Purification of a liver DNA-synthesis promoter from plasma of partially hepatectomized rats

J J Díaz-Gil; P Escartín; R García-Cañero; C Trilla; J J Veloso; G Sánchez; A Moreno-Caparrós; C Enrique de Salamanca; R Lozano; J G Gavilanes

doi:10.1042/bj2350049

Purification of a liver DNA-synthesis promoter from plasma of partially hepatectomized rats

Biochemical Journal ◽

10.1042/bj2350049 ◽

1986 ◽

Vol 235 (1) ◽

pp. 49-55 ◽

Cited By ~ 41

Author(s):

J J Díaz-Gil ◽

P Escartín ◽

R García-Cañero ◽

C Trilla ◽

J J Veloso ◽

...

Keyword(s):

Dna Synthesis ◽

Mitotic Index ◽

Gel Electrophoresis ◽

Deae Cellulose ◽

Single Band ◽

Organ Specificity ◽

Purification Procedure

A protein was isolated from plasma of partially (70%) hepatectomized rats that, injected in mice, increases the uptake of [3H]thymidine by liver DNA by 200-300% over that by injected control saline. The purification procedure consists essentially of three chromatography steps, employing Sephadex G-75, DEAE-cellulose and hydroxyapatite. The hepatic promoter (HP) preparation shows a single band in SDS/polyacrylamide (15%)-gel electrophoresis (silver stained), with an Mr of 64 000; its activity is suppressed by trypsin or pepsin and is unaffected by deoxyribonuclease or ribonuclease. On injection into mice (150 ng/mouse), it increases the mitotic index of the liver. It shows organ-specificity, acting on liver but not on spleen, kidney, lung or brain. In primary liver cultures, it produces an increase in uptake of [3H]thymidine into DNA in the range 1-10 ng/ml. In this system in vitro, it increases the uptake of 22Na+ immediately after addition.

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Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties

Biochemical Journal ◽

10.1042/bj1750391 ◽

1978 ◽

Vol 175 (2) ◽

pp. 391-406 ◽

Cited By ~ 51

Author(s):

R Jones ◽

M B Wilkins ◽

J R Coggins ◽

C A Fewson ◽

A D B Malcolm

Keyword(s):

Phosphoenolpyruvate Carboxylase ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Maximum Velocity ◽

Single Band ◽

Kinetic Properties ◽

Subunit Structure

Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.

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Purification and characterization of mouse α-lactalbumin and preparation of its antibody

Biochemical Journal ◽

10.1042/bj1850227 ◽

1980 ◽

Vol 185 (1) ◽

pp. 227-237 ◽

Cited By ~ 25

Author(s):

Y Nagamatsu ◽

T Oka

Keyword(s):

Concanavalin A ◽

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Polyacrylamide Gel ◽

Single Band ◽

Major Species ◽

Alpha Lactalbumin

alpha-Lactalbumin was purified to apparent homogeneity from mouse milk by combined use of gel filtration, chromatography on DEAE-cellulose and hydroxyapatite, and concanavalin A-Sepharose affinity chromatography. Mouse alpha-lactalbumin exists in several species with different charges and in two molecular-size forms. The smaller form, which constituted over 90% of total alpha-lactalbumin, included two major and two minor species, each of which showed different electrophoretic mobility on polyacrylamide-gel electrophoresis, but gave the same single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in two different buffer systems and over the range 10-15% acrylamide concentrations. The molecular weight was estimated as 14 100. The two major species of the smaller form had the same amino acid composition and contained no significant amount of carbohydrate. The larger form of alpha-lactalbumin, consisting of two species with different charges, was present in a small amount (less than 10%) in the milk and was isolated by its ability to interact with concanavalin A-Sepharose. Each of the two species also gave the same single band of apparent mol.w.t 18 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, this value may be anomalous, since this larger form appears to be glycosylated, and glycoproteins can behave anomalously on sodium dodecyl sulphate/polyacrylamide gels by binding less sodium dodecyl sulphate. All species of mouse alpha-lactalbumin from milk were active in the lactose synthase reaction and showed identical immunological properties, as determined by the mono-specific antibody prepared against the small major species. The presence of both the larger and the smaller forms, each in a percentage concentration similar to that found in milk, was also demonstrated in alpha-lactalbumin induced by hormones in organ cultureof pregnant-mouse mammary gland.

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Purification and properties of arginase from human liver and erythrocytes

Biochemical Journal ◽

10.1042/bj1750449 ◽

1978 ◽

Vol 175 (2) ◽

pp. 449-454 ◽

Cited By ~ 56

Author(s):

J Berüter ◽

J P Colombo ◽

C Bachmann

Keyword(s):

Gel Electrophoresis ◽

Human Liver ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Deae Cellulose ◽

Sedimentation Coefficient ◽

Polyacrylamide Gel ◽

Kinetic Properties ◽

Purification Procedure

Arginase was isolated from human liver and erythrocytes. The purification procedure used acetone precipitation, heat-treatment, (NH4)2SO4 precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-200 in the presence of 2-mercaptoethanol. Both enzymes migrated to the anode at pH8.3 on polyacrylamide-gel electrophoresis. After incubation at pH8.0 and 37 degrees C the purified anionic liver arginase migrated to the cathode on polyacrylamide-gel electrophoresis. It is assumed that the multiple forms of the enzyme reported in the literature are partly artifacts of the purification procedure. The liver arginase showed a mol.wt. of 107000 determined by gel filtration and a sedimentation coefficient of 5.9S. Treatment of the liver enzyme with 0.25% sodium dodecyl sulphate at pH10 demonstrated an oligomeric structure of the enzyme with a mol.wt. of the subunit of 35000. The kinetic properties determined for the purified liver arginase showed an optimum pH of 9.3 and an optimal MnCl2 concentration of 2mM. The Km for L-arginine was 10.5 mM and for L-canavanine 50mM, and L-lysine exhibited a competitive type of inhibition with a Ki of 4.4mM. L-Homoarginine was not a substrate for liver arginase.

