Tryptophan synthase: purification, and some properties of an inhibitor from pea roots

1971 ◽  
Vol 49 (6) ◽  
pp. 821-832 ◽  
Author(s):  
James Chen ◽  
W. G. Boll
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Absorption Spectrum ◽  
Neurospora Crassa ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Ultraviolet Absorption Spectrum ◽  
Tryptophan Synthase ◽  
Shoot Tissue ◽  
Pea Roots

An undialyzable, thermostable inhibitor of tryptophan synthase from pea plants, Escherichia coli, and Neurospora crassa occurs in the filtrate from acetone extraction of pea tissue. A procedure is described for purification of the inhibitor extracted from roots. The final, purified preparation is homogeneous as shown by disc electrophoresis on acrylamide gel. The molecular weight, estimated by gel filtration on Sephadex G-200, is about 18 000. The inhibitor is stable even on heating at pH 7.0 in a water bath at 100 °C and has a characteristic ultraviolet absorption spectrum. The inhibitor shows some protein-like properties and acid hydrolysates were found to contain substances sensitive to ninhydrin. However, the hydrolysates also contain substances, presumably sugars, sensitive to benzidine reagent, appreciable sugar in galactose equivalents, and some fluorescent substances. The inhibitor was also found in shoot tissue. In both root and shoot the inhibitor increases, on a protein basis, with age of the tissue.

Inhibition of tryptophan synthase by extracts from pea roots

1969 ◽  
Vol 47 (5) ◽  
pp. 649-653 ◽  
Author(s):  
James Chen ◽  
W. G. Boll
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Inhibitory Factor ◽  
Heat Inactivation ◽  
Tryptophan Synthase ◽  
Fresh Tissue ◽  
Low Concentration ◽  
Root Extracts ◽  
Pea Roots ◽  
Inhibitory Components

Low tryptophan synthase (L-serine hydro-lyase (adding indole), EC 4.2.1.20) activity in extracts of pea roots is a consequence of both a low concentration of the enzyme in roots and the presence of inhibitors at least some of which may be removed by homogenizing the fresh tissue with acetone. Inhibition by root extracts, and fractions thereof, was assayed against tryptophan synthase from seedling buds. At least three inhibitory components were detected, namely (1) a dialyzable, thermostabile component, (2) an undialyzable, thermolabile component, and (3) an undialyzable, thermostabile component. A dialyzable factor which protects an undialyzable inhibitory factor against heat inactivation is also indicated.Fractionation of crude root extracts by gel filtration on a column of Sephadex G-50 shows inhibitor with a molecular weight of about 10 000 or larger.

scholarly journals Rat liver alcohol dehydrogenase. Purification and properties

1971 ◽  
Vol 125 (4) ◽  
pp. 1039-1047 ◽  
Author(s):  
M J Arslanian ◽  
E Pascoe ◽  
J G Reinhold
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Rat Liver ◽  
Gel Filtration ◽  
Rabbit Antiserum ◽  
Minor Component ◽  
Disc Electrophoresis ◽  
Supernatant Fraction ◽  
Liver Alcohol Dehydrogenase ◽  
A Minor

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD+ and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn2+ at concentrations above 0.1mm.

Isolation of specific protease inhibitors from Neurospora crassa

1976 ◽  
Vol 54 (8) ◽  
pp. 699-703 ◽  
Author(s):  
Peter H. Yu ◽  
Maria R. Kula ◽  
Hsin Tsai
Keyword(s):  
Neurospora Crassa ◽  
Protease Inhibitors ◽  
Gel Filtration ◽  
Physiological Role ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Tryptophan Synthase ◽  
Molecular Weights ◽  
Specific Protease

Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography and gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000 – 24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed.

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa

1978 ◽  
Vol 56 (10) ◽  
pp. 927-933 ◽  
Author(s):  
W. S. Lin ◽  
M. Kapoor
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamine Synthetase ◽  
Neurospora Crassa ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Affinity Column ◽  
Subunit Structure ◽  
Α Helix

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate–Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate α-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360 000 and as having a sedimentation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate α-helix content of 23–24%. The subunit generated by treatment with urea was found to be 45 000 daltons by gel filtration methods and a molecular weight of 46 000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45 000 daltons.

scholarly journals Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain)

1973 ◽  
Vol 131 (4) ◽  
pp. 833-841 ◽  
Author(s):  
Donald Stribling ◽  
Richard N. Perham
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Metal Ions ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Class Ii ◽  
Class I ◽  
E Coli ◽  
Km Value ◽  
Bivalent Metal Ions

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca2+, Zn2+. It is a dimer with a molecular weight of approx. 70000, and the Km value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH4 in the presence of substrate. The Km value for fructose diphosphate is about 20μm (although the Lineweaver–Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.

