Purification and partial characterization of a plasma inhibitor of tonin

1981 ◽  
Vol 59 (4) ◽  
pp. 256-261 ◽  
Author(s):  
J. Tremblay ◽  
G. Thibault ◽  
J. Gutkowska ◽  
R. Boucher ◽  
J. Genest
Keyword(s):  
Molecular Weight ◽  
Plasma Proteins ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Angiotensin I ◽  
Stokes Radius

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/f0) of 1.95 was calculated from the molecular weight and Stokes radius.Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat α1-macroglobulin or human α2-macroglobulin

scholarly journals The isolation and partial characterization of low-molecular-weight phosphorylated component of the non-histone proteins of mouse nuclei

1978 ◽  
Vol 175 (1) ◽  
pp. 35-46 ◽  
Author(s):  
A J MacGillivray ◽  
C Johnston ◽  
R MacFarlane ◽  
D Rickwood
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Liver Nuclei ◽  
Acidic Proteins

After labelling of mouse liver nuclei with [gamma-32P]ATP in vitro, 10-20% of the radioactivity incorporated into the saline-soluble nuclear and HAP2 chromatin fractions was located in a low-molecular-weight component (component 10) with pI near 4.5 in urea. By using combinations of ion-exchange chromatography, preparative thin-layer isoelectric focusing and gel filtration, this component was isolated from both nuclear fractions. Recovery from the saline-soluble fraction was poor under conditions that allow endogenous phosphatases to be active. Component 10 was shown to be a phosphoprotein on the basis of enzyme-digestion experiments and the detection of phosphoserine and phosphothreonine. The 32P radioactivity did not appear to be associated with phosphorylated basic amino acids. Its molecular weight was determined by gel chromatography and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels as approx. 10000, and tryptic digestion of the reduced carboxymethylated protein in urea yielded two 32P-labelled peptides. It has not been possible as yet to assign a function to component 10, though its similarity to other low-molecular-weight acidic proteins is discussed.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

scholarly journals Purification and some properties of rabbit antiovalbumin

1973 ◽  
Vol 135 (4) ◽  
pp. 705-711 ◽  
Author(s):  
Aftab A. Ansari ◽  
A. Salahuddin
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Acid Media ◽  
Stokes Radius ◽  
Antigen Antibody ◽  
Two Peaks ◽  
Complete Dissociation ◽  
Phosphate Groups

Unlike previous reports that the ovalbumin–anti-ovalbumin complex did not dissociate completely in acid media, our results showed complete dissociation of the complex both in 1.2m-acetic acid, pH2.3, and in KCl–HCl, pH2.2, I 0.06. Thus Sephadex chromatography of the solution obtained by dissolving the antigen–antibody precipitate in these media repeatedly gave two peaks corresponding to anti-ovalbumin and ovalbumin. Further, gel-diffusion and immunoelectrophoresis experiments showed that the phosphate groups of ovalbumin are not involved in the antigenic sites. The antibody thus purified was more easily precipitated than previous preparations. The molecular weight and Stokes radius of the antibody were calculated from its gel-filtration behaviour and were found to be 148000 and 4.8nm respectively. The molecular weight determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis was essentially similar (about 0.7% lower).

scholarly journals PREPARATION AND CHARACTERIZATION OF AN IMMUNOELECTRON MICROSCOPE TRACER CONSISTING OF A HEME-OCTAPEPTIDE COUPLED TO Fab

1974 ◽  
Vol 139 (1) ◽  
pp. 208-223 ◽  
Author(s):  
J. P. Kraehenbuhl ◽  
R. E. Galardy ◽  
J. D. Jamieson
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Antibody Fragments ◽  
Amino Groups ◽  
Electron Microscope Level ◽  
Ph Optimum ◽  
Peroxidatic Activity ◽  
Stokes Radius ◽  
Trypsin Hydrolysis

