scholarly journals Studies on extracellular ribonucleases of Ustilago sphaerogena. Purification and properties

1968 ◽  
Vol 106 (3) ◽  
pp. 601-607 ◽  
Author(s):  
Terukatsu Arima ◽  
Tsuneko Uchida ◽  
Fujio Egami
Keyword(s):  
Culture Media ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Ribonuclease T1 ◽  
Molecular Weights ◽  
Purification And Properties

1. Four ribonucleases were isolated from culture media of Ustilago sphaerogena. They were designated ribonucleases U1, U2, U3 and U4. 2. They were purified about 1600-, 3700-, 1100- and 16-fold respectively. 3. It was shown by gel filtration that ribonucleases U1, U2 and U3 have molecular weights about 10000 like ribonuclease T1, and that ribonuclease U4 is much larger. 4. Ribonucleases U1, U2 and U3 are thermostable, but ribonuclease U4 is not. 5. The pH optimum of ribonucleases U1 and U4 is pH8·0–8·5, and that of ribonucleases U2 and U3 is pH4·5.

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.

scholarly journals Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues

1978 ◽  
Vol 175 (3) ◽  
pp. 1051-1067 ◽  
Author(s):  
K K Mäkinen ◽  
P L Mäkinen
Keyword(s):  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Ph Optimum ◽  
Preparative Scale ◽  
Molecular Weights ◽  
Gel Permeation ◽  
Lysine Residues ◽  
Enzyme I

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

Partial purification and properties of an L-arabinose dehydrogenase from Azospirillum brasiliense

1983 ◽  
Vol 29 (2) ◽  
pp. 242-246 ◽  
Author(s):  
Norman J. Novick ◽  
Max E. Tyler
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Gel Filtration ◽  
Reducing Agent ◽  
Partial Purification ◽  
Ph Optimum ◽  
Glycine Buffer ◽  
Purification And Properties ◽  
Filtration Technique

An L-arabino-aldose dehydrogenase responsible for the oxidation of L-arabinose to L-arabino-γ-lactone has been purified 59-fold from L-arabinose grown cells of Azospirillum brasiliense. The dehydrogenase was found to be specific for substrates with the L-arabino-configuration at carbons 2, 3, and 4. Km values for L-arabinose of 75 and 140 μM were found with NADP and NAD as coenzymes, respectively. The enzyme had a pH optimum of 9.5 in glycine buffer and was stable when heated to 55 °C for 5 min. No enhancement of activity in the presence of any divalent cation or reducing agent tested was found. L-Arabinose dehydrogenase had a molecular weight of 175 000 as measured by the gel filtration technique.

scholarly journals Studies on the porphobilinogen deaminase–uroporphyrinogen cosynthetase system of cultured soya-bean cells

1971 ◽  
Vol 121 (2) ◽  
pp. 327-340 ◽  
Author(s):  
Elena B. C. Llambías ◽  
Alcira M. Del C. Batlle
Keyword(s):  
Culture Media ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Dicarboxylic Acids ◽  
Soya Bean ◽  
Porphobilinogen Deaminase ◽  
Starch Gel Electrophoresis ◽  
Ammonium Ions ◽  
Filtration Method ◽  
Molecular Weights

1. Porphobilinogenase was isolated and purified from soya-bean callus tissue; its components, porphobilinogen deaminase and uroporphyrinogen isomerase, were separated and purified. 2. The purified porphobilinogenase was resolved into two bands on starch-gel electrophoresis. The molecular weights of porphobilinogenase, deaminase and isomerase fractions were determined by the gel-filtration method. Porphobilinogenase activity was affected by the presence of air; uroporphyrinogens were only formed under anaerobic conditions, although substrate consumption was the same in the absence of oxygen as in its presence. 3. pH-dependence of both porphobilinogenase and deaminase was the same and a sharp optimum at pH 7.2 was obtained. Isomerase was heat-labile, but the presence of ammonium ions or porphobilinogen afforded some protection against inactivation. The action of several compounds added to the system was studied. Cysteine, thioglycollate, ammonium ions and hydroxylamine inhibited porphobilinogenase; certain concentrations of sodium and magnesium salts enhanced activity; some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited the deaminase. 4. δ-Aminolaevulate and ethionine in the culture media stimulated porphyrin synthesis and increased porphobilinogenase activity, whereas iron deficiency resulted in porphyrin accumulation. 5. The development of chlorophyll and porphobilinogenase on illumination of dark-grown callus was followed. 6. A hypothetical scheme is suggested for the enzymic synthesis of uroporphyrinogens from porphobilinogen.

