scholarly journals Chromatography of transforming deoxyribonucleic acid on methylated albumin and by gel filtration

1974 ◽  
Vol 137 (3) ◽  
pp. 543-546
Author(s):  
G. R. Barker ◽  
P. Hodges
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
High Molecular Weight ◽  
Deoxyribonucleic Acid ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Transforming Activity ◽  
Different Strains ◽  
Two Peaks

1. Native DNA from two strains of Bacillus subtilis was chromatographed by stepwise elution from MAK (methylated albumin on kieselguhr). 2. Transforming activity was confined to two out of the three main fractions, activity being distributed between the two peaks differently for DNA from the different strains. 3. Fractionation of DNA from both strains on 2% agarose gel gave two components. Approx. 75% of the material was eluted within the void volume of the column. Approx. 25% of the material consisted of degradation products of lower molecular weight. 4. Chromatography on MAK of the material of high molecular weight eluted from agarose gel gave a number of peaks differing in molecular weight, indicating that degradation of the DNA takes place during chromatography on MAK. 5. The distribution of transforming activity among the fractions from MAK suggests that degradation occurs preferentially in certain regions of the DNA.

Fibrin-I Degradation Products with a Molecular Weight Higher than That of Fibrinogen

1973 ◽  
Vol 29 (01) ◽  
pp. 122-129 ◽  
Author(s):  
René von Hugo ◽  
Henner Graeff
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Two Dimensional ◽  
Plasma Clot ◽  
Two Dimensional Gel Electrophoresis ◽  
Parent Molecule

SummaryThe observation of intravascular lysis of fibrin deposits and of fibrinogen derivatives with a molecular weight higher than the parent molecule in human cases of disseminated intravascular coagulation (DIC) initiated the following in vitro study. Following streptokinase induced plasma clot solubilization fibrinogen derivatives were investigated after ß-alanine precipitation of the plasma samples by polyacrylamide (PAA) gel electrophoresis, intra gel immunoprecipitation, two dimensional gel electrophoresis and by agarose gel filtration. Three fibrin-i degradation products were observed and characterized according to their relative electrophoretic mobility in 5% PAA gel: 0.23, 0.35, 0.46 (fibrinogen: 0.43) x 10-5 cm2/V x sec. They could also be demonstrated after electrophoresis in the presence of 5 M urea. Agarose gel filtration yielded one peak at 180 ml of effluent volume. The 0.23 derivative was eluted in the peak fractions, whilst the 0.35 and 0.46 derivatives were eluted together at approximately 201 ml of the effluent volume (fibrinogen: 225 ml). This indicates, that the three fibrin-i degradation products described are molecular entities with molecular weights higher than fibrinogen and, that the 0.46 derivative has an increased charge/molecular size ratio in comparison with fibrinogen. Corresponding data were obtained by two dimensional gel electrophoresis in gels of different pore size.

scholarly journals Plasmin Inhibitors in Human Urine

1977 ◽  
Author(s):  
H. Sumi ◽  
Y. Takada ◽  
A. Takada
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Molecular Form ◽  
Membrane Filter ◽  
Gel Filtration ◽  
Total Activity ◽  
Native Form ◽  
Molecular Weights ◽  
Two Peaks

By the gel filtration of urinary protein on Sephadex G-200, two peaks of activity to hydrolyze Nα-acetylglycyl-L-lysine methyl ester (AGLMe) were detected. One was the native form of urokinase, and the molecular weight was about 54,000. The other was of high molecular weight, and eluted in void fraction. The high molecular form was thought to be a complex of urokinase and urinary plasmin inhibitor (UPI). By using Arg-Sepharose, membrane filter (M.W. 10,000), and pevikon block electrophoresis, we could isolate four types of UPI from normal human urine. One UPI was positively charged at pH 8.6, and of high molecular weight. Other types were negatively charged, and the molecular weights by gel filtration on Sephadex G-200 were about 67,000 (UPI6.7), 45,000 (UPI4.5), and 22, 000 (UPI2.2), respectively. In acrylamide disc gel electrophoresis, UPI57 migrated to serum prealbumin fraction, and UPI45 and UPI? 2 were less anionic. Negative-charged UPIs could be adsorbed on trypsin-Sepharose, and were thought to be identical to urinary trypsin inhibitors. Purified UPIs showed strong inhibition on caseinolytic- and esterolytic-activities of plasmin, and the total activity was about 16 UPIU(inhibited 16 casein U of plasmin)/liter of urine.