A heme-octapeptide (mol wt 1,550) has been obtained from cytochrome c by successive pepsin and trypsin hydrolysis and purified by gel filtration and countercurrent distribution. It possesses peroxidatic activity characterized by an apparent Km of 0.2 M, an apparent vmax of 4 mmol/min per mg of peptide, and a pH optimum of 7.0. Using a novel two-step conjugation procedure, the heme-octapeptide was coupled to rabbit Fab antibody fragments by first derivatizing it with the N-hydroxysuccinimide ester of p-formylbenzoic acid and subsequently allowing it to form a Schiff base with the amino groups of Fab. Stable covalent linkages were then obtained by reduction of the Schiff bases with sodium borohydride. The conjugate consists of ∼2 heme-octapeptides attached to each Fab molecule. The molecular weight is 45,000 daltons when coupled to sheep Fab and 50,000 daltons with a Stokes radius of 32 Å, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent Km of 0.4 M, an apparent vmax of 0.4 mmol/min and per mg of attached heme-octapeptide and a pH optimum of 7.0. The conjugate has been used for the localization at the electron microscope level of secretory immunoglobulins in the mammary gland of lactating rabbits.

Characterization of Two Soluble Basic Cytochromes Isolated from the Anoxygenic Phototrophic Sulfur Bacterium Chlorobium phaeobacteroides

1989 ◽  
Vol 44 (1-2) ◽  
pp. 71-76 ◽  
Author(s):  
Ulrich Fischer
Keyword(s):  
Molecular Weight ◽  
Cytochrome C ◽  
Redox Potential ◽  
Isoelectric Point ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Chlorobium Phaeobacteroides ◽  
Flavocytochrome C ◽  
Reduced State

Abstract Chlorobium phaeobacteroides contains two soluble basic c-type cytochromes, a flavocytochrome c-552 and a small cytochrome c-555. Both electron transfer proteins were highly purified by ion exchange chromatography and gel filtration. The flavocytochrome c-552 exhibits maxima at 552 nm, 523 nm and 416 nm in the reduced state and at 409.5 nm with two shoulders at 440 nm and 480 nm in the oxidized form. The best purity index (A280/A416)obtained was 0.65. The molecular properties of this flavocytochrome are as follows: isoelectric point, pH 9.5 - 10; redox potential, +63 mV; molecular weight, 56,000. Cytochrome c-555 is a small basic hemoprotein with an isoelectric point of pH 9.5 - 10, a molecular weight of 9,500 and a midpoint redox potential of +105 mV. The best purity index {A280/A418) obtained was 0.176. The oxidized form of this cytochrome has a maximum at 411.5 nm, while the reduced state shows three maxima (α-band at 554.5 nm; β-band at 523 nm, and γ-band at 418 nm). The a-band is asymmetrical with a typical shoulder at 551 nm.

scholarly journals The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and β-glucosidases

1979 ◽  
Vol 177 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Gabriel M. Umezurike
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Cellulolytic Enzymes ◽  
Botryodiplodia Theobromae ◽  
Molecular Weights ◽  
Low Concentrations ◽  
High Concentrations

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000–48000 (C1), 30000–35000 (C2), 15000–18000 (C3), 10000–11000 (C4) and 4800–5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2–C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2–C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight β-glucosidase (component B1, mol.wt. 350000–380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000–47000) predominated in older filtrates. 9. Intermediate molecular species of β-glucosidase (B2, mol.wt. 170000–180000; B3, mol.wt. 83000–87000) were also found. 10. Cellulases C2–C5 acted in synergism with C1, particularly in the presence of β-glucosidase.

Purification and characterization of an acid phosphatase from peanut (Arachis hypogaea) seed

1984 ◽  
Vol 62 (2) ◽  
pp. 385-391 ◽  
Author(s):  
Sheikh Mehboob Basha
Keyword(s):  
Molecular Weight ◽  
Acid Phosphatase ◽  
Arachis Hypogaea ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Arachis Hypogaea L ◽  
Nitrophenyl Phosphate ◽  
Phosphorylated Sugars

An acid phosphatase (EC 3.1.3.2) from peanut (Arachis hypogaea L.) seed has been purified 433-fold by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. The purified preparation was found to be homogeneous by electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be approximately 240 000 and it was found to be composed of six identical subunits, each with an apparent molecular weight of 42 500. Following isoelectric focusing, the isolectric point (pI) of the enzyme was found to be around pH 5.6. The apparent Km value with p-nitrophenyl phosphate as substrate was 2 ? 10−1 μM. The enzyme was inhibited by Hg2+, Fe2+, Cu2+, Zn2+, Pb2+, and F−. Higher concentrations (2–50 μM) and long incubation periods (60–90 min) with Ca2+ and Mg2+ ions were shown to activate the enzyme. This enzyme showed no effect toward phosphorylated sugars but appear to hydrolyze ATP, ADP, AMP, and β-glycerophosphate.

