scholarly journals Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver

1973 ◽  
Vol 135 (4) ◽  
pp. 683-693 ◽  
Author(s):  
María Campos-Cavieres ◽  
Edward A. Munn
Keyword(s):  
Aspartate Aminotransferase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Antiserum ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Molar Ratio ◽  
Agar Gel ◽  
Sheep Liver

1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s20,w of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25°C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25°C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from sheep liver

1969 ◽  
Vol 115 (4) ◽  
pp. 639-643 ◽  
Author(s):  
R. H. Villet ◽  
K. Dalziel
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Equilibrium Studies ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver ◽  
Equilibrium Sedimentation ◽  
Purification And Properties

A method is described for the isolation of 6-phosphogluconate dehydrogenase from sheep liver. The product appears to be homogeneous in polyacrylamide-gel electrophoresis and in sedimentation-velocity and sedimentation-equilibrium studies in the ultracentrifuge. The molecular weight is estimated as 129000 from equilibrium sedimentation.

scholarly journals Purification of 2-oxoaldehyde dehydrogenase and its dependence on unusual amines

1975 ◽  
Vol 149 (3) ◽  
pp. 609-617 ◽  
Author(s):  
J Dunkerton ◽  
S P James
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
General Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Ph Values ◽  
Sheep Liver ◽  
Living Tissues

1. 2-Oxoaldehyde dehydrogenase was purified from sheep liver and gave one band on polyacrylamide-gel electrophoresis. 2. The enzyme was completely dependent for its activity on the presence of Tris or one of a number of related amines, all of general structure: (See article). When more than one R group was hydrogen no enzyme activity was observed. 3. Only one of these amines is known to exist in living tissues and large concentrations of all amines were required for maximum activity. L-2-Aminopropan-1-ol was the most effective amine on the basis of substrate Km and Vmax. values and the amine Km values. 4. The enzyme was activated by phosphate which lowered the Km values for methylglyoxal, amine and NAD+. 5. The pH optimum of the enzyme was 9.3 and there was no activity at pH values below 7.8. A search for activators that might produce activity at pH 7.4 proved unsuccessful. 6. The enzyme was inhibited by rather large concentrations of barbiturates (6-46 mM) and nitro-alcohol analogues of the activating amines (66-139 mM).

The isolation and characterization of the tropomyosin binding component (TN-T) of bovine cardiac troponin

1976 ◽  
Vol 54 (6) ◽  
pp. 546-552 ◽  
Author(s):  
L. D. Burtnick ◽  
W. D. McCubbin ◽  
C. M. Kay
Keyword(s):  
Cardiac Muscle ◽  
Gel Electrophoresis ◽  
Calcium Binding ◽  
Cardiac Troponin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chemical Properties ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Isolation And Characterization ◽  
Binding Component

The tropomyosin binding component (TN-T) of troponin was purified from bovine cardiac muscle using a combination of ion exchange chromatographies in the presence of urea. Sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-T of 36 300 ± 2 000, consistent with a value of 37 000 ± 1 000 determined by polyacrylamide gel electrophoresis. Calculations based upon circular dichroism spectra indicate an apparent α-helical content of 43 ± 3% for TN-T. Polyacrylamide gel electrophoresis and the effects of the calcium binding component (TN-C) upon the solubility of TN-T suggest that the two cardiac troponin components can interact with each other. Cosedimentation analysis of solutions containing cardiac tropomyosin and TN-T provide evidence for complex formation involving these two proteins. The data presented on the physical and chemical properties of TN-T, as well as the interaction studies indicate that the cardiac muscle regulatory system operates in a manner similar to that proposed for skeletal muscle.

scholarly journals Amelogenins. Purification and partial characterization of proteins from developing bovine dental enamel

1973 ◽  
Vol 131 (3) ◽  
pp. 471-484 ◽  
Author(s):  
F. Michael Eggert ◽  
Grania A. Allen ◽  
Ralph C. Burgess
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dental Enamel ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Amino Acid Compositions ◽  
Small Proteins ◽  
Equilibrium Ultracentrifugation

1. Procedures are described for the purification of amelogenin electrophoretic components and their analysis for homogeneity by polyacrylamide-gel electrophoresis at both acidic and alkaline pH values. 2. Most of these components belonged to two main groups, termed the J group and the C group after their major electrophoretic components. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis indicated that, within each group, proteins were of similar size, but the C-group proteins were larger than those of the J group. 3. By sedimentation-equilibrium ultracentrifugation and amino acid analysis, the four J-group components were found to be very small proteins (mol. wt. 5500–3000) and, except for one, similar in amino acid composition. The components of the C group were found to be proteins of moderate size (mol. wt. 16800–16100) with very similar amino acid compositions. A third minor amelogenin group of intermediate size was also found, but not further analysed. Details of the results of the ultracentrifuge studies are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50014 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. 4. Two of the J-group components were similar to amelogenins isolated by other workers. 5. All amelogenins analysed were rich in proline, glutamic acid, histidine and methionine, and contained no half-cystine. Their amino acid compositions, combined with their molecular weights, serve to distinguish the amelogenins from both collagens and keratins.

