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.

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.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

Partial characterization of an abnormal lactate dehydrogenase isoenzyme, LDH-1ex, in serum from a patient with hepatocellular carcinoma.

1989 ◽  
Vol 35 (5) ◽  
pp. 844-848
Author(s):  
D L Kalpaxis ◽  
E E Giannoulaki
Keyword(s):  
Hepatocellular Carcinoma ◽  
Lactate Dehydrogenase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Heat Stable ◽  
Single Polypeptide ◽  
Polypeptide Chains

Abstract Serum from a patient with hepatocellular carcinoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), LDH-1ex, that on electrophoresis on 10-g/L agarose gel migrated anodally to the LDH-1 band. This isoenzyme was partly purified by ultrafiltration and preparative electrophoresis. Gel chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies of the resulting LDH-1ex preparation suggested that this isoenzyme is probably a tetramer made up of four single polypeptide chains (monomers), each having a molecular mass of about 32,000 Da. LDH-1ex was heat stable and reacted more readily with 2-hydroxybutyrate than did the slower migrating LDH-4 and LDH-5 isoenzymes. LDH-1ex showed no activity when lactate was omitted from the substrate solution or replaced by ethanol.

scholarly journals A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage

1981 ◽  
Vol 197 (2) ◽  
pp. 355-366 ◽  
Author(s):  
D Heinegård ◽  
M Paulsson ◽  
S Inerot ◽  
C Carlström
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Chondroitin Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Partial Specific Volume ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Cscl Density Gradient

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40-1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.

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.

Quantitative analysis of serum proteins separated by capillary electrophoresis

1993 ◽  
Vol 39 (4) ◽  
pp. 689-692 ◽  
Author(s):  
J W Kim ◽  
J H Park ◽  
J W Park ◽  
H J Doh ◽  
G S Heo ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Gel Electrophoresis ◽  
Serum Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Serum Samples ◽  
Polyclonal Gammopathy ◽  
Quantitative Analyses

Abstract The possibility of open tubular capillary electrophoresis for clinical diagnostic use is examined. Capillary electrophoresis was performed in an untreated 50 microns (i.d.) x 100 cm (65 cm to detector) capillary with detection of absorbance at 200 nm. Conditions for the separation of serum proteins without adsorption to the capillary surface were established. Quantitative analyses of serum samples from 38 patients with liver cirrhosis, nephrotic syndrome, or polyclonal gammopathy by capillary electrophoresis were done and the results were compared with those by conventional agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All samples were analyzed in duplicate. We evaluated linearity of response, within-run CV, and the correlation between capillary electrophoresis and agarose gel electrophoresis.

Immunoadsorbent isolation of pregnancy-specific β1-glycoprotein from maternal serum

1983 ◽  
Vol 61 (2-3) ◽  
pp. 130-136 ◽  
Author(s):  
Bertram W. Griffiths ◽  
André Godard
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maternal Serum ◽  
Polyacrylamide Gel ◽  
Relative Mass ◽  
Gel Chromatography ◽  
Polymeric Form ◽  
Step Procedure ◽  
New Findings

A three-step procedure for the purification of pregnancy-specific β1-glycoprotein (PSβ1G) on a milligram scale from maternal serum has been developed. The principal purification was achieved by the use of an immunoadsorbent and the remaining impurities were removed by hydroxylapatite chromatography and negative affinity chromatography. The overall procedure resulted in the purification of approximately 10 mg of PSβ1G which represented about 21% of PSβ1G in 300 mL of serum. The PSβ1G was of high purity as shown by analytical polyacrylamide gel electrophoresis, sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and immunochemical tests. Experiments by immunoelectrophoresis and gel chromatography indicate that the electrophoretic mobility and relative mass of the purified PSβ1G are very similar to those of the native serum protein. Structural analysis of PSβ1G suggests that it is composed of two identical subunit chains bonded noncovalently. However, a trimeric structure for PSβ1G cannot be ruled out based on the uncertainty of relative mass estimates by gel chromatography in nondenaturing solvent. The anomalous characteristics of a previous purified polymeric form of PSβ1G (PSβ1G-I) are discussed in relation to the new findings presented here.

scholarly journals Human plasma kallikrein. A rapid purification method with high yield

1981 ◽  
Vol 193 (1) ◽  
pp. 187-192 ◽  
Author(s):  
H Nagase ◽  
A J Barrett
Keyword(s):  
Human Plasma ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Gel Chromatography ◽  
Purification Method ◽  
Simple Method ◽  
Bean Trypsin Inhibitor ◽  
Rapid Purification ◽  
Sepharose 4B

A simple method for isolation of kallikrein from human plasma is described. Before activation of the enzyme with acetone, the plasma was treated with 0.2 M-methylamine at pH 8.2 to inactivate alpha 2-macroglobulin and thus prevent the irreversible binding of the active enzyme to the inhibitor. The enzyme was adsorbed on soya-bean trypsin inhibitor-Sepharose 4B and eluted with 5 mM-NaOH, pH 11.3. It was further purified by immunoadsorption of contaminating proteins, and gel chromatography on Ultrogel AcA 44. About 3 mg of kallikrein was obtained from 400 ml of plasma (35% yield). The purified enzyme was shown to be homogeneous by electrophoretic and immunological criteria. The specific activities against benzyloxycarbonylphenylalanylarginine methylcoumarylamide, prolylphenylalanylarginine methylcoumarylamide and tosylarginine methyl ester were higher than any previously reported. The purified enzyme was resolved into two forms of mol.wts. 88 000 and 86 000 in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis without reduction. Each consisted of three chains linked by disulphide bonds, one containing the reactive serine residue (mol.wt. 36 000 or 34 000), and two additional chains (mol.wt. 28 000 and 22 000).

Structural Studies on Antigen E of Ragweed Pollen

1973 ◽  
Vol 51 (9) ◽  
pp. 1275-1280 ◽  
Author(s):  
B. W. Griffiths
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reducing Agent ◽  
Ragweed Pollen ◽  
Gel Chromatography ◽  
Structural Studies ◽  
Molecular Weights ◽  
Polypeptide Chains

The analysis of antigen E of ragweed pollen by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) resulted in the demonstration of two subunits of molecular weights of about 20 000 and 14 500. These results confirm earlier studies on antigen E by gel chromatography that demonstrated a two-polypeptide subunit composition. Treatment of antigen E with 1% SDS (in the absence of reducing agent) resulted in the dissociation of the two polypeptide chains, which indicates that the bonds bridging the subunits are non-covalent in nature.

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