Solubilization and purification of rat liver 5′-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent

1. A variety of detergents were used to solubilize 5′-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5′-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.

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

Canadian Journal of Biochemistry ◽

10.1139/o77-143 ◽

1977 ◽

Vol 55 (9) ◽

pp. 958-964 ◽

Cited By ~ 6

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.

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Inhibition of the thrombin–fibrinogen reaction by a macromolecular factor from rat liver

Biochemical Journal ◽

10.1042/bj1290083 ◽

1972 ◽

Vol 129 (1) ◽

pp. 83-89 ◽

Cited By ~ 5

Author(s):

Ragnar Flengsrud ◽

Bjarne Østerud ◽

Hans Prydz

Keyword(s):

Molecular Weight ◽

Rat Liver ◽

Gel Electrophoresis ◽

Competitive Inhibition ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Liver Homogenate ◽

Kinetic Studies ◽

Disc Electrophoresis ◽

Nitrobenzoic Acid

1. The supernatant obtained by centrifugation of a rat liver homogenate at 100000g for 1h contained a heat-labile macromolecular inhibitor of the thrombin–fibrinogen reaction. 2. The inhibitor was purified to electrophoretic homogeneity by repeated preparative polyacrylamide disc electrophoresis. Inhibition was observed with purified inhibitor equivalent to about 1μg of protein/ml. 3. The inhibitor had a pI of 3.50–3.75, a molecular weight (from sodium dodecyl sulphate–polyacrylamide-gel electrophoresis) of 72000±3000 and was inactivated by p-hydroxymercuribenzoate or 5,5′-dithiobis-(2-nitrobenzoic acid). 4. Kinetic studies revealed a non-competitive inhibition, with the inhibitor probably acting on the thrombin–fibrinogen complex.

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

Canadian Journal of Biochemistry ◽

10.1139/o82-054 ◽

1982 ◽

Vol 60 (4) ◽

pp. 463-470 ◽

Cited By ~ 24

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.

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Purification and characterization of a new cytosolic glutathione S-transferase (glutathione S-transferase X) from rat liver

Biochemical Journal ◽

10.1042/bj2150617 ◽

1983 ◽

Vol 215 (3) ◽

pp. 617-625 ◽

Cited By ~ 28

Author(s):

T Friedberg ◽

U Milbert ◽

P Bentley ◽

T M Guenther ◽

F Oesch

Keyword(s):

Rat Liver ◽

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Polyacrylamide Gel ◽

Glutathione S Transferase ◽

Cytosolic Glutathione ◽

Glutathione S Transferases

A hitherto unknown cytosolic glutathione S-transferase from rat liver was discovered and a method developed for its purification to apparent homogeneity. This enzyme had several properties that distinguished it from other glutathione S-transferases, and it was named glutathione S-transferase X. The purification procedure involved DEAE-cellulose chromatography, (NH4)2SO4 precipitation, affinity chromatography on Sepharose 4B to which glutathione was coupled and CM-cellulose chromatography, and allowed the isolation of glutathione S-transferases X, A, B and C in relatively large quantities suitable for the investigation of the toxicological role of these enzymes. Like glutathione S-transferase M, but unlike glutathione S-transferases AA, A, B, C, D and E, glutathione S-transferase X was retained on DEAE-cellulose. The end product, which was purified from rat liver 20 000 g supernatant about 50-fold, as determined with 1-chloro-2,4-dinitrobenzene as substrate and about 90-fold with the 1,2-dichloro-4-nitrobenzene as substrate, was judged to be homogeneous by several criteria, including sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, isoelectric focusing and immunoelectrophoresis. Results from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration indicated that transferase X was a dimer with Mr about 45 000 composed of subunits with Mr 23 500. The isoelectric point of glutathione S-transferase X was 6.9, which is different from those of most of the other glutathione S-transferases (AA, A, B and C). The amino acid composition of transferase X was similar to that of transferase C. Immunoelectrophoresis of glutathione S-transferases A, C and X and precipitation of various combinations of these antigens by antisera raised against glutathione S-transferase X or C revealed that the glutathione S-transferases A, C and X have different electrophoretic mobilities, and indicated that transferase X is immunologically similar to transferase C, less similar to transferase A and not cross-reactive to transferases B and E. In contrast with transferases B and AA, glutathione S-transferase X did not bind cholic acid, which, together with the determination of the Mr, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyse the reaction of menaphthyl sulphate with glutathione, and was in this respect dissimilar to glutathione S-transferase M; however, it conjugated 1,2-dichloro-4-nitrobenzene very rapidly, in contrast with transferases AA, B, D and E, which were nearly inactive towards that substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

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Studies on the purification of rat liver uridine diphosphate glucuronyltransferase

Biochemical Journal ◽

10.1042/bj1610543 ◽

1977 ◽

Vol 161 (3) ◽

pp. 543-549 ◽

Cited By ~ 34

Author(s):

B Burchell

Keyword(s):

Enzyme Activity ◽

Rat Liver ◽

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Uridine Diphosphate ◽

Enzyme Protein ◽

Epoxide Hydratase

1. A stable, more highly purified, preparation of UDP-glucuronyltransferase was obtained than previously reported. 2. Enzyme activity towards o-aminophenyl and p-nitrophenyl was increased 43- and 46-fold respectively. 3. The final preparation contains only three staining polypeptide bands visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The only known major accompanying protein appears to be epoxide hydratase. 5. The purified enzyme activity towards o-aminophenol can still be activated 3 fold by diethylnitrosamine. 6. On evidence from purification, o-aminophenol and p-nitrophenol appear to be glucuronidated by the same enzyme protein. The possible recognition of the UDP-glucuronyltransferase enzyme is discussed.

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Synthesis of 3-ketoacyl-CoA thiolase of rat liver peroxisomes on free polyribosomes as a larger precursor. Induction of thiolase mRNA activity by clofibrate

Biochemical Journal ◽

10.1042/bj2260697 ◽

1985 ◽

Vol 226 (3) ◽

pp. 697-704 ◽

Cited By ~ 45

Author(s):

Y Fujiki ◽

R A Rachubinski ◽

R M Mortensen ◽

P B Lazarow

Keyword(s):

Rat Liver ◽

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Immunoblot Analysis ◽

Polyacrylamide Gel ◽

Proteolytic Processing ◽

Translation Product ◽

Free Translation

The site of synthesis and induction by clofibrate of peroxisomal 3-ketoacyl-CoA thiolase (acetyl-CoA acyltransferase; EC 2.3.1.16) was investigated. Free and membrane-bound polyribosomal RNA species from the livers of normal rats and rats treated with clofibrate, a hypolipidaemic drug that causes marked proliferation of peroxisomes, were translated in a nuclease-treated rabbit reticulocyte-lysate cell-free protein-synthesizing system with [35S]methionine as label. The cell-free translation products were immunoprecipitated with monospecific X rabbit anti-thiolase serum and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. Thiolase mRNA was found predominantly in free polyribosomes, in both normal and clofibrate-treated rats. Clofibrate treatment increased mRNA activity for thiolase approx. 20-fold. The translation product of clofibrate-induced thiolase mRNA migrated slightly faster in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than did the translation product of normal thiolase mRNA. Both the normal and the clofibrate-induced translation products were approx. 6000 Da larger than the 41000-Da subunit of the purified enzyme. Immunoblot analysis of liver homogenates, isolated peroxisomes and the purified enzyme indicated that the thiolase subunit was approx. 41000 Da in all samples, ruling out proteolysis during the purification of thiolase. Thiolase biogenesis thus differs from that of rat liver peroxisomal proteins studied previously in that it is synthesized as a larger precursor, implying post-translational import of thiolase into peroxisomes with proteolytic processing. Clofibrate apparently alters the size as well as the amount of the translation product.

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Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

YARSI Medical Journal ◽

10.33476/jky.v26i2.394 ◽

2018 ◽

Vol 26 (2) ◽

pp. 058

Author(s):

Anna P. Roswiem ◽

Triayu Septiani

Keyword(s):

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Sds Page

Bahan baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.

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