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.

scholarly journals A study of maturation events in jackbeans (Canavalia ensiformis)

1984 ◽  
Vol 222 (1) ◽  
pp. 265-268 ◽  
Author(s):  
S E Marcus ◽  
J Burgess ◽  
P R Maycox ◽  
D J Bowles
Keyword(s):  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Molecular Forms ◽  
Con A ◽  
Canavalia Ensiformis ◽  
Immature Seeds

Maturation events have been studied in developing jackbean (Canavalia ensiformis) cotyledons by using a combination of analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, overlays with 125I-concanavalin A (Con A) and the use of anti-(Con A) after Western transfer. The number of polypeptides recognized by 125I-Con A varies during maturation, until at maturity only one remains. Several molecular forms of the lectin occur during development; one, corresponding to Mr 33 000 and found only in immature seeds, interacts with 125I-Con A, suggesting that it is glycosylated.

A Sensitive Peroxidase Staining Immunoblotting Method for Measuring Total Protein S in Human Plasma

1993 ◽  
Vol 69 (04) ◽  
pp. 331-334 ◽  
Author(s):  
Xiangan Li ◽  
Kaoru Hatanaka ◽  
Ling Guo ◽  
Motoo Tsushima ◽  
Yukihiko Kitamura ◽  
...  
Keyword(s):  
Human Plasma ◽  
Total Protein ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Protein S ◽  
Polyacrylamide Gel ◽  
Free Form

SummaryIn plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added tonormal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.

scholarly journals Activation of Human Factor X

1977 ◽  
Author(s):  
Carolyn L. Orthner ◽  
Sam Morris ◽  
David P. Kosow
Keyword(s):  
Molecular Weight ◽  
Heavy Chain ◽  
Gel Electrophoresis ◽  
Human Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Coagulation Cascade ◽  
Molecular Forms ◽  
Factor X

Factor X is the zymogen of the proteolytic coagulation enzyme Factor Xa. Since the activation of Factor X to Factor Xa may be a rate limiting step of the coagulation cascade we have begun investigations of the mechanism of this reaction. Human Factor X has been purified 6000-fold from human plasma and the final product is over 95% pure as judged by Polyacrylamide gel electrophoresis. Human Factor X has a monomeric molecular weight of 75,000 and consists of two chains held together by a disulphide bridge. The molecular weight of the heavy chain is 56,000 and that of the light chain is 17,500. The venom coagulant protein of V. russelli (RVV-X) activates human Factor X by cleaving the heavy chain. When fully activated, human Factor Xa shows two bands on Polyacrylamide gel electrophoresis indicating that human Factor Xa like the bovine enzyme has two molecular forms.The kinetic mechanism of the activation reaction has been investigated utilizing the chromogenic Factor Xa substrate Bz-Ile-Glu-Gly-Arg-p-Nitroanilide (S-2222). The reaction has an absolute requirement for Ca; Mg cannot substitute for Ca, however Mg can increase the Vmax of Xa formation in the presence of suboptimal concentrations of Ca. Both Ca and Mg effects exhibit positive cooperativity. Our data indicate that human Factor X has at least three cooperative metal binding sites some of which are specific for Ca.

scholarly journals Physical and chemical properties of human plasma β2-macroglobulin

1978 ◽  
Vol 173 (1) ◽  
pp. 27-38 ◽  
Author(s):  
P K Hall ◽  
R C Roberts
Keyword(s):  
Human Plasma ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chemical Properties ◽  
Sedimentation Coefficient ◽  
Binding Activity ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium

Alpha2-M (alpha2-macroglobulin) was purified from human plasma by two different procedures. As well as having no detectable impurities by the usual criteria for testing the homogeneity of protein preparations, these alpha2M preparations showed a single component, after reduction in urea, of 185000 daltons by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weight of the alpha2M was found to be 718000 by sedimentation equilibrium experiments using the gravimetrically determined -v of 0.731 ml/g. The interaction of several proteinases with alpha2M was studied by using a novel discontinuous polyacrylamide-gel system, which showed clear separation of the enzyme-complexed alpha2M from the free alpha2M. These studies indicated that urokinase, as well as trypsin, chymotrypsin, plasmin and thrombin forms complexes with alphaM. The cleavage of the 185000-dalton subunit to a 85000-dalton species on interaction of trypsin with alpha2M was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction of the alpha2M-trypsin complex in urea. The amino acid composition, carbohydrate content, absorption coefficient at 280 nm, the specific refractive increment and the sedimentation coefficient for these alpha2M preparations were measured. The stability of the trypsin-binding activity of the alpha2M preparations was also studied under several storage situations.

Glucagon from the bovine fetal pancreas: chromatographic and electrophoretic characterizations of high molecular weight immunoreactive species

1980 ◽  
Vol 58 (9) ◽  
pp. 707-714 ◽  
Author(s):  
A. K. Tung ◽  
J. L. Ruse ◽  
E. Cockburn
Keyword(s):  
Molecular Weight ◽  
Acetic Acid ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Con A ◽  
Fetal Bovine ◽  
Bovine Pancreas

Fetal bovine pancreas was extracted for glucagon using (A) ethanol–HCl after trichloroacetic acid (TCA) treatment of the pancreas, (B) ethanol–HCl and (C) urea–acetic acid. Fractionation of the acetic acid soluble proteins via Sephadex G-50 columns yielded glucagon immunoreactivity in the void volume, high molecular weight glucagon immunoreactivities (HMW-IRGs), "proglucagon" [Formula: see text], and true glucagon (3.5 K Δ) regions. HMW-IRGs were obtainable using all three methods of extraction. The material obtained from the ethanol–HCl–TCA method appeared stable on Sephadex G-100 (1 M acetic acid) rechromatography. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis analysis showed immunoreactive species corresponding to [Formula: see text] and [Formula: see text]. HMW-IRGs did not bind to concanavalin A (Con A) – agarose. SDS–polyacrylamide gel electrophoresis of the Con A – agarose filtered IRG again showed a major immunoreactive peak of [Formula: see text]. Dose–response RIA studies indicated that the HMW-IRGs from both the gel filtration and SDS–polyacrylamide gel experiments were immunochemically indistinguishable from glucagon. HMW-IRGs bind to antiglucagon antibody agarose, further indicating their reactivity towards glucagon antibodies. When HMW-IRGs are incubated with guanidinium hydrochloride and gel filtered in the same system, a significant fraction of HMW-IRG (representing up to 25% of the total IRG analysed) was found to resist disruption. Our data support the contention that a significant portion of the HMW-IRGs (molecular weight > 20 K Δ) extracted from fetal bovine pancreas are composed of glucagon covalently linked to larger protein unit(s).

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

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