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

1979 ◽  
Vol 44 (2) ◽  
pp. 626-630 ◽  
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.

Glycoproteins, enzymatic activities, and b proteins in intercellular fluid extracts from hypersensitive Nicotiana species infected with tobacco mosaic virus

1985 ◽  
Vol 63 (5) ◽  
pp. 928-931 ◽  
Author(s):  
Jean-Guy Parent ◽  
Richard Hogue ◽  
Alain Asselin
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Electrophoretic Pattern ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Intercellular Fluid ◽  
Nicotiana Tabacum L ◽  
The One

Intercellular fluid b proteins from hypersensitive Nicotiana tabacum L. cv. Xanthi-nc and N. sylvestris Speg. and Comes infected with tobacco mosaic virus were compared by two-dimensional (2-D) polyacrylamide gel electrophoresis. Except for missing bands b2, b6a, b6b, and b7b, the overall 2-D electrophoretic pattern of N. sylvestris intercellular fluid proteins was similar to the one observed with 'Xanthi-nc' tobacco. Intercellular proteins were also studied by chromatography on con-canavalin A. Glycoproteins corresponding to b6a and b7a proteins of N. tabacum and the [Formula: see text] analog of N. sylvestris were identified. These proteins are probably peroxidase isozymes, as peroxidase activities with the same electrophoretic mobility were detected after polyacrylamide gel electrophoresis. No esterase activity was associated with any b protein band in gels. Esterase activities decreased upon virus infection, but accumulation of b proteins and peroxidase activities increased.

scholarly journals Glycosidases induced in Aspergillus tamarii. Secreted α-d-galactosidase and β-d-mannanase

1984 ◽  
Vol 219 (3) ◽  
pp. 857-863 ◽  
Author(s):  
A Civas ◽  
R Eberhard ◽  
P Le Dizet ◽  
F Petek
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Preceding Paper ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Aspergillus Tamarii ◽  
Electrophoretic Method ◽  
Single Protein

An alpha-D-galactosidase (EC 3.2.1.22) and a beta-D-mannanase (EC 3.2.1.78), which were secreted into the growth medium when Aspergillus tamarii was cultivated in the presence of galactomannan, were purified by a procedure including chromatography on hydroxyapatite and DEAE-cellulose columns. Each of these enzymes showed a single protein band, corresponding to their respective activities, on polyacrylamide-gel electrophoresis. Both enzymes were shown to be glycoproteins containing N-acetylglucosamine, mannose and galactose, with molar proportions of 1:6:1.5 for alpha-D-galactosidase and 1:13:8 for beta-D-mannanase. Mr values as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and by the electrophoretic method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164] were 56000 and 53000 respectively. The alpha-D-galactosidase differed markedly from the mycelial forms I and II studied in the preceding paper [Civas, Eberhard, Le Dizet & Petek (1984) Biochem. J. 219, 849-855] with regard to both its kinetic and structural properties.

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 Co-purification of galactosyltransferases from chick-embryo liver

1985 ◽  
Vol 227 (2) ◽  
pp. 573-582 ◽  
Author(s):  
K Furukawa ◽  
S Roth
Keyword(s):  
Chick Embryo ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Cellulose Acetate Electrophoresis ◽  
Km Value ◽  
Alpha Lactalbumin

Two galactosyltransferases with nearly identical Mr values were purified 5000-7000-fold from microsomal membranes of chick-embryo livers by using several affinity columns. One enzyme transfers galactose from UDP-galactose to form a β-(1→4)-linkage to GlcNAc (N-acetylglucosamine) or AsAgAGP [asialo-agalacto-(alpha 1-acid glycoprotein)]. The other enzyme forms a β-(1→3)-linkage to AsOSM [asialo-(ovine submaxillary mucin)]. Both enzymes were solubilized (85%) from a microsomal pellet by using 1% Triton X-100 in 0.1 M-NaCl. The supernatant activities were subjected to DEAE-Sepharose chromatography and four affinity columns: UDP-hexanolamine-Sepharose, alpha-lactalbumin-Sepharose, GlcNAc-Sepharose and either AsAgAGP-Sepharose or AsOSM-Sepharose. The AsAgAGP enzyme [(1→4)-transferase] and the AsOSM enzyme [(1→3)-transferase] behave identically on the DEAE-Sepharose and UDP-hexanolamine-Sepharose columns, and similarly on the alpha-lactalbumin-Sepharose column. Final separation of the two enzymes, however, could only be achieved on affinity columns of their immobilized respective acceptors. Both purified enzymes migrate as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after silver staining, and both have an apparent Mr of 68 000. The enzymes were radioiodinated and again subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioautographic analyses showed only one, intensely radioactive, band. Activity stains performed for both transferases after cellulose acetate electrophoresis indicate that, with this system too, both activities have identical mobilities, and co-migrate, as well, with the major, silver-stained, protein band. Kinetic studies with the purified enzymes show that the Km value for GlcNAc, for the (1→4)-transferase, is 4mM; for the (1→3)-transferase the Km value for AsOSM is 5mM, in terms of GalNAc (N-acetylgalactosamine) equivalents. Both enzymes have a Km value of 25 microM for UDP-galactose.

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