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.

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.

Rat liver ribonucleotide reductase: separation, purification, and properties of two nonidentical subunits

1982 ◽  
Vol 60 (4) ◽  
pp. 463-470 ◽  
Author(s):  
T. Youdale ◽  
J. P. MacManus ◽  
J. F. Whitfield
Keyword(s):  
Rat Liver ◽  
Ribonucleotide Reductase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Relative Mass ◽  
Purification And Properties ◽  
Exclusion Chromatography

Two nonidentical subunits of mammalian ribonucleotide reductase, L1 and L2, from regenerating rat liver have been extensively purified for the first time. They were separated by dATP-Sepharose affinity chromatography. Subunit L1, which bound to dATP-Sepharose, was eluted with 50 mM ATP and purified to homogeneity (as demonstrated by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis) by molecular exclusion high-pressure liquid chromatography (HPLC). This subunit had an apparent relative mass (Mr) of 45 000 and a Km of 0.9 × 10−4 for CDP. Subunit L2, which did not bind to dATP-Sepharose, was purified by pH 5.2 precipitation followed by chromatography on CM-Sephadex, molecular exclusion HPLC, and DEAE-cellulose. This subunit contained iron and had an apparent Mr of 120 000 by HPLC molecular exclusion chromatography, but showed two bands (Mr 75 000 and Mr 47 000) on SDS–polyacrylamide gel electrophoresis. Neither L1 nor L2 separately had any enzyme activity but when combined they reduced CDP to dCDP.

Production of antibody to phosphoprotein associated with nucleosome structure

1982 ◽  
Vol 60 (3) ◽  
pp. 356-363 ◽  
Author(s):  
M. S. Zhao ◽  
C. C. Liew
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Methyl Cellulose ◽  
Polyacrylamide Gel ◽  
Relative Mass ◽  
Nucleosome Structure ◽  
The Relationship

Antibodies were produced to phosphoprotein fraction, and a phosphoprotein B2 obtained by carboxyl methyl cellulose column chromatography and sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis as described previously (Biochem. J. 183, 147 (1979)). Production of the specific antibody was confirmed by double immunodiffusion. The phosphoprotein B2 (relative mass 68 000), which was isolated from the phosphoprotein fraction by SDS–polyacrylamide gel electrophoresis, specifically reacted with the antisera, as identified by "rocket" immunoelectrophoresis. Further characterization of the antibody to the phosphoprotein was carried out by isoelectrofocusing gel electrophoresis. The phosphoprotein, previously identified in the isoelectric point (pI) region 6.2–8.5, was subsequently reacted with antisera and 125I-labelled protein A. A prominent radioactive peak was identified in the region in which the phosphoprotein was focused. The radioimmunoactivity was proportional to the amount of phosphoproteins present in the isoelectric focusing gel. The presence of phosphoprotein in two types of mononucleosomes (MN1 and MN2) was demonstrated immunologically by use of the phosphoprotein antibody. The relationship between the phosphoprotein and chromatin structure, and possible role in gene regulation is discussed.

scholarly journals Spectrophotometric assay, solubilization and purification of brain 2′:3′-cyclic nucleotide 3′-phosphodiesterase

1980 ◽  
Vol 191 (1) ◽  
pp. 71-82 ◽  
Author(s):  
Y Nishizawa ◽  
T Kurihara ◽  
Y Takahashi
Keyword(s):  
White Matter ◽  
Cyclic Nucleotide ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Spectrophotometric Assay ◽  
Polyacrylamide Gel ◽  
Hexadecyltrimethylammonium Bromide ◽  
Gel Chromatography ◽  
Brain White Matter

1. A spectrophotometric assay of 2′:3′-cyclic nucleotide 3′-phosphodiesterase (EC 3.1.4.37) based on the use of an acid-base indicator and a buffer having identical pKa values is described. The assay is simple and rapid; it was particularly convenient for monitoring the enzyme activity at various stages of purification. 2. Several proteinases were examined for their ability to solubilize 2′:3′-cyclic nucleotide 3′-phosphodiesterase from delipidated brain white matter. Trypsin (EC 3.4.21.4) and elastase (EC 3.4.21.11) appeared to be more effective than the other proteinases examined. Trypsin, however, caused inactivation; elastase was therefore chosen to solubilize 2′:3′-cyclic nucleotide 3′-phosphodiesterase. When a partially purified preparation of 2′:3′-cyclic nucleotide 3′-phosphodiesterase was treated with elastase, 2′:3′-cyclic nucleotide 3′-phosphodiesterase was solubilized nearly quantitatively. Elastatinal, a specific inhibitor of elastase, specifically inhibited the solubilization with elastase. 3. 2′:3′-cyclic nucleotide 3′-phosphodiesterase was purified from bovine brain white matter by: (i) delipidation; (ii) solubilization with hexadecyltrimethylammonium bromide; (iii) gel chromatography on Sepharose; (iv) ethanol precipitation and resolubilization by digestion with elastase; (v) chromatography on DEAE-Sephadex; (vi) affinity chromatography on 8-(6-aminohexyl)amino-2′-AMP-Sepharose. 4. The purified enzyme migrated as a single protein band on polyacrylamide-gel electrophoresis at pH 4.3 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the estimated mol.wt. in the latter electrophoresis was 27000-31000. Gel filtration of the purified enzyme through Sephadex G-150 indicated a mol.wt. of 31000. Therefore the purified enzyme is a monomer protein with a mol.wt. of approx. 30000.