Purification and characterization of exo-β-1,3-glucanase from a hatching supernatant of Strongylocentrotus intermedius

1983 ◽  
Vol 61 (1) ◽  
pp. 54-62 ◽  
Author(s):  
Kiyoshi Takeuchi
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Protein Band ◽  
Divalent Ions ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Single Protein

Exo-β-1,3-glucanase from the sea urchin embryos was purified 114-fold from the initial hatching supernatant by the following procedures: (a) gel filtration on Sephadex G-100; (b) hydrophobic chromatography on 4-phenylbutylamine-Sepharose (PBA-Sepharose); (c) two ion-exchange chromatographic steps on DEAE-cellulose; (d) gel filtration on Ultrogel AcA 34; (e) gel filtration on Sephadex G-100. The purified enzyme contained 2.2% carbohydrate and gave a single protein band corresponding to a molecular weight of 136 000 following electrophoresis on sodium dodecyl sulfate (SDS) – urea – polyacrylamide gel. Gel filtration on Ultrogel AcA 34 with a nondenaturing solvent gave a molecular weight of 130 000 ± 6000. The enzyme displayed an optimum pH at 5.0–5.5 and hydrolysed laminarin and PS(curdlan)-beads at the nonreducing ends, releasing glucose. Although activity of the purified enzyme was not affected by SDS, urea, some divalent ions, and 2-mercaptoethanol, both dithiothreitol and Hg2+ were markedly inhibitory.

Separation and Characterization of Two Fibrinolytic Inhibitors from Human Placenta

1971 ◽  
Vol 25 (03) ◽  
pp. 580-589 ◽  
Author(s):  
M Uszynski ◽  
U Abildgaard
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Isoelectric Focusing ◽  
Basic Protein ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Coagulation Inhibitor ◽  
Tissue Thromboplastin

SummaryProcedures for the separation of two inhibitors of the activation of plasminogen to plasmin by urokinase are described. Tissue thromboplastin was removed by adsorption to Al(0H)3 gel followed by ultracentrifugation. Plasminogen, plasminogen activator, a coagulation inhibitor and hemoglobin were removed by ion exchange chromatography (CM- or DEAE-Sephadex with NaCl gradients). The minor UK inhibitor is a relative basic protein with a pI of about 5.8. The major inhibitor was purified further by isoelectric focusing, preparative electrophoresis in polyacrylamide gel, and gel filtration. This inhibitor has α1-motility, the pI is about 5.2, and the molecular weight about 100,000. It inactivates urokinase progressively, but does not inhibit streptokinase, plasmin or thrombin.

Isolation and partial characterization of bovine liver aminopeptidase B

1985 ◽  
Vol 50 (5) ◽  
pp. 1249-1257 ◽  
Author(s):  
Ján Blahovec ◽  
Michal Bartík ◽  
Evžen Kasafírek
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Bovine Liver ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Original Material ◽  
Purification Procedure ◽  
Michaelis Constant ◽  
Derivatives Of

Aminopeptidase B, specifically hydrolyzing the L-lysine and L-arginine derivatives of p-nitroaniline and β-naphthylamine, was isolated from bovine liver. A multistep purification procedure involving fractionation with ammonium sulfate, gel filtration on Sephadex, ion exchange chromatography on Ecteola-cellulose, and adsorption chromatography on hydroxylapatite, afforded an enzyme whose activity was approximately 240 times higher than the activity of the original material. The molecular weight of the enzyme determined by gel filtration on Sephadex G-200 was approximately 55 000. The Michaelis constant with respect to L-lysyl-p-nitroanilide was 1.2 . 10-3 mol/l.

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