Site of synthesis of metallothionein in rat liver

1981 ◽  
Vol 59 (4) ◽  
pp. 301-306 ◽  
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.

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.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

scholarly journals Purification and characterization of rat liver glycosylasparaginase

1989 ◽  
Vol 260 (1) ◽  
pp. 101-108 ◽  
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.

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

1983 ◽  
Vol 215 (3) ◽  
pp. 617-625 ◽  
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)

scholarly journals The purification and molecular properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa

1981 ◽  
Vol 197 (2) ◽  
pp. 427-436 ◽  
Author(s):  
G A Nimmo ◽  
J R Coggins
Keyword(s):  
Molecular Weight ◽  
Neurospora Crassa ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Phosphate Synthase

Neurospora crassa contains three isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, which are inhibited by tyrosine, tryptophan and phenylalanine respectively, and it was estimated that the relative proportions of the total activity were 54%, 14% and 32% respectively. The tryptophan-sensitive isoenzyme was purified to homogeneity as judged by polyacrylamide-gel electrophoresis and ultracentrifugation. The tyrosine-sensitive and phenylalanine-sensitive isoenzymes were only partially purified. The three isoenzymes were completely separated from each other, however, and can be distinguished by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and Ultrogel AcA-34 and polyacrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the tryptophan-sensitive isoenzyme contained one type of subunit of molecular weight 52000. The molecular weight of the native enzyme was found to be 200000 by sedimentation-equilibrium centrifugation, indicating that the enzyme is a tetramer, and the results of cross-linking and gel-filtration studies were in agreement with this conclusion.

scholarly journals Immunologically reactive tryptic fragments of human prostatic acid phosphatase

1984 ◽  
Vol 223 (3) ◽  
pp. 871-877 ◽  
Author(s):  
C L Lee ◽  
S S L Li ◽  
T M Chu
Keyword(s):  
Acid Phosphatase ◽  
Inhibitory Activity ◽  
Gel Electrophoresis ◽  
Active Sites ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Prostatic Acid Phosphatase ◽  
Peptide Fragments

Three peptide fragments (designated II, III and IV) of human prostatic acid phosphatase (PAP) were isolated to homogeneity from a limited tryptic hydrolysate of PAP by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose and Sephadex G-75. The homogeneity was confirmed by disc poly-acrylamide-gel electrophoresis. The Mr values were 32 500, 25 000 and 11 000 as estimated by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Immunoprecipitation study revealed that only fragment II formed an immune precipitate with anti-PAP antibodies. Fragment II exhibited 45% of maximum inhibitory activity on the reaction between PAP and goat anti-PAP IgG (immunoglobulin G) antibodies (or rabbit anti-PAP antibodies), whereas fragments III and IV demonstrated 24% (or 23%) and 29% (or 27%) inhibition respectively. A mixture of these three tryptic fragments of PAP result in 96% (for goat anti-PAP antibodies) and 94% (for rabbit anti-PAP antibodies) inhibitory activities, which were equivalent to the sum of maximum inhibitory activity of the three fragments individually. The results demonstrated that these three tryptic peptide fragments carried all the antigenic active sites of the native PAP, and suggested that the entire molecule of human PAP comprised a minimum of four distinguishable, nonoverlapping antigenic determinants. These three fragments also were shown to retain all the disulphide bonds of the native PAP, and thus were useful reagents for the elucidation of PAP molecular structure.

scholarly journals Purification and properties of arginase from human liver and erythrocytes

1978 ◽  
Vol 175 (2) ◽  
pp. 449-454 ◽  
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.

scholarly journals Purification and properties of S-adenosylmethionine: aldoxime O-methyltransferase from Pseudomonas sp. N.C.I.B. 11652

1985 ◽  
Vol 226 (1) ◽  
pp. 147-153 ◽  
Author(s):  
D B Harper ◽  
J T Kennedy
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Pseudomonas Sp ◽  
Ph Optimum

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.

scholarly journals Purification of uroporphyrinogen decarboxylase from human erythrocytes. Immunochemical evidence for a single protein with decarboxylase activity in human erythrocytes and liver

1983 ◽  
Vol 215 (1) ◽  
pp. 45-55 ◽  
Author(s):  
G H Elder ◽  
J A Tovey ◽  
D M Sheppard
Keyword(s):  
Gel Electrophoresis ◽  
Solid Phase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Human Erythrocytes ◽  
Decarboxylase Activity ◽  
Uroporphyrinogen Decarboxylase

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified 4419-fold to a specific activity of 58.3 nmol of coproporphyrinogen III formed/min per mg of protein (with pentacarboxyporphyrinogen III as substrate) from human erythrocytes by adsorption to DEAE-cellulose, (NH4)2SO4 fractionation, gel filtration, phenyl-Sepharose chromatography and polyacrylamide-gel electrophoresis. Progressive loss of activity towards uroporphyrinogens I and III occurred during purification. Experiments employing immunoprecipitation, immunoelectrophoresis and titration with solid-phase antibody indicated that all the uroporphyrinogen decarboxylase activity of human erythrocytes resides in one protein, and that the substrate specificity of this protein had changed during purification. The purified enzyme had a minimum mol.wt. of 39 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gel filtration gave a mol.wt. of 58 000 for the native enzyme. Isoelectric focusing showed a single band with a pI of 4.60. Reaction with N-ethylmaleimide abolished both catalytic activity and immunoreactivity. Incubation with substrates or porphyrins prevented inactivation by N-ethylmaleimide. An antiserum raised against purified erythrocyte enzyme precipitated more than 90% of the uroporphyrinogen decarboxylase activity from human liver. Quantitative immunoprecipitation and crossed immunoelectrophoresis showed that the erythrocyte and liver enzymes are very similar but not identical. The differences observed may reflect secondary modification of enzyme structure by proteolysis or oxidation of thiol groups, rather than a difference in primary structure.

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

1978 ◽  
Vol 175 (3) ◽  
pp. 1069-1077 ◽  
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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