Purification of Porcine and Human Tissue Activator

Tissue activator from pig heart: A highly purified activator preparation from pig heart has been prepared, essentially using two affinity adsorbtion steps. 1) Affinity adsorbtion to fibrin and elution with potassium thiocyanate. 2) Affinity chromatography on Sepharose-arginine. The final product, which is obtained by gel filtration on Sephacryl S-200 contains according to SDS-polyacrylamide gel electrophoresis one main band with Mw 64000. Reduced samples still appear as one component but with Mw 31000. The specific activity is about 500000 IU/mg (WHO Reference Preparation for Urokinase) and the yield 15-25 %. Tissue activator from human uterus: A highly purified preparation of human tissue activator has been prepared from uterus by an immunosorbent technique using antibodies produced in goats against the porcine tissue activator and coupled to Sepharose. A crude preparation from 1 kg uterus tissue and containing about 100000 IU tissue activator was adsorbed on 30 g of the immunosorbent. The activity was eluted with a KSCN-gradient. Further purification was obtained by affinity chromatography on Sepharose-arginine. The yield in the active fraction was 30-35 % and the specific activity 200000 to 300000 IU/mg. SDS-polyacrylamide gel electrophoresis showed one main band and 1-2 additional trace components.

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Isolation of carboxypeptidase N by affinity chromatography on column of CNBr-activated Sepharose with immobilized antibody

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19790626 ◽

1979 ◽

Vol 44 (2) ◽

pp. 626-630 ◽

Cited By ~ 1

Author(s):

Eva Simonianová ◽

Marie Petáková

Keyword(s):

Affinity Chromatography ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Rabbit Antiserum ◽

Serum Analysis ◽

Polyacrylamide Gel ◽

Rat Serum ◽

Carboxypeptidase N

The isolation of rat serum carboxypeptidase N (EC 3.4.2.2) by affinity chromatography on a column of CNBr-activated Sepharose with immobilized antibody is described. The ligands used were either rabbit antiserum to rat carboxypeptidase N or the IgG fraction prepared from this serum. The coupling of the isolated antibodies to CNBr-activated Sepharose increased the capacity of the column approximately three times. The specific activity of the enzyme prepared by this method was 109-times higher than the activity of the serum. Analysis of the final product by polyacrylamide gel electrophoresis showed carboxypeptidase N and traces of albumin.

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Purification and characterization of rat liver glycosylasparaginase

Biochemical Journal ◽

10.1042/bj2600101 ◽

1989 ◽

Vol 260 (1) ◽

pp. 101-108 ◽

Cited By ~ 26

Author(s):

O K Tollersrud ◽

N N Aronson

Keyword(s):

Rat Liver ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Deae Cellulose ◽

Polyacrylamide Gel ◽

Oligosaccharide Chain ◽

Molecular Masses ◽

High Mannose

1. Rat liver glycosylasparaginase [N4-(beta-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. 2. The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS/polyacrylamide-gel electrophoresis. 3. The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain. 4. N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit. 5. The enzyme exhibited a broad pH maximum above 7. Two major isoelectric forms were found at pH 6.4 and 6.6. 6. Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0.

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Isolation of a human hepatic 60 kDa aspartylglucosaminidase consisting of three non-identical polypeptides

Biochemical Journal ◽

10.1042/bj2620189 ◽

1989 ◽

Vol 262 (1) ◽

pp. 189-194 ◽

Cited By ~ 11

Author(s):

M Baumann ◽

L Peltonen ◽

P Aula ◽

N Kalkkinen

Keyword(s):

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Polyacrylamide Gel ◽

Inherited Disease ◽

Ph Optimum ◽

Native Polyacrylamide Gel Electrophoresis ◽

Molecular Masses ◽

Polypeptide Chains

We have characterized the properties of human aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which is deficient in the human inherited disease aspartylglucosaminuria. The purification procedure from human liver included affinity chromatography, gel filtration, strong-anion- and strong-cation-exchange h.p.l.c., chromatofocusing and reverse-phase h.p.l.c. In a denaturing SDS/polyacrylamide-gel electrophoresis, the 6600-fold purified enzyme was shown to be composed of three non-identical inactive polypeptide chains of molecular masses 24, 18 and 17 kDa. In a native polyacrylamide-gel electrophoresis, these polypeptide chains ran as one active enzyme complex. As judged from the elution position of the native enzyme in a Biogel P-100 gel filtration, the approximate molecular mass of this complex was 60 kDa. The enzyme had a pI of 5.7, a pH optimum at 6, of 0.48 mM and a specific activity of 200,000 nkat for the substrate 2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-D-glucose. The enzyme showed a 57% loss of activity at 60 degrees C after 45 h but was practically inactive after incubation at 72 degrees C for a few minutes. The molecular structure, Km and specific activity as well as the thermostability of the enzyme described here are different from those reported previously for human aspartylglucosaminidase.

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Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

Biochemical Journal ◽

10.1042/bj2130187 ◽

1983 ◽

Vol 213 (1) ◽

pp. 187-191 ◽

Cited By ~ 37

Author(s):

A Lewendon ◽

J R Coggins

Keyword(s):

Escherichia Coli ◽

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Polyacrylamide Gel ◽

Purification Step ◽

Phosphate Synthase

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.

