Characterization of orthophosphate release from dissolved organic phosphorus by gel filtration and several hydrolytic enzymes

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Download Full-text

The Immunological Characterization of Human Antibodies to Factor VIII Isolated by Immuno-Affinity Chromatography

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650129 ◽

1981 ◽

Vol 45 (01) ◽

pp. 060-064 ◽

Cited By ~ 10

Author(s):

M L Kavanagh ◽

C N Wood ◽

J F Davidson

Keyword(s):

Factor Viii ◽

Affinity Chromatography ◽

Gel Filtration ◽

Immunological Characterization ◽

Human Antibodies ◽

Heavy Chains ◽

Light Chain Type ◽

Antibody Preparations ◽

Chain Type

SummaryNine human antibodies to factor VIII were isolated from haemophilic plasmas by affinity chromatography and gel filtration and six were subsequently subjected to immunological characterization. Three partially purified preparations were similarly characterized. Eight of the antibodies were characterized as being exclusively IgG and one preparation was found to contain IgM. Seven of the antibodies contained only a single light chain type, four being of type lambda and three of type kappa. Two antibody preparations contained both kappa and lambda light chains. In four of the preparations, only a single heavy chain sub-class could be demonstrated, three of IgG3 and one of IgG4. Of the remainder, three were a mixture of IgG3 and IgG4 sub-classes and one contained both IgG2 and IgG4. IgG sub-classification could not be achieved with the IgM-containing preparation. These results demonstrate a restricted heterogeneity of light and heavy chains in human antibodies to factor VIII.

Download Full-text

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660999 ◽

1984 ◽

Vol 51 (01) ◽

pp. 016-021 ◽

Cited By ~ 2

Author(s):

S Birken ◽

G Agosto ◽

B Lahiri ◽

R Canfield

Keyword(s):

High Performance ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Reverse Phase ◽

Human Fibrinogen ◽

Human Volunteers ◽

Cnbr Cleavage ◽

Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

Download Full-text

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657167 ◽

1982 ◽

Vol 47 (03) ◽

pp. 197-202 ◽

Cited By ~ 23

Author(s):

Kurt Huber ◽

Johannes Kirchheimer ◽

Bernd R Binder

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Human Urine ◽

Gel Filtration ◽

Chain Structure ◽

The Other ◽

Single Chain ◽

Apparent Homogeneity ◽

Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

Download Full-text

Purification and Characterization of Tissue-Type Plasminogen Activator in Maxillary Mucosa with Chronic Inflammation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657879 ◽

1985 ◽

Vol 54 (02) ◽

pp. 485-489 ◽

Cited By ~ 3

Author(s):

Yukiyoshi Hamaguchi ◽

Masuichi Ohi ◽

Yasuo Sakakura ◽

Yasuro Miyoshi

Keyword(s):

Plasminogen Activator ◽

Chronic Inflammation ◽

Gel Filtration ◽

Potassium Acetate ◽

Tissue Type ◽

Purification And Characterization ◽

Tissue Type Plasminogen Activator ◽

Urokinase Inhibitor ◽

Type Plasminogen Activator

SummaryTissue-type plasminogen activator (TPA) was purified from maxillary mucosa with chronic inflammation and compared with urokinase. Purification procedure consisted of the extraction from delipidated mucosa with 0.3M potassium acetate buffer (pH 4.2), 66% saturation of ammonium sulfate, zinc chelate-Sepharose, concanavalin A-Sepharose and Sephadex G-100 gel filtration chromatographies.The molecular weight of the TPA was approximately 58,000 ± 3,000. Its activity was enhanced in the presence of fibrin and was quenched by placental urokinase inhibitor, but not quenched by anti-urokinase antibody. The TPA made no precipitin line against anti-urokinase antibody, while urokinase did.All these findings indicate that the TPA in maxillary mucosa with chronic inflammation is immunologically dissimilar to urokinase and in its affinity for fibrin.

Download Full-text