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Gel electrophoresis and immunoblotting for the detection of casein proteolysis in cheese

Journal of Dairy Research ◽

10.1017/s0022029900030995 ◽

1995 ◽

Vol 62 (2) ◽

pp. 297-309 ◽

Cited By ~ 28

Author(s):

Francesco Addeo ◽

Giuseppina Garro ◽

Nunziatina Intorcia ◽

Luisa Pellegrino ◽

Pierpaolo Resmini ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Isoelectric Focusing ◽

Gel Electrophoresis ◽

Polyclonal Antibodies ◽

Deae Cellulose ◽

Casein Hydrolysis ◽

Gel Isoelectric Focusing ◽

Parmigiano Reggiano

SUMMARYThe whole N fraction of six samples of hard and semi-hard pressed cheeses was analysed using PAGE, polyacrylamide gel isoelectric focusing and immuno blotting with polyclonal antibodies against β- and αs1-casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzymic degradation of the casein fractions was established. A number of these peptides were also present in the in vitro hydrolysates of casein with plasmin and chymosin. Thus, it was also possible to determine which casein was the source of each peptide and which enzymes were active in cheese. Compared with the traditional Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the state of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4·6-insoluble N fraction of Parmigiano-Reggiano using DEAE-cellulose chromatography and identified, from the amino acid sequence of the N- and C-terminal ends, as γ3-casein (β-casein(f108–209)) and αs1-PL1 (αs1-casein(f80–199)). In both cases, a Lys–X bond was hydrolysed, indicating the action of a trypsin-like enzyme in β- and αs1-casein hydrolysis during the ripening of this variety of hard pressed cheese.

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Purification Of Factors IX And X From Clinical Concentrate

10.1055/s-0038-1652638 ◽

1981 ◽

Author(s):

G C Russell ◽

G Kemble ◽

E G D Tuddenham

Keyword(s):

Molecular Weight ◽

Affinity Chromatography ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Deae Cellulose ◽

Polyacrylamide Gel ◽

Single Band ◽

Factor Ix ◽

Factor X ◽

Clinical Factor

Human factors IX and X have been purified to homogeneity from clinical factor IX concentrate that had been rejected for therapeutic use due to particulate contamination. (It was necessary to start with this material since in the UK, plasma is not commercially available). The procedure involved barium citrate adsorption followed by ammonium sulphate elution, DEAE- cellulose chromatography, gel filtration on Sephacryl S-200 and affinity chromatography on heparin sepharose gel. The preparation of factor IX at this stage showed a single band on SDS-polyacrylamide gel electrophoresis, of molecular weight 58,000. No change in molecular weight was observed in the presence of 2-mercaptoethanol. A further affinity chromatography column - poly (homoarginine) Sepharose or dextran sulphate sepharose - was necessary to obtain homogeneous factor X. The preparation obtained showed a single band on SDS-polyacrylamide gel electrophoresis of molecular weight 67,000. In the presence of 2-mercaptoethanol, two bands were obtained of molecular weights 49000 and 17000 representing the heavy and light chains respectively of factor X. The purified coagulation proteins contained no activated species detectable by nonactivated partial thromboplastin time or by chromogenic substrate (S2222) assay. Prothrombin protein Sand protein C are by-products of this purification procedure.

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α-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro

Biochemical Journal ◽

10.1042/bj1751069 ◽

1978 ◽

Vol 175 (3) ◽

pp. 1069-1077 ◽

Cited By ~ 25

Author(s):

J Williams ◽

H Villarroya ◽

F Petek

Keyword(s):

Trifolium Repens ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Disc Electrophoresis ◽

Polyacrylamide Gel ◽

Ph Optimum

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).

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Site of synthesis of metallothionein in rat liver

Canadian Journal of Biochemistry ◽

10.1139/o81-041 ◽

1981 ◽

Vol 59 (4) ◽

pp. 301-306 ◽

Cited By ~ 18

Author(s):

M. George Cherian ◽

Sharon Yu ◽

Colvin M. Redman

Keyword(s):

Rat Liver ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Deae Cellulose ◽

Adult Rat ◽

Cadmium Treatment ◽

Reticulocyte Lysate

Free and membrane-attached polysomes were isolated from the livers of normal and cadmium-treated rats, and were translated using L-[35S]cysteine and a nuclease-treated reticulocyte lysate system. The translation products were analyzed for radioactive metallothionein by immunoprecipitation with antibodies to rat cadmium metallothionein followed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. In both normal and cadmium-treated rats, radioactive metallothionein was produced by free polysomes but not by membrane-attached polysomes. Cadmium treatment did not increase the in vitro ability of polysomes to synthesize metallothionein. As a control, the translation products of these two classes of polysomes were also analyzed for radioactive albumin and it was confirmed that membrane-attached polysomes produce albumin but do not synthesize metallothionein. The cell-free synthesis of metallothionein by free polysomes was also demonstrated by isolation of nascent metallothionein by Sephadex gel filtration and DEAE-cellulose chromatography. In adult rat liver there are two forms of metallothionein and both were produced in vitro by free polysomes.

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Platelet Microtubule Subunit Proteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657068 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1630-1633 ◽

Cited By ~ 3

Author(s):

A G Castle ◽

N Crawford

Keyword(s):

Epithelial Cells ◽

Gel Electrophoresis ◽

Blood Platelets ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Lens Epithelial Cells ◽

Molecular Weights ◽

Kinetics Of ◽

Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

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