scholarly journals Purification and properties of l-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus

1987 ◽  
Vol 248 (3) ◽  
pp. 871-876 ◽  
Author(s):  
M E Hoey ◽  
N Allison ◽  
A J Scott ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Potential Inhibitors ◽  
Triton X

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a ‘wall + membrane’ fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.

scholarly journals Purification and properties of α-galactosidases from Vicia faba seeds

1969 ◽  
Vol 113 (1) ◽  
pp. 49-55 ◽  
Author(s):  
P. M. Dey ◽  
J. B. Pridham
Keyword(s):  
Amino Acid ◽  
Vicia Faba ◽  
Aromatic Amino Acid ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Thermal Stabilities ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Enzyme I ◽  
Carbohydrate Contents

Two forms of α-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2·8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. α-Galactosidases I and II showed different pH optima and Km and Vmax. values with p-nitrophenyl α-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.

Malate dehydrogenase from thermophilic Humicola lanuginosa and Mucor pusillus: purification and comparative properties of the enzymes with differing thermostabilities

1984 ◽  
Vol 62 (7) ◽  
pp. 559-565 ◽  
Author(s):  
Anupam S. Wali ◽  
Autar K. Mattoo
Keyword(s):  
Malate Dehydrogenase ◽  
Conformational Changes ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
The Other ◽  
Heat Inactivation ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Other Hand ◽  
Humicola Lanuginosa

Malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) was purified from the thermophiles Humicola lanuginosa and Mucor pusillus. The H. lanuginosa enzyme was homogeneous on sodium dodecyl sulphate – polyacrylamide gels, while the M. pusillus enzyme was more than 95% pure. The two enzymes appeared to be composed of two subunits of equal size, each of 36 000 daltons (H. lanuginosa) or 33 000 daltons (M. pusillus). The native enzymes revealed molecular weights of 68 000 as determined by gel filtration. The isoelectric points of malate dehydrogenase from H. lanuginosa and M. pusillus were 3.9 and 4.2, respectively. The reduction of oxaloacetate by the H. lanuginosa enzyme was optimum at pH 8.5–9 with apparent Km's of 0.12 mM for oxaloacetate and 0.027 mM for NADH. On the other hand, M. pusillus enzyme snowed a pH optimum of 7.8–8.5 with apparent Km's of 0.075 mM for oxaloacetate and 0.1 mM for NADH. The L-malate oxidation reaction was catalyzed optimally at pH 10 by the H. lanuginosa enzyme with apparent Km's of 5.8 mM for malate and 0.1 mM for NAD, while the M. pusillus enzyme catalyzed it optimally between pH 9.5 and 10 with apparent Km's of 4.44 mM for malate and 0.16 mM for NAD. The optimum temperature for reduction of oxaloacetate was 50 °C for both the enzymes. The H. lanuginosa enzyme was resistant to heat inactivation at 40 °C, but lost 60% of its activity after 15 min at 50 °C. Mucor pusillus enzyme, on the other hand, retained 90% activity at 60 °C after 10 min. The two enzymes were protected from heat inactivation by monovalent cations (viz Na+, K+, and NH4+), as well as citrate, which may possibly involve conformational changes.

scholarly journals Purification and properties of acid phosphatase from Avena elatior L. seeds

2015 ◽  
Vol 46 (3) ◽  
pp. 481-488 ◽  
Author(s):  
E. Wieczorek ◽  
I. Lorenc-Kubis ◽  
B. Morawiecka
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Sepharose 4B

Acid phosphatase F1 from <i>Avena elatior</i> seeds was isolated and partially purified by means of alcohol precepitation, DEAE-, CM-column chromatography, Sephadex G-150, Sephadex G-200 and Sepharose 4B - gel filtration. The enzyme was stable at 50°C, pH 5.1. The pH optimum for phosphatase activity was 4.2. Fluoride, Zn<sup>2+</sup>, molybdate were effective inhibitors. EDTA and l, 10-phenanthroline activated the enzyme.

scholarly journals Purification and properties of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase