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.

Interactions between Factors XII and XI and Activating Agents in Purified Systems

1975 ◽  
Author(s):  
B. Binder ◽  
G. Krug ◽  
Th. Vukovich
Keyword(s):  
Complex Formation ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Coagulation Factors ◽  
Polyacrylamide Gel ◽  
The Other ◽  
Ionic Exchange ◽  
Solid Complex ◽  
No Activation

To obtain a better understanding of the activating mechanism of F XI we studied the interaction of highly purified F XII and F XI with activating agents (kaolin, cephalin) F XII as well as F XI were purified from ACD-plasma (ionic exchange procedures, gel fractionation). Both coagulation factors were obtained with a specific activity per mg protein of approximately 4000 times the value of the original plasma, showing a single band in polyacrylamide gel electrophoresis. The preparations contained less than 1 per cent of their activated forms.F XII and F XI as well as the mixture of both were exposed to kaolin, cephalin, or a kaolin-cephalin mixture, respectively. F XII was adsorbed to about 90 per cent by kaolin alone and the adsorbed fraction was activated completely within minutes; F XI on the other hand was neither adsorbed nor activated by kaolin. The kaolin-activated F XII effected practically no activation of F XI. By cephalin F XII was activated only within hours, but the so activated F XII produced activation of F XI corresponding to F XIIa available in a ratio F XIIa/F XI a of 10/1. A mixture of kaolin and cephalin activated F XII within minutes; ca 20 to 30 per cent of the resulting F XIIa activity became not adsorbed to kaolin and this F XII a activates F XI in the same ratio as in the case of cephalin-activated F XII. Gel fractionation of F XII and F XI together with cephalin revealed that only F XIIa is bound to the phospholipid; no solid complex formation was found between F XI and cephalin or between F XI and F XII.(Supp. by a Grant from the Eisner-Foundation.)

scholarly journals Apoprotein B of lipoprotein(a) of human plasma

1982 ◽  
Vol 208 (2) ◽  
pp. 393-398 ◽  
Author(s):  
P Mondola ◽  
D Reichl
Keyword(s):  
Human Plasma ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Low Density Lipoproteins ◽  
Gel Chromatography ◽  
Agarose Gel ◽  
Fatty Meal ◽  
Lipoprotein A ◽  
Apoprotein B

Lipoprotein(a) was purified by agarose-gel chromatography from human plasma from which lipoproteins of Sf greater than 0 had been removed either by sequential or by density-gradient ultracentrifugation. After delipidation, the apoprotein B of this lipoprotein was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It could not be distinguished from the apoprotein B of low-density lipoproteins (rho 1.019-1.063 g/ml). A significant increase in the concentration of apoprotein B in plasma from which the Sf greater than 0 lipoproteins had been removed was observed in six subjects 4 h after a fatty meal.

scholarly journals Purification of Human Factor

1977 ◽  
Author(s):  
V.P.A. Bolhuis ◽  
T.B.M. Hakvoort ◽  
K. Breederveld ◽  
I.A. Mochtar ◽  
J.W. ten Cate
Keyword(s):  
Polyethylene Glycol ◽  
Gel Electrophoresis ◽  
Human Factor ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Factor V ◽  
Native Protein ◽  
Sepharose 4B ◽  
Polyethylene Glycol 6000

Human factor V has been purified from cryo-supernatant by fractionated precipitation with polyethylene glycol (6000, 16-24% w/v, yield 65%), followed by gelfiltration on AcA 44 in Michaelis buffer with 50mM Ca++ (elution in Vo, yield 90%), adsorption of contaminating haptoglobinpolymers on hemoglobin bound to Sepharose-4B (yield 95%) and adsorption of the glycoproteins (mainly factor V) onto concanavaline A bound to Sepharose-4B, elution with α-methylpyranoside (4M) and gelfiltration of the factor V eluted. The purified factor V hasa specific activity of about 28 u/mg and thetotal yield is 16%. The native protein and the subunitsobtained by reduction in the presence of dodecyl sulphate and urea have been characterized by Polyacrylamide gel electrophoresis.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

scholarly journals High Resolution Microsatellite Marker Analysis of Some Rice Landraces Using Metaphor Agarose Gel Electrophoresis

2016 ◽  
Vol 20 (1) ◽  
pp. 54 ◽  
Author(s):  
K. Kristamtini ◽  
T. Taryono ◽  
Panjisakti Basunanda ◽  
Rudi Hari Murti
Keyword(s):  
Microsatellite Markers ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Molecular Techniques ◽  
Agarose Gel ◽  
Chain Reaction ◽  
Rice Landraces ◽  
Polymorphic Pattern ◽  
Polymerase Chain ◽  
Microsatellite Marker Analysis

Microsatellite markers or simple sequences repeats are DNA - based molecular techniques that areused to see the different among accessions and inbred lines. There are three methods to analysis the results ofthe polymerase chain reaction of microsatellite markers namely polyacrylamide gel electrophoresis (PAGE),capillary electroforesis, and Metaphor Agarose Gel Electroforesis (MAGE), and the Use of MAGE assessedmore easily and economically the polymorphic pattern of DNA markers. This study aimed to obtain fast,effective and efficient in term of easy and cheap technique to identify microsatellite markers of some blackrice cultivars and F2 populations from crosses between black with white rice. The results showed that MAGEsuccessfully separated clearly SSRs alleles with different sizes of less than 25 bp .

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

scholarly journals Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 389-397 ◽  
Author(s):  
D A Wiginton ◽  
M S Coleman ◽  
J J Hutton
Keyword(s):  
Molecular Weight ◽  
Adenosine Deaminase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Periodic Acid Schiff ◽  
Apparent Homogeneity

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.

EFFECTS OF 5α-ANDROSTANE-3β,17β-DIOL AND 5β-DIHYDROTESTOSTERONE ON ACID PHOSPHATASE ACTIVITY IN THE PROSTATE GLAND OF THE CASTRATED ADULT RAT

1978 ◽  
Vol 79 (1) ◽  
pp. 9-16 ◽  
Author(s):  
M. P. TENNISWOOD ◽  
PAMELA P. ABRAHAMS ◽  
C. E. BIRD ◽  
A. F. CLARK
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Gel Electrophoresis ◽  
Acid Phosphatase Activity ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Prostate Gland ◽  
Intraperitoneal Administration ◽  
Adult Rat ◽  
Secretory Acid Phosphatase

Polyacrylamide gel electrophoresis of filtrates from adult rat prostatic tissue showed two bands of acid phosphatase activity. These corresponded to the lysosomal and secretory acid phosphatases. After castration the secretory acid phosphatase disappeared. The specific activity of the enzyme increased from the time of castration to a maximum on day 7 before declining steadily, while the percentage inhibition by tartrate of acid phosphatase increased from control levels to a maximum on day 7 and then decreased to a new steady state by day 15. When 5α-androstane-3β,17β-diol was administered i.p. at a dose of 2 mg/day, starting immediately after castration, the secretory acid phosphatase was retained but the percentage inhibition and the specific activity were both raised above control levels. When this steroid was administered daily starting 7 days after castration the secretory acid phosphatase band on the gels returned more rapidly than with the classical androgens, but the percentage inhibition and specific activity were once again raised. Intraperitoneal administration of 5β-dihydrotestosterone, at a dose of 2 mg/day, did not maintain the secretory acid phosphatase activity which disappeared by day 5. However, the specific activity of acid phosphatase and the percentage inhibition by tartrate were both raised throughout the experiment. If this steroid was given 7 days after castration, the percentage inhibition by tartrate did not respond and fell to the level seen in castrated rats. The specific activity, however, remained significantly above the level found in castrated control rats.

scholarly journals Effects of some antibiotics on glucose 6-phosphate dehydrogenase in sheep liver

2012 ◽  
Vol 47 (No. 10 - 11) ◽  
pp. 283-288 ◽  
Author(s):  
M. Çiftci ◽  
V. Turkoglu ◽  
S. Aldemir
Keyword(s):  
Gel Electrophoresis ◽  
Enzyme Purification ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sheep Liver ◽  
Sds Page ◽  
In Vitro Effects ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

In vitro effects of penicillin, sulbactam, cefazolin, and amikacine on the activity of the enzyme glucose-6-phosphate dehydrogenase in sheep liver were investigated. Glucose 6-phosphate dehydrogenase was purified from sheep liver, using a simple and rapid method. The purification consisted of two steps, preparation of homogenate and 2’, 5’-ADP Sepharose 4B affinity chromatography. As a result of the two consecutive procedures, the enzyme, having the specific activity of 11.76 EU/mg proteins, was purified with a yield of 35.72% and 1.913 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS-PAGE showed a single band for the enzyme. In addition, I50 values of the antibiotics were determined by plotting activity % vs. antibiotic concentrations. I50 values were 17.71 mM for penicillin, 27.38 mM for sulbactam, 28.88 mM for cefazolin, and 30.59 mM for amikacine.

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