Effects of Irradiated and Reconstituted Uridine on Growth of a Neurospora Crassa Mutant and Synergism Between Uridine and Uracil

1956 ◽  
Vol 187 (2) ◽  
pp. 383-388 ◽  
Author(s):  
Attilio Canzanelli ◽  
Rhea Sossen ◽  
David Rapport
Keyword(s):  
Absorption Spectrum ◽  
Latent Period ◽  
Neurospora Crassa ◽  
Ultraviolet Light ◽  
Ultraviolet Absorption ◽  
Ultraviolet Absorption Spectrum ◽  
Progressive Loss

Solutions of uridine were irradiated with ultraviolet light for varying periods, resulting in a progressive loss of the selective ultraviolet absorption spectrum. Such absorption can be restored (‘reconstitution’) by treatment of the irradiated material with heat, acid, a combination of the two, and alkali, though the restored spectrum is not identical with spectrum of uridine. Further study of the ‘reconstituted’ material shows that both uridine and uracil were present in varying percentages, depending on the duration of irradiation, as was a small amount of ribose, and small amounts of two other unidentified U.V.-absorbing substances. The growth of a uridine-requiring mutant of Neurospora crassa is well supported by the ‘reconstituted’ material. Uracil will also support growth of the mutant, but only in much larger concentrations than is the case with uridine and after a latent period of several days. Uracil, in concentrations which alone are not adequate for immediate growth, will support growth if ‘catalytic’ amounts of uridine are added either simultaneously or previously. The possible mechanisms of this synergism are discussed.

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.

Purification and some properties of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Neurospora crassa

1979 ◽  
Vol 25 (12) ◽  
pp. 1381-1386 ◽  
Author(s):  
Kenji Yamamoto ◽  
Mitsuaki Moriguchi ◽  
Hiroyasu Kawai ◽  
Tatsurokuro Tochikura
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Neurospora Crassa ◽  
Isoelectric Point ◽  
Enzyme Preparation ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Uridine Diphosphate ◽  
Chromatographic Techniques

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (EC. 2.7.7.23) of Neurospora crassa has been purified approximately 210-fold with dithiothreitol as the stabilizing agent by use of chromatographic techniques. The enzyme preparation appeared to be homogeneous when subjected to electrophoresis. The molecular weight was estimated as approximately 37 000 by gel filtration. The enzyme had an isoelectric point around pH 4.4.Maximum activity of the enzyme was observed at pH7.5. The enzyme required Mg2+, which may be replaced by other divalent cations such as Mn2+ and Co2+ for lesser degrees of effectiveness. The enzyme was strictly specific for UDP-N-acetylglucosamine as the substrate. The estimated values of Km were 2.2 mM for UDP-N-acetylglucosamine and 5.4 mM for inorganic pyrophosphate.The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol but was lost by sulfhydryl inhibitory reagents.

scholarly journals The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa

1988 ◽  
Vol 251 (2) ◽  
pp. 313-322 ◽  
Author(s):  
P J White ◽  
G Millar ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Neurospora Crassa ◽  
Gel Filtration ◽  
Cross Linking ◽  
Amino Acid Residues ◽  
Chorismate Synthase ◽  
E Coli ◽  
A Subunit

The enzyme chorismate synthase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroC gene and confirmed by determining the N-terminal amino acid sequence of the purified enzyme. The complete polypeptide chain consists of 357 amino acid residues and has a calculated subunit Mr of 38,183. Cross-linking and gel-filtration experiments show that the enzyme is tetrameric. An improved purification of chorismate synthase from Neurospora crassa is also described. Cross-linking and gel-filtration experiments on the N. crassa enzyme show that it is also tetrameric with a subunit Mr of 50,000. It is proposed that the subunits of the N. crassa enzyme are larger because they contain a diaphorase domain that is absent from the E. coli enzyme.

scholarly journals The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes

1970 ◽  
Vol 118 (1) ◽  
pp. 15-23 ◽  
Author(s):  
K. Balasingam ◽  
W. Ferdinand
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Disc Electrophoresis ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Major Form ◽  
Purification And Properties ◽  
Minimal Molecular Weight

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.

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