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.

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.

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.

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.

scholarly journals Partial purification of topoisomerase IB from serum of diabetic patients and study it's kinetic properties and molecular weight

2019 ◽  
Vol 23 (10) ◽  
pp. 46
Author(s):  
Saif M. Hasan ◽  
Firas T. Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Maximum Velocity ◽  
Partial Purification ◽  
Diabetic Patients ◽  
Kinetic Properties ◽  
Topoisomerase Ib ◽  
Electrophoresis Method

This study was done to partially purification of  topoisomerase IB from serum of diabetic patients using Gel filtration technique, by using Sephadex G 100 gel. A single peak in fraction four has been obtained, and the degree of purification (17.1) fold, enzyme yield (108.2%) and specific activity (0.189ng/mg). Kinetics studies for the partial purified enzyme were carried out which showed optimal concentration of  substrate which was (0.1ng/ml), Michael's - Menten constant (Km=0.033ng) and maximum velocity (Vmax=0.90 ng/ml), while optimum Temperature was (37C°) and optimum pH was (7.5). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method, in presence of polyacrylamide  gel and sodium dodecyl sulphate (SDS-PAGE) which showed that the approximated molecular weight was (66KD).   http://dx.doi.org/10.25130/tjps.23.2018.168 

Antithrombotic Properties Of Low Molecular Weight Heparin Fractions From Porcine Mucosal Heparin

1981 ◽  
Author(s):  
J Choay ◽  
Jean C Lormeau ◽  
Harry L Messmore ◽  
Jawed Fareed ◽  
J Stulc ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Prothrombin Complex Concentrate ◽  
Specific Activity ◽  
Weight Fraction ◽  
Low Molecular Weight ◽  
Prothrombin Complex Concentrates ◽  
Heparin Fractions ◽  
Russell’S Viper ◽  
Molecular Weight Fractions

A previous report from our laboratories has described the extraction and physicochemical properties of a low molecular weight fraction (mol wt 4-8 × 103 daltons) from porcine mucosal heparin (Choay et. al. thrombosis Res 18, 573, 1980). Beside exhibiting a strong anti Xa (>250 u/mg) activity, this product possessed strong antithrombotic properties in a modified rabbit stasis thrombosis model. At a 125 anti Xa u/kg it protected the thrombotic effects of activated prothrombin complex concentrate (20 u/kg) and Prothrombin Complex Concentrate/Russell’s Viper Venom challenge in both the pretreatment and post-treatment regiments. At 1250 anti Xa u/kg SC it also showed antithrombotic effects for various periods. We have also obtained another low molecular weight fraction from porcine mucosal heparin by controlled depolymerization with nitrous acid. This product possessed saccharides with molecular weight ranging 3-6 × 103 daltons and exhibited a specific activity of >200 anti Xa u/mg. At a 125 anti Xa u/kg this product also showed antithrombotic activity against the thrombotic effects of activated prothrombin complex concentrates, prothrombin complex concentrates and Russell’s Viper Venom. In contrast to these two low molecular weight fractions porcine mucosal heparin in identical anti Xa units failed to produce protection against the thrombogenic stimuli. Our studies suggest that low molecular weight heparin fractions with strong anti Xa and antithrombotic activities can be obtained by chemical depolymerization. Furthermore, their biologic properties are found to be similar to the naturally occuring low molecular weight fractions present in native porcine mucosal heparin.

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.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

scholarly journals Purification and partial characterization of superoxide dismutase from the thermophilic bacteria Thermothrix sp

2004 ◽  
Vol 69 (1) ◽  
pp. 9-16 ◽  
Author(s):  
Svetlana Seatovic ◽  
Ljubinka Gligic ◽  
Zeljka Radulovic ◽  
Ratko Jankov
Keyword(s):  
Molecular Weight ◽  
Superoxide Dismutase ◽  
Specific Activity ◽  
Thermophilic Bacteria ◽  
Noncovalent Interactions ◽  
Gel Filtration ◽  
Antioxidant Defense System ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Sod Activity

Superoxide dismutase (SOD; EC 1.15.1.1), a high molecular weight component of the antioxidant defense system, provided promising results in the treatment of oxidative damage. Thermothrix sp., isolated from thermal spa water in Serbia, showed high superoxide dismutase activity. The SOD, from cell free extract, was purified to homogenity by ammonium sulfate precipitation, Sephadex G 75 gel filtration chromatography and QAE Sephadex ion exchange chromatography. The specific activity of the purified enzyme was 9191 U/mg. The purified enzyme was analyzed and partially characterized. SOD was localized in polyacrylamide gel by activity staining, based on the reduction of nitroblue tetrazolium (NBT) by superoxide. The enzyme molecular weight determined by gel chromatography is 37 kD. According to SDS PAGE it is composed of two subunits of equal size, joined by noncovalent interactions. The isoelectric point, assessed by isoelectric focusing is 5.3. The optimum pH for enzyme activity was in the range of 8 to 10. The optimum temperature for SOD activity was 60 ?C. After one hour of incubation at 40, 50 and 60 ?C the SOD activity increases, but at 80 ?C, the SOD is denaturated. After 24 hours of incubation at 25 ?C SO Dactivity only slightly decreases.

scholarly journals Study of some hormones and Partial purification of prolidase from serum of women with polycystic ovary syndrome

2019 ◽  
Vol 24 (7) ◽  
pp. 59
Author(s):  
Reemy M. Mohamed Saleh ◽  
Firas T . Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Polycystic Ovary Syndrome ◽  
Specific Activity ◽  
Polycystic Ovary ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Ovary Syndrome ◽  
Electrophoresis Method ◽  
High Level

This study was done by partially purification of prolidase from serum of patients with polycystic ovary syndrome by Gel filtration technique, and using sephadex G100 gel as a stationary phase. The degree of purification (15.1) fold, enzyme yield (95.5%) and specific activity (0.00176 IU/I), were carried out .Kinetics studies for the partial purified enzyme technique showed optimal concentration of substrate which was 5 mmol/l Km = 0.66ng and Vmax =0.80 mM, while optimum Temperature was (35C°) and optimum pH was (8). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method , in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS_PAGE) which showed that the approximates molecular weight was (54KD),we found high level of prolactin in the patient with polycystic ovary syndrome which was(24.03) when in the control was( 10.09),the value of TSH in the patient was( 17.08) which is high value and in the control was( 1.49), the value of T4 in the patient was (100.2) and in control was (118.4),the value of T3 in the patient was (0.3)and in control was (1.3).Testosterone in patient was (0.391) and in control was (0.206).    http://dx.doi.org/10.25130/tjps.24.2019.130

scholarly journals PREPARATION, PURIFICATION AND PROPERTIES OF LIPASE FROM HEPATOPANCREAS OF TRA (PANGASIUS) CATFISH

2011 ◽  
Vol 14 (3) ◽  
pp. 5-11
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Heavy Metals ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Crude Extract ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Purification And Properties ◽  
Temperature Optima

Lipase from the hepatopancreas of Tra (Pangasius) catfish was purified by ammonium sulfate fractionation, followed by ion-exhange chromatography on DEAE Cellulose and gel filtration Sephadex G-75. The preparation was homogeneous on polyacrylamide disc gel electrophoresis. The specific activity of the purified enzyme was 37.95 times higher than that of the crude extract. The enzyme showed a molecular weight of 57000 Da. The pH and temperature optima of purified lipase were 8 and 500C respectively. Enzyme activity was enhanced by Ca2+ but inhibited by heavy metals Zn2+, Cd2+, Mg2+.

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