Characterization Of Soluble DES-A And DES-AB Fibrin In Human Plasma At 37°C By Agarose-Gel Filtration

1981 ◽  
Author(s):  
G Müller-Berghaus ◽  
J-C Bernhard ◽  
I Mahn
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Void Volume ◽  
Agarose Gel ◽  
High Molecular Weight Material ◽  
Different Temperatures ◽  
Lower Temperature

In two consecutive steps, thrombin cleaves the fibrinopeptides A and B from fibrinogen producing des-A fibrin and des-AB fibrin. Labeled des-A fibrin was prepared by batroxobin and labeled des-AB fibrin by clotting of 125I-fibrinogen with thrombin. Fibrin solubilized in buffered urea was mixed with plasma containing 131I-fibrinogen (fibrin:fibrinogen ratio = 1:20). These fibrinfibrinogen mixtures were applied to sepharose CL- 6B columns eq ui librated with buffered plasma (0.0025 M EDTA, 0.1 M NaCl, 0.05 M tris, 0.005 M EACA, 2 AT U hirudin/ml, 500 KIU a protinin/ml, 0.003 M NaN3, pH 7.4). Plasma was used as an equilibration and elution medium to prevent precipitation of fibrin in the columns. At 20°C, labeled des-A fibrin as well as des-AB fibrin were eluted in the void volume as high-molecular weight aggregates (peak A) and separated from m onomeric labeled fibrinogen (peak B). At 37°C, however, des- A fibrin was eluted at the same position as monomeric fibrinogen (peak B), whereas des-AB fibrin was eluted in the void volume as at 20°C. Rechromatography of isolated fractions of peak A and peak B at different temperatures showed that monomeric fibrin isolated at 37°C formed high molecular weight material at 20°C, and high-molecular weight fibrin isolated at 20°C dissociated at 37 ° C. The results suggest that des-A fibrin solubilized in plasma in the absence of calcium ismonomeric at 37°C but forms high-molecular weight aggregates at lower temperature, whereas des-AB fibrin is oligomeric at 20°C as well as at 37°C.

In Vitro Formation of Fibrin-Monomer Complexes in the Presence of Isotope Labelled Fibrinogen-Fibrin Degradation Products. An Agarose Gel Filtration Study

1975 ◽  
Author(s):  
F. Asbeck ◽  
van de J. Loo
Keyword(s):  
Molecular Weight ◽  
Complex Formation ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Fibrinogen Degradation Products ◽  
Molecular Weights ◽  
Fibrin Degradation Products ◽  
Fibrin Monomer

Human citrated plasmas were mixed with purified 131I-fibrinogen and 131I-fibrinogen degradation products (FDP) or 125I-fibrin degradation products (fdp). After incubation with small amounts of thrombin (0.01–0.02 imits/ml Pl.), these mixtures were gel filtrated on Biogel A5m columns and the elution patterns of the 131I- and -labelled materials were determined.In control experiments without thrombin incubation, no complex formation between fibrinogen, FDP or fdp in citrated plasmas could be detected. This was even true for fdp with a higher molecular weight than fibrinogen.After thrombin incubation, up to 11% fibrin-monomer complexes were formed. Irrespective of their molecular weights, labelled fdp were not incorporated into these complexes.Only large FDP – presumably derivative X – did partially copolymerize with fibrin-monomer complexes in citrated plasmas.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
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.

scholarly journals Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

1984 ◽  
Vol 32 (7) ◽  
pp. 690-696 ◽  
Author(s):  
J Fischer ◽  
G Uhlenbruck ◽  
P J Klein ◽  
M Vierbuchen ◽  
R Fischer
Keyword(s):  
Molecular Weight ◽  
Gastric Mucosa ◽  
High Molecular Weight ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Gastric Cancer Cells ◽  
Tissue Sections ◽  
Gradient Gel Electrophoresis ◽  
Triton X

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

Interaction of high molecular weight kininogen binding proteins on endothelial cells

2004 ◽  
Vol 91 (01) ◽  
pp. 61-70 ◽  
Author(s):  
Baby Tholanikunnel ◽  
Berhane Ghebrehiwet ◽  
Allen Kaplan ◽  
Kusumam Joseph
Keyword(s):  
Molecular Weight ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Surface Proteins ◽  
Factor Xii ◽  
High Molecular Weight Kininogen ◽  
Dot Blot ◽  
Cell Plasma ◽  
Molecular Weight Peak

SummaryCell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation. The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endothelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular complexes of gC1qR-cytokeratin 1 and u-PAR-cytokeratin 1, with gC1qR binding being favored.

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