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The purification of porcine haptoglobin by affinity chromatography with concanavalin A – Sepharose

Canadian Journal of Biochemistry ◽

10.1139/o76-144 ◽

1976 ◽

Vol 54 (11) ◽

pp. 999-1001 ◽

Cited By ~ 8

Author(s):

Frank A. Terpstra ◽

David B. Smith

Keyword(s):

Affinity Chromatography ◽

Concanavalin A ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Polyacrylamide Gel ◽

Small Scale ◽

Scale Separation

Methods designed for the isolation of human haptoglobin (Hp) were found insufficient when applied to pig plasma due to the formation of a material tentatively identified as albumin dimer. Small scale separation is possible by preparative polyacrylamide gel electrophoresis. Larger scale purification from nonglycoprotein contaminants such as albumin dimer is achieved by affinity chromatography using immobilized concanavalin A. Porcine haptoglobin is microheterogeneous. More than 14 components, partially resolveable by gel filtration, were detected.

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Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase

Biochemical Journal ◽

10.1042/bj1830531 ◽

1979 ◽

Vol 183 (3) ◽

pp. 531-537 ◽

Cited By ~ 18

Author(s):

P N Srivastava ◽

A A Farooqui

Keyword(s):

Hyaluronic Acid ◽

Affinity Chromatography ◽

Seminal Plasma ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Single Band ◽

National Formulary ◽

Turnover Number

Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C.

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Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis

Biochemical Journal ◽

10.1042/bj2610483 ◽

1989 ◽

Vol 261 (2) ◽

pp. 483-488 ◽

Cited By ~ 40

Author(s):

K Takazawa ◽

H Passareiro ◽

J E Dumont ◽

C Erneux

Keyword(s):

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Bovine Brain ◽

Polyacrylamide Gel ◽

Maximal Velocity ◽

Inositol 1,4,5 Trisphosphate ◽

Maximal Activation

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). A method is presented for the rapid purification of InsP3 3-kinase from bovine brain by calmodulin (CaM)-Sepharose affinity chromatography. Maximal activation of the purified InsP3 3-kinase by Ca2+/CaM was 6-7-fold as compared with the activity measured in the presence of EGTA (1 mM) and 10 microM-InsP3. At 10 microM-InsP3 and 0.1 mM free Ca2+, half-maximal activation required about 2 nM-CaM. The mechanism of activation by CaM appeared to be an increase in the maximal velocity of the enzyme without a substantial change in the Km for InsP3. Further purification was achieved by phosphocellulose chromatography eluted with ATP. Specific activity of the purified enzyme at 37 degrees C and 10 microM-InsP3 was 10-20 mumol/min per mg. The apparent Mr of the enzyme, determined by f.p.l.c.-gel filtration, was estimated as about 44,000. The purified InsP3 3-kinase was subjected to SDS/10%-polyacrylamide-gel electrophoresis. InsP3 3-kinase activity was associated with three silver-stained bands, which migrated with apparent Mr values of approx. 52,000, 38,000 and 35,000.

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Purification, Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase fromAspergillus oryzae

Applied and Environmental Microbiology ◽

10.1128/aem.64.10.3607-3614.1998 ◽

1998 ◽

Vol 64 (10) ◽

pp. 3607-3614 ◽

Cited By ~ 225

Author(s):

Christine Riou ◽

Jean-Michel Salmon ◽

Marie-Jose Vallier ◽

Ziya Günata ◽

Pierre Barre

Keyword(s):

Molecular Mass ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Polyacrylamide Gel ◽

Glucose Tolerant

ABSTRACT Aspergillus oryzae was found to secrete two distinct β-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3′,4′,5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total β-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant β-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-β-d-glucoside as the substrate, we found that the enzyme was optimally active at 50°C and pH 5.0 and had a specific activity of 1,066 μmol min−1mg of protein−1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki , 1.36 M) or glucono-δ-lactone (Ki , 12.5 mM), another powerful β-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal β-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1→3)- and (1→6)-β-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-β-d-glucosides in a grape must (pH 2.9, 90 g of glucose liter−1). Other flavor precursors (benzyl- and 2-phenylethyl-β-d-glucosides) and prunin (4′,5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel β-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.

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Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

Biochemical Journal ◽

10.1042/bj1970629 ◽

1981 ◽

Vol 197 (3) ◽

pp. 629-636 ◽

Cited By ~ 19

Author(s):

J L McKenzie ◽

A K Allen ◽

J W Fabre

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Human Brain ◽

Affinity Chromatography ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Antibody Affinity ◽

Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

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Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with α2-macroglobulin

Biochemical Journal ◽

10.1042/bj1910799 ◽

1980 ◽

Vol 191 (3) ◽

pp. 799-809 ◽

Cited By ~ 41

Author(s):

R G Sutcliffe ◽

B M Kukulska-Langlands ◽

J R Coggins ◽

J B Hunter ◽

C H Gore

Keyword(s):

Molecular Weight ◽

Affinity Chromatography ◽

Plasma Protein ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Protein A ◽

Carbohydrate Content ◽

Polyacrylamide Gel ◽

Gel Chromatography ◽

Tryptic Fragment

Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000–820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A.

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