scholarly journals A comparative study on the preparation of immunoglobulin-galactosidase conjugates.

1981 ◽  
Vol 29 (11) ◽  
pp. 1273-1280 ◽  
Author(s):  
A M Deelder ◽  
R de Water
Keyword(s):  
Molecular Weight ◽  
Coupling Agent ◽  
Gel Filtration ◽  
The Other ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chromogenic Substrate ◽  
Fluorogenic Substrate ◽  
Detection Level ◽  
Lower Detection ◽  
Molecular Weight Fractions

For the application of beta-D-galactosidase-immunoglobulin conjugates in the enzyme-linked immunosorbent assay (ELISA), four techniques for the preparation of such conjugates were compared. Sheep immunoglobulin (Ig) (against soluble egg antigens of the trematode Schistosoma mansoni) was coupled to beta-D-galactosidase by means of 1) glutaraldehyde treatment, 2) the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), and 3,4) two different procedures using the coupling agent m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS). The prepared conjugates were then fractionated by gel filtration on Sepharose 6B and the resultant molecular weight fractions were tested in an ELISA for the detection of S. mansoni antigen. Optimal results were obtained with a conjugate that was synthesized according to one of the two techniques using MBS. With this conjugate, 10(-9) g antigen/ml could still be detected in an ELISA with a chromogenic substrate, which was at least ten times as sensitive as with the other conjugates. Application of a fluorogenic substrate resulted in a lower detection level of 10(-10) g antigen/ml.

Fractionation of the Color of Pure Maple and Other Sirups by Gel Filtration

1965 ◽  
Vol 48 (3) ◽  
pp. 493-497
Author(s):  
E E Stinson ◽  
C O Willits
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Cane Sugar ◽  
The Other ◽  
Low Molecular Weight ◽  
Brown Sugar ◽  
Maple Sugar ◽  
Molecular Weight Fractions ◽  
Color Fraction

Abstract The colorants of pure maple, cane and maple, refined cane sugar, and light brown sugar sirups were separated into two fractions, one of high- and the other of lowmolecular weights, by means of gel filtration. The ratio of the amounts of high- to the low-molecular weight fractions of pure maple was the lowest of the four sirups and serves as a means of differentiation from these sirups. The color fraction ratio was highest for blended cane-maple sugar sirup. Many maple sirups are also distinguished by a pink band formed on the gel filtration column.

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.

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

1992 ◽  
Vol 12 (1) ◽  
pp. 15-21
Author(s):  
S. Kojima ◽  
K. Nara ◽  
Y. Inada ◽  
S. Hirose ◽  
Y. Saito
Keyword(s):  
Molecular Weight ◽  
Endothelial Cell ◽  
Specific Activity ◽  
Gel Filtration ◽  
Platelet Activating Factor ◽  
Lipid Vesicles ◽  
Specific Factor ◽  
Cell Lysate ◽  
Aggregation Activity ◽  
Molecular Weight Fractions

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [3H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [3H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.

scholarly journals α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

In Vivo Studies On The Inhibition Of Coagulation By Fractionated Heparin

1981 ◽  
Author(s):  
U Schmitz-Huebner ◽  
L Balleisen ◽  
F Asbeck ◽  
J van de Loo
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Gel Filtration ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
Chromogenic Substrate ◽  
Serotonin Content ◽  
Heparin Activity ◽  
Heparin Fractions

Recent investigations suggest that low molecular weight heparin may have advantages over conventional heparin with regard to the prevention of venous thrombosis and haemorrhagic side effects.High (HMW) and low (LMW) molecular weight heparin fractions with mean MWs of 16,000 and 8,800 respectively, obtained by gel filtration chromatography of sodium mucosal heparin (B. Braun Melsungen), were injected subcutaneously into six volunteers and compared with the unfractionated substance in a cross-over trial. Doses of 5,000 U were administered twice daily over a period of three days and heparin activity was controlled before injection and 2,4,8 hours afterwards by means of the APTT, the anti-Xa clotting test and a chromogenic substrate assay. In addition, the in vivo effect of fractionated heparin on platelet function was examined. The results show that the LMW fraction induced markedly higher anti-Xa activity than the other preparations. At the same time, APTT results did not significantly differ. Unfractionated heparin and the HMW fraction enhanced ADP-induced platelet aggregation and collagen-mediated MDA-production, while the LMW fraction hardly affected these assays, but potently inhibited thrombin-induced MDA production. All heparin preparations stimulated the release of PF IV, whereas the serotonin content of platelets determined at the same time increased.It is concluded that s.c. injections of LMW heparin induce relatively high levels of anti-Xa activity without leading to sensitive platelet activation or to major effects on overall clotting tests.

