scholarly journals Molecular forms of myeloperoxidase in human plasma

1986 ◽  
Vol 237 (2) ◽  
pp. 559-565 ◽  
Author(s):  
R L Olsen ◽  
T K Steigen ◽  
T Holm ◽  
C Little
Keyword(s):  
Ion Exchange ◽  
Human Plasma ◽  
Gel Electrophoresis ◽  
Large Subunit ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoaffinity Chromatography ◽  
Molecular Forms ◽  
Active Components ◽  
Protein Blotting ◽  
Catalytically Active

A radioimmunoassay for myeloperoxidase was established with the use of affinity-purified anti-(human myeloperoxidase) immunoglobulins. By the use of ion-exchange followed by immunoaffinity chromatography a preparation of immunoreactive, catalytically active myeloperoxidase was obtained from fresh human plasma. In non-denaturing gel electrophoresis, the plasma preparation showed about four catalytically active components of mobility very similar to that of the granulocyte enzyme. SDS/polyacrylamide-gel electrophoresis combined with protein blotting showed that the two polypeptides of strongest antigenicity in the plasma preparation corresponded in Mr to the large and the small subunits of the granulocyte enzyme. In addition, the plasma preparation contained a higher-Mr immunoreactive polypeptide, possibly a precursor form of the enzyme, together with another of Mr similar to that of the large subunit of eosinophil peroxidase.

scholarly journals Identification of molecular forms of plasminogen and plasmin-inhibitor complexes in urokinase-activated human plasma

1984 ◽  
Vol 223 (1) ◽  
pp. 169-177 ◽  
Author(s):  
S Müllertz ◽  
S Thorsen ◽  
L Sottrup-Jensen
Keyword(s):  
Acetic Acid ◽  
Human Plasma ◽  
Heavy Chain ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Molecular Forms ◽  
Low Grade ◽  
Plasmin Inhibitor

Urokinase-activated human plasma was analysed by acetic acid/urea/polyacrylamide-gel electrophoresis. The bands representing plasminogen, the plasmin-alpha 2-plasmin inhibitor and plasmin-alpha 2-macroglobulin complexes were identified by immunoprecipitation with specific antibodies and by comparison with purified components. Plasminogen and the plasmin-inhibitor complexes were isolated from plasma or thrombin-clotted plasma containing 125I-labelled Glu-plasminogen (residues 1-790) and urokinase. The plasma was kept at 37 degrees C for 0.5 and 10 times the lysis time of the clotted plasma, the clotted plasma until lysis. The plasmin heavy chain from the plasmin-inhibitor complexes was subsequently prepared. Only in one case could a low-grade proteolytic conversion of Glu- forms into Lys/Met/Val-forms (residues 77-790, 68-790 and 78-790 respectively) during the preparations be detected. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and N-terminal sequence analysis of the purified plasminogen and plasmin heavy chain showed the following. The plasminogen in plasma was on the Glu- form. Glu-plasmin constituted 0.74 and 0.58 of the plasmin bound to the alpha 2-plasmin inhibitor in plasma after brief and prolonged activation respectively. The rest was Lys/Met/Val-plasmin. The clotted plasma contained about equal amounts of Glu-plasminogen and Lys/Met/Val-plasminogen, and predominantly Lys/Met/Val-plasmin complexed to alpha 2-plasmin inhibitor and alpha 2-macroglobulin. The results of the analysis of the purified material substantiated the identity of radioactive protein bands in the gel after acetic acid/urea/polyacrylamide-gel electrophoresis.

Incorporation of Uridine in Cleavage Stage Eggs of the Sea Urchin Paracentrotus Lividus

1971 ◽  
Vol 26 (8) ◽  
pp. 816-821 ◽  
Author(s):  
Larry E. Bockstahler
Keyword(s):  
Ion Exchange ◽  
Sea Urchin ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Paracentrotus Lividus ◽  
Cleavage Stage ◽  
Early Cleavage ◽  
Cell Stage ◽  
Cleavage Stages ◽  
Layer Chromatography

Incorporation of uridine in cleavage stage eggs of the sea urchin Paracentrotus lividus was investigated. It was shown by ion exchange and thin layer chromatography that most of the uridine taken up during the 16-cell stage was converted into UTP with some incorporation into UDP and UMP. Conversion of uridine to these phosphorylated nucleosides occurred throughout early cleavage stages. A very small amount of uridine taken up by cleavage stage eggs is incorporated into RNA heterogeneous in size. This RNA was examined by polyacrylamide gel electrophoresis.

Rapid separation of bovine caseins by mass ion exchange chromatography

1989 ◽  
Vol 56 (3) ◽  
pp. 391-397 ◽  
Author(s):  
K. F. Ng-Kwai-Hang ◽  
J. P. Pélissier
Keyword(s):  
Liquid Chromatography ◽  
Ion Exchange ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Rapid Separation ◽  
Rapid Isolation ◽  
Time Required

SummaryThe rapid isolation of major bovine caseins in gram quantities was investigated. Whole casein was precipitated from individual cow's milk by adjusting the pH to 4·6 and the precipitated casein was suspended in 4·5 M urea (pH 8·0) containing 0·02 M imidazole and 0·03 M β-mercaptoethanol, and bound on a QAE Zeta Prep 250 cartridge. Stepwise elution with the urea/imidazole β-mercaptoethanol buffer and varying amounts of NaCl gave five well resolved peaks, which were identified by polyacrylamide gel electrophoresis and fast protein liquid chromatography to be pure γ-casein, κ-casein. β-casein, β-casein and αs-casein, respectively. The ion exchange cartridge was regenerated by flushing with buffer containing 0·50 Μ-NaCl followed by equilibration with starting buffer before separation of next sample. The time required to run each sample including cartridge regeneration and equilibration was 4 hours.

