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.

Purification and properties of mercuric reductase from Yersinia enterocolitica 138A14

1993 ◽  
Vol 39 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Mohamed Blaghen ◽  
Dominique J. M. Vidon ◽  
Mohamed Said El Kebbaj
Keyword(s):  
Molecular Weight ◽  
Yersinia Enterocolitica ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Mercury Resistance ◽  
Thiol Compounds ◽  
Mercuric Reductase ◽  
Purification And Properties ◽  
Layer Chromatography

A mercuric ion-reducing flavoprotein was purified from Yersinia enterocolitica 138A14 using dye matrix affinity chromatography. The purified enzyme had a characteristic absorption spectrum similar to those of flavin compounds, and FAD was detected as a part of the purified enzyme by thin-layer chromatography. Freshly purified preparations of the enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a molecular weight of 70 000. The isolated enzyme had a molecular weight of about 200 000 as determined by gel filtration and disc gel electrophoresis. These results suggest an apparently trimeric structure of the enzyme. Dithiothreitol treatment disrupted the trimer into a dimeric structure of 140 000. Along with ageing, as well as limited proteolytic digestion, the enzyme evolved to give a dimeric molecule of 105 000 composed of two identical subunits of 52 000. The combination of the purified enzyme with HgCl2, or unexpectedly with merthiolate, oxidised the NADPH, which was followed spectrophotometrically. The Km for HgCl2 was dependent on the concentration of exogenous thiol compounds. A comparison of physical properties as well as kinetic characteristics indicated that the enzyme from Y. enterocolitica 138A14 is similar to mercuric reductases isolated from other mercury-resistant bacteria.Key words: Yersinia enterocolitica, mercury resistance, mercuric reductase.

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

Physicochemical Studies of Conglutin γ, a Storage Globulin From Seeds of Lupinus angustifolius

1980 ◽  
Vol 7 (1) ◽  
pp. 1 ◽  
Author(s):  
RJ Blagrove ◽  
JM Gillespie ◽  
GG Lilley ◽  
EF Woods
Keyword(s):  
Molecular Weight ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Optical Rotatory Dispersion ◽  
Sedimentation Equilibrium ◽  
Native Protein ◽  
Equilibrium Studies ◽  
Physicochemical Studies ◽  
Α Helix

Physicochemical studies are reported for conglutin �, the minor globulin isolated from seeds of L. angustifolius cv. Uniwhite. Isoelectric focusing of the native protein in polyacrylamide gel slabs resolved major and minor broad bands near pH 8.0 and 7.8 respectively. Following reduction of disulfide bonds with β-mercaptoethanol in 8 M urea, the smaller polypeptide chain of known sequence focused near pH 6.9 while the larger chain focused near pH 8.0. Sedimentation equilibrium studies showed that the major component in aqueous buffers at neutral pH is a hexamer of molecular weight 280 000 which dissociates to the monomer of molecular weight 47 000 at pH 4.8. The sequence molecular weight of the small subunit polypeptide is 16 517 [Elleman, T.C. (1977). Aust. J. Biol. Sci. 30, 33-45]. The molecular weights determined for the larger chain by sedimentation equilibrium or column chromatography in 6 M guanidine hydrochloride, and by dodecyl sulfate-polyacrylamide gel electrophoresis, were in the range 28 000-30 000. Optical rotatory dispersion and circular dichroism measurements have been used to establish the approximate proportions of α-helix (15%), β-structure (35%), β-turns (18%) and unordered regions (32%) in the native protein. The denaturation curve for guanidine hydrochloride and the proportions of α-helix (50%), β-turns (18%) and unordered regions (32%) in 80 % trifluoroethanol have been determined.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

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 The purification and properties of isocitrate lyase from Chlorella

1967 ◽  
Vol 105 (1) ◽  
pp. 409-416 ◽  
Author(s):  
P. C. L. John ◽  
P. J. Syrett
Keyword(s):  
Molecular Weight ◽  
Diffusion Coefficient ◽  
Gel Electrophoresis ◽  
Isocitrate Lyase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Coefficient ◽  
Chlorella Pyrenoidosa ◽  
Turnover Number ◽  
Purification And Properties ◽  
Stokes Radius

1. Isocitrate lyase (threo-ds-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified from acetate-adapted cells of Chlorella pyrenoidosa. 2. The final preparation was homogeneous by the criteria of sedimentation, diffusion and polyacrylamide-gel electrophoresis. 3. The sedimentation coefficient (S20,w) was 9·04×10−13sec. and the diffusion coefficient (D20,w) 4·62×10−7cm.2/sec.; from these values the molecular weight of the enzyme was calculated to be 170000 and its Stokes radius to be 4·63×10−7cm. 4. The elution of the enzyme from Sephadex G-100 was studied and estimates of molecular weight and Stokes radius were obtained from the elution data. 5. The turnover number of the enzyme was 5950moles of glyoxylate formed/min./mole of enzyme at 30°. 6. With threo-ds(+)-isocitrate as substrate, the Km of the enzyme was 0·023mm.

scholarly journals The purification and properties of urocanase from Pseudomonas testosteroni

1978 ◽  
Vol 171 (1) ◽  
pp. 41-50 ◽  
Author(s):  
A J Hacking ◽  
M V Bell ◽  
H Hassall
Keyword(s):  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Equilibrium Analysis ◽  
Sedimentation Equilibrium ◽  
Carbonyl Groups ◽  
Prosthetic Group ◽  
Purification And Properties ◽  
Pseudomonas Testosteroni

Urocanase (urocanate hydratase, EC 4.2.1.49) purified from Pseudomonas testosteroni has a mol.wt. of 118000 determined by sedimentation-equilibrium analysis. Ultracentrifugation in 6M-guanidine hydrochloride and polyacrylamide-gel electrophoresis in sodium dodecyl sulphate show that the enzyme consists of two identical or very similar subunits. It is, like urocanase isolated from other sources, inhibited by reagents that react with carbonyl groups. Although urocanase from Ps. testosteroni is strongly inhibited by NaBH4, no evidence could be obtained for the presence of covalently bound 2-oxobutyrate as a prosthetic group; this is in contrast with findings elsewhere for urocanase from Pseudomonas putida. Urocanase from Ps. testosteroni does not contain pyridoxal 5′-phosphate as a coenzyme and in this respect is similar to all urocanases studied in purified form.

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.

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