scholarly journals Purification of rat fibrinogen and its constituent chains

1978 ◽  
Vol 169 (3) ◽  
pp. 653-658 ◽  
Author(s):  
Irina A. M. Van Ruijven-Vermeer ◽  
Willem Nieuwenhuizen
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rat Plasma ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gel Chromatography

Rat fibrinogen was purified from rat plasma by using lysine–Sepharose chromatography, repeated precipitation with 25%-satd. (NH4)2SO4 and gel chromatography on Sepharose 6B. To minimize proteolytic activity, rats were injected intravenously with Trasylol before bleeding and the collected blood was treated with Trasylol and di-isopropyl phosphorofluoridate. A 95%-clottable preparation was obtained in 70–75% yield; it proved to be free of factor XIII and plasminogen. It showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and on disc electrophoresis in 8m-urea. Alanine was the only detectable N-terminal amino acid. After reduction and modification of the thiol groups, the material could be separated into three distinct chains (Aα, Bβ and γ) by pore-limit polyacrylamide slab-gel electrophoresis in the presence of sodium dodecyl sulphate. The amino acid compositions of the whole fibrinogen and of the separated modified chains were determined. The molecular weights were 61000, 58000 and 51000 for Aα-, Bβ- and γ-chains respectively. Our results for the chains are in contrast with previous reports on rat fibrinogen [Bouma & Fuller (1975) J. Biol. Chem.250, 4678–4683; Stemberger & Jilek (1976) Thromb. Res.9, 657–660], in which no separation between Aα- and Bβ-chains was achieved on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis for 3h. Evidence is presented that this is probably due to Aα-chain degradation as a result of incomplete inhibition of proteolytic enzymes during the purification. Complete inhibition of proteolytic activities is essential in all steps of the present purification procedure.

scholarly journals Isolation and characterization of a variant of ovomucoid

1975 ◽  
Vol 147 (1) ◽  
pp. 139-144 ◽  
Author(s):  
A Waheed ◽  
A Salhuddin
Keyword(s):  
Gel Electrophoresis ◽  
Simple Procedure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Preparative Scale ◽  
Isolation And Purification ◽  
Isolation And Characterization ◽  
Fluorescence Characteristics

A simple procedure, which can be used on a preparative scale, for the isolation and purification of a major variant of ovomucoid from egg white is described. Ovomucoid was precipitated by salt, and further fractionated by chromatography on sulphoethyl-Sephadex. It showed size homogeneity as revealed by gel chromatography and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis where the mobility was consistent with a molecular weight of 28 300+/-2300. The inhibitor showed full antiryptic but no antichymotryptic activity. The u.v.-absorption and fluorescence characteristics indicated the absence of tryptophan. Polyacrylamide-gel electrophoresis in the presence of 9M-urea demonstrated absence of charge heterogeneity. The intrinsic viscosity of ovomucoid was 5.36ml/g which yielded an equivalent hydrodynamic radius (2.9nm), axial ratio (6.0) and frictional ratio (1.31) of the molecule. The Stokes radius (3.5nm), diffusion coefficient (7.8 times 10(-7 cm2/s) and frictional ratio (1.35) were calculated from gel-filtration data. These results suggest that ovomucoid exists in non-globular conformation under native conditions and that the deviation from the behaviour of a typical globular protein seems to be due both to asymmetry and hydration.

scholarly journals Purification of eosinophil peroxidase and studies of biosynthesis and processing in human marrow cells

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1143-1148 ◽  
Author(s):  
I Olsson ◽  
AM Persson ◽  
K Stromberg ◽  
I Winqvist ◽  
PC Tai ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Proteolytic Processing ◽  
Gel Chromatography ◽  
Human Eosinophil ◽  
Eosinophil Peroxidase ◽  
Marrow Cells

Abstract Human eosinophil peroxidase (EPO) was purified from leukocytes obtained from a patient with hypereosinophilia. EPO was extracted from the granule fraction using 0.2 mol/L sodium acetate pH 4.0, and the extract was subjected to gel chromatography on Sephadex G-75 and ion exchange chromatography on Biorex 70. The mol wt calculated from gel chromatography was approximately 50,000. However, under reducing and denaturing conditions, polyacrylamide gel electrophoresis revealed two subunits with mol wt of 50,000 and 15,000. The biosynthesis of EPO was studied in marrow cells from patients with eosinophilia using labeling with (14C)-leucine, followed by immunoprecipitation with anti-EPO, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography for visualization of labeled EPO. Biosynthesis of an Mr 53,000 subunit was demonstrated. Biosynthetic labeling of the Mr 15,000 subunit was not demonstrated. A labeled Mr 25,000 chain was detected and may represent a degradation product or a chain that, after further modification, produces the Mr 15,000 subunit. Labeling was also detected in two polypeptides with mol wt of 78,000 and 72,000. These forms of EPO seem to represent precursor polypeptides subjected to proteolytic processing in a similar manner as has been reported for myeloperoxidase (MPO). However, Monensin, a proton ionophore, which blocks the processing of MPO, did not inhibit processing of EPO, indicating separate mechanisms by which MPO and EPO are directed to granules.

scholarly journals Purification of eosinophil peroxidase and studies of biosynthesis and processing in human marrow cells

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1143-1148
Author(s):  
I Olsson ◽  
AM Persson ◽  
K Stromberg ◽  
I Winqvist ◽  
PC Tai ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Proteolytic Processing ◽  
Gel Chromatography ◽  
Human Eosinophil ◽  
Eosinophil Peroxidase ◽  
Marrow Cells

Human eosinophil peroxidase (EPO) was purified from leukocytes obtained from a patient with hypereosinophilia. EPO was extracted from the granule fraction using 0.2 mol/L sodium acetate pH 4.0, and the extract was subjected to gel chromatography on Sephadex G-75 and ion exchange chromatography on Biorex 70. The mol wt calculated from gel chromatography was approximately 50,000. However, under reducing and denaturing conditions, polyacrylamide gel electrophoresis revealed two subunits with mol wt of 50,000 and 15,000. The biosynthesis of EPO was studied in marrow cells from patients with eosinophilia using labeling with (14C)-leucine, followed by immunoprecipitation with anti-EPO, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography for visualization of labeled EPO. Biosynthesis of an Mr 53,000 subunit was demonstrated. Biosynthetic labeling of the Mr 15,000 subunit was not demonstrated. A labeled Mr 25,000 chain was detected and may represent a degradation product or a chain that, after further modification, produces the Mr 15,000 subunit. Labeling was also detected in two polypeptides with mol wt of 78,000 and 72,000. These forms of EPO seem to represent precursor polypeptides subjected to proteolytic processing in a similar manner as has been reported for myeloperoxidase (MPO). However, Monensin, a proton ionophore, which blocks the processing of MPO, did not inhibit processing of EPO, indicating separate mechanisms by which MPO and EPO are directed to granules.

scholarly journals Proteoglycans of bovine periodontal ligament and skin. Occurrence of different hybrid-sulphated galactosaminoglycans in distinct proteoglycans

1982 ◽  
Vol 201 (1) ◽  
pp. 27-37 ◽  
Author(s):  
C H Pearson ◽  
G J Gibson
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Periodontal Ligament ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Self Association

A proteoglycan purified from 4 M-guanidinium chloride extracts of bovine periodontal ligament closely resembled that of bovine skin, except for a rather lower protein content and a higher molecular weight (120 000 compared with about 90 000) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The latter difference was explained by the molecular weights (29 000 and 16 000) of the respective dermatan sulphate components, each of which was rich in L-iduronate (about 75% of the total hexuronate). Significant amounts of other glycosaminoglycans did not occur in these proteoglycans, which were homogenous on gel chromatography and agarose/polyacrylamide-gel electrophoresis. Polydispersity was observed in sedimentation equilibrium experiments, but proteolysis or self-association of the proteodermatan sulphates may have affected these results. Ligament proteoglycans that were almost completely extracted with 0.1 M-NaCl contained less protein of a completely different amino acid composition than the proteodermatan sulphates. They were heterogeneous in size but generally smaller than cartilage proteoglycans and L-iduronate was a component, comprising about 7% of the total hexuronate of the sulphated galactosaminoglycan chains. The latter consisted of two fractions differing in molecular weight, but a dermatan sulphate with a high L-iduronate content was not present. These proteoglycans had some resemblance to D-glucuronate-rich proteoglycans of other non-cartilaginous tissues. Such compounds, however, are difficult to categorize at present.

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.

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