

S-protein (S) is a newly discovered 80,000 MW plosma glycoprotein. It functions as an inhibitor of the membrane attack complex of complement. We now wish to report that S also functions as thrombin protecting factor in coagulation; S forms a reversible complex with thrombin which is more resistant to inactivation by antithrombin III (AT III) than thrombin alone. An S-thrombin complex and on S-throm-bin-AT III complex were formed in clotted plasma and with isolated proteins as demonstrated by two dimensional Immunoelectrophoresis. Functional studies measuring the esterolytic or clotting activity of thrombin showed that S in the presence and absence of heparin decreased the rate of inactivation of thrombin by AT III. Similar results were observed using plasma. For example, in the presence of 0.04 u/ml heparin and 1.6 u/ml thrombin, the thrombin time of plasma depleted in S was 150 sec. as opposed to 15 sec. when the plasma was reconstituted with purified S. That this effect of S was due to a decreased inactivation of thrombin by AT III was demonstrated directly by SDS-PAGE analysis of plasma containing 125l-thrombin. In the presence of S the rate of formation of the 95,000 dalton 125I-thrombin-AT III complex was markedly decreased compared to the rate of complex formation in the S-depleted plasma. These data suggest that S may modulate the interactions of thrombin and AT III.

scholarly journals Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

2005 ◽  
Vol 37 (6) ◽  
pp. 363-370 ◽  
Author(s):  
Ye-Yun Li ◽  
Chang-Jun Jiang ◽  
Xiao-Chun Wan ◽  
Zheng-Zhu Zhang ◽  
Da-Xiang Li
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Optimum Activity ◽  
Development Of Resistance ◽  
Sds Page ◽  
C 50 ◽  
Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

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