scholarly journals Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin

1974 ◽  
Vol 143 (2) ◽  
pp. 273-283 ◽  
Author(s):  
Sten Müllertz
Keyword(s):  
Human Plasma ◽  
Protease Inhibitors ◽  
Gel Electrophoresis ◽  
Protease Activity ◽  
Gel Filtration ◽  
Molecular Forms ◽  
Crossed Immunoelectrophoresis ◽  
Low Concentrations ◽  
Α2 Macroglobulin ◽  
Esterase Inhibitor

Urokinase-activated human plasma was studied by gel electrophoresis, gel filtration, crossed immunoelectrophoresis and electroimmunoassay with specific antibodies and by assay of esterase and protease activity of isolated fractions. Urokinase induced the formation of different components with plasminogen+plasmin antigenicity. At low concentrations of urokinase, a component with a KD value of 0.18 by gel filtration and post β1 mobility by gel electrophoresis was detected. The isolated component had no enzyme or plasminogen activity. In this plasma sample fibrinogen was not degraded for 10h, but when fibrin was formed, by addition of thrombin, fibrin was quickly lysed, and simultaneously a component with a KD value of 0 and α2 mobility appeared, which was probably plasmin in a complex with α2 macroglobulin. This complex showed both esterase and protease activity. After gel filtration with lysine buffer of the clotted and lysed plasma another two components were observed with about the same KD value by gel filtration as plasminogen (0.35), but β1 and γ mobilities by gel electrophoresis. They appeared to be modified plasminogen molecules, and possibly plasmin with γ mobility. Similar processes occurred without fibrin at higher urokinase concentrations. Here a relatively slow degradation of fibrinogen was correlated to the appearance of the plasmin–α2 macroglobulin complex. The fibrin surface appeared to catalyse the ultimate production of active plasmin with a subsequent preferential degradation of fibrin and the formation of a plasmin–α2 macroglobulin complex. The gel filtration and electrophoresis of the plasma protease inhibitors, α1 antitrypsin, inter-α-inhibitor, antithrombin III, and C1-esterase inhibitor indicated that any complex between plasmin and these inhibitors was completely dissociated. The β1 and post β1 components appear to lack correlates among components occurring in purified preparations of plasminogen and plasmin.

scholarly journals Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens

1988 ◽  
Vol 253 (1) ◽  
pp. 263-267 ◽  
Author(s):  
I Vancurová ◽  
J Volc ◽  
M Flieger ◽  
J Neuzil ◽  
J Novotná ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Size Exclusion ◽  
Streptomyces Aureofaciens ◽  
Hydrophobic Chromatography ◽  
Step Procedure

Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.

scholarly journals The membrane attack mechanism of complement: the three polypeptide chain structure of the eigth component (C8).

1976 ◽  
Vol 143 (5) ◽  
pp. 1131-1139 ◽  
Author(s):  
W P Klob ◽  
H J Müller-Eberhard
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Ammonium Sulfate ◽  
Gel Electrophoresis ◽  
Polypeptide Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chain Structure ◽  
Ammonium Sulfate Precipitation ◽  
Ion Exchange Column ◽  
Polypeptide Chains

The purification of human C8 in milligram quantities from outdated human serum was achieved by ammonium sulfate precipitation (37.5-50% saturation) and ion exchange column chromatography employing CM-32 cellulose and QAE-Sephadex. The yield of C8 activity ranged from 2-9%, and the average purification was 1,700-fold. Fully reduced C8 was shown by SDS polyacrylamide gel electrophoresis to have three polypeptide chains which were present in equimolor ratios: alpha, 77,000 daltons; beta, 63,000 daltons; and gamma, 13,700 daltons. C8 denaturation by SDS and urea in the absence of reducing agents revealed two noncovalently linked subunits: alpha-gamma, 99,000 daltons, and beta, 75,000 daltons.

The Fractionation of Factor V from Human Plasma

1974 ◽  
Vol 31 (03) ◽  
pp. 457-468 ◽  
Author(s):  
John C. Giddings
Keyword(s):  
Human Plasma ◽  
Gel Electrophoresis ◽  
Chloride Solution ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Coagulation Factors ◽  
Polycythaemia Vera ◽  
Agarose Gel ◽  
Factor V ◽  
Fresh Plasma

SummaryHuman plasma from normal donors and from patients with polycythaemia vera was fractionated in order to obtain a preparation of highly purified factor V. Fresh plasma was initially treated with aluminium hydroxide to remove factors II, VII, IX and X. Fibrinogen, factor VIII and most of the immunoglobulins were removed by precipitation with ethanol at -5° C. Crude factor V was precipitated with dilute acetic acid at pH 5.1, and then further purified by precipitation with acridine lactate (Rivanol), extraction with sodium chloride solution and column chromatography on agarose gel. Factor V was purified four hundred times with specific activity of 2.5-6.0 units per mg. protein. The final concentrate was devoid of activity of other coagulation factors but was heterogeneous on disc Polyacrylamide gel electrophoresis and Immunoelectrophoresis against anti human serum. On rechromatography on agarose gel there was a single peak of factor V activity, the molecular weight of which was estimated to be 300,000.

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