Purification and characterization of exocellular proteases produced by a clinical isolate and a laboratory strain of Pseudomonas aeruginosa

1980 ◽  
Vol 26 (1) ◽  
pp. 77-86 ◽  
Author(s):  
S. E. Jensen ◽  
L. Phillippe ◽  
J. Teng Tseng ◽  
G. W. Stemke ◽  
J. N. Campbell
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Aeruginosa ◽  
Clinical Isolate ◽  
Protease Activity ◽  
Gel Filtration ◽  
Laboratory Strain ◽  
Exchange Chromatography ◽  
The Other ◽  
Protease Production ◽  
Peptide Bonds

Exocellular protease production was examined in two separate strains of Pseudomonas aeruginosa, one a clinical isolate and the other a laboratory strain. Both strains produced two separate proteases (proteases 1 and 2) which were indistinguishable from one strain to the other. The two proteases were purified by a two-step procedure of gel filtration chromatography followed by ion-exchange chromatography. Proteases 1 and 2 were shown to be distinct serologically and unrelated by physicochemical parameters examined. Protease 1 was the major exocellular protein produced and contributed about 95% of the total protease activity of the culture. It was estimated to have a molecular weight of 34 850 and was also shown to contain 10% glucosamine by weight. Protease 2, in contrast, had an estimated molecular weight of 52750 and contained no detectable carbohydrate. Proteases 1 and 2 were both stimulated by Ca2+, and Mg2+ and inhibited by Co2+Zn2+, and 1,10-o-phenanthroline. Protease 1 was also inhibited by EDTA. In addition to protease activity, both proteases 1 and 2 demonstrated elastase activity as well as a limited collagenase activity. Specificity of the two proteases against synthetic peptides was, however, quite different. Protease 1, but not protease 2, showed a preference for peptide bonds in which the amino group was contributed by an amino acid with a hydrophobic R group.

scholarly journals Macromolecules stimulating human granulocytic colony-forming cells, precursors of these cells, and primitive erythroid progenitors: some apparent nonidentities

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 960-966 ◽  
Author(s):  
T Hoang ◽  
NN Iscove ◽  
N Odartchenko
Keyword(s):  
Molecular Weight ◽  
Conditioned Medium ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
The Other ◽  
Erythroid Progenitors ◽  
Granulocytic Colony ◽  
Ph Range ◽  
Direct Primary ◽  
The Relationship

Abstract The relationship between molecules having granulocyte colony- stimulating activity (G-CSA), erythroid burst-promoting activity (E- BPA), and activity promoting increase in the number of granulocytic progenitors in liquid culture (delta GPA) was explored in conditioned medium from human leukocytes (HLCM) and human placenta (HPCM). As tested on human hemopoietic progenitors in culture, G-CSA eluted from Sephadex G100 as a single peak with apparent molecular weight of 25,000, separating partially from E-BPA and delta GPA, which both had an apparent molecular weight of 45,000. All three activities eluted together from hydroxyapatite at low molarity phosphate. Their charge properties were also similar and all three electrofocused in flat gel beds in the pH range near 5.4. On both hydroxyapatite and isoelectric focusing, delta GPA sometimes separated partially from the other two activities but not consistently. The gel filtration result shows that in conditioned medium of human origin, molecules having G-CSA are not the same as those having delta GPA, suggesting a dual factor requirement in the granulocytic lineage reminiscent of that in the erythroid pathway. The results suggesting that delta GPA might differ from E-BPA, on the other hand, were not consistent enough to establish their nonidentity. Single micromanipulated cells proved capable of forming erythroid or granulocytic colonies in the presence of either crude or partially purified activity. The results establish that human colony-forming cells are direct primary targets of growth factors in HLCM and HPCM.

scholarly journals New Exfoliative Toxin Produced by a Plasmid-Carrying Strain of Staphylococcus hyicus

1999 ◽  
Vol 67 (8) ◽  
pp. 4014-4018 ◽  
Author(s):  
Hisaaki Sato ◽  
Takao Watanabe ◽  
Yasuko Murata ◽  
Ayumi Ohtake ◽  
Mayumi Nakamura ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
The Other ◽  
Exfoliative Toxin ◽  
Sds Page ◽  
Serotype B

ABSTRACT A new serotype of Staphylococcus hyicus exfoliative toxin (SHET), serotype B, was isolated from the culture filtrate of a plasmid-carrying strain of S. hyicus. The new SHET was purified by precipitation with 70% saturated ammonium sulfate, gel filtration on a Sephadex G-75 column, column chromatography on DEAE–Cellulofine A-500, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The new SHET caused exfoliation of the epidermis as determined by the so-called Nikolsky sign when inoculated into 1-day-old chickens. The new SHET was serologically different fromStaphylococcus aureus exfoliative toxins (ETs) (ETA, ETB, and ETC) and from the SHET from the plasmidless strain but showed the same molecular weight as the other serotypes of toxins on SDS-PAGE. It was thermolabile and lost its toxicity after being heated at 60°C for 30 min. We propose that the new SHET be designated SHETB and that the SHET produced by the plasmidless strain be designated SHETA.

Regulation of two aspartokinase isozymes in Streptococcus bovis

1994 ◽  
Vol 40 (3) ◽  
pp. 224-227 ◽  
Author(s):  
E. O. Kalcheva ◽  
M. M. Faiziev ◽  
V. O. Shanskaya ◽  
S. S. Maluta
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Growth Conditions ◽  
Growth Cycle ◽  
The Other ◽  
Streptococcus Bovis ◽  
Gel Filtration Chromatography ◽  
Regulatory Properties ◽  
Second Peak

Streptococcus bovis has been found to contain two distinct aspartokinases that can be separated by gel filtration chromatography. One of these isozymes elutes on Sephadex G-200 gel filtration at a molecular weight greater than 250 000. The molecular weight of the other isozyme is approximately 125 000. The earlier peak of aspartokinase activity is slightly inhibited by meso-diaminopimelate, while the second peak is sensitive to inhibition by lysine. The latter aspartokinase is not formed when the organism is grown in a medium containing more than 1 mM lysine. The level of lysine-sensitive aspartokinase is decreased during the growth cycle, whereas diaminopimelate-sensitive activity is little affected by the growth conditions. The regulatory properties of the two aspartokinases suggest that they may play different physiological roles.Key words: aspartokinase activity, isozymes, repression, inhibition.

scholarly journals Isolation and fractionation of glycopeptides from porcine thyroglobulin

1971 ◽  
Vol 123 (3) ◽  
pp. 407-414 ◽  
Author(s):  
Minoru Fukuda ◽  
Fujio Egami
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Chemical Composition ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Alkaline Treatment ◽  
The Other

1. Glycopeptides were isolated by gel filtration on Sephadex G-25 and Sephadex G-50 from a Pronase digest of porcine thyroglobulin. 2. Isolated glycopeptides were separated into five main fractions on a column of DEAE-Sephadex A-25. Of these fractions I to III were further purified by SE-Sephadex C-25 or DEAE-Sephadex A-25 column chromatography. Several of the purified glycopeptides were homogeneous on paper electrophoresis. 3. Based on the chemical composition and molecular weight of the fractionated glycopeptides, two distinct types of heterosaccharide chain were demonstrated. 4. One type of the heterosaccharide unit consisted of four to eight residues of mannose and two residues of glucosamine and had a molecular weight of 1000–1700. The other type of unit contained sialic acid, fucose and galactose in addition to mannose and glucosamine and had a molecular weight of about 3600. 5. Mild alkaline treatment of the glycopeptide did not result in the destruction of threonine and serine. 2-Acetamido-1-N-(4-l-aspartyl)-2-deoxy-β-d-glucopyranosylamine was isolated from partial acid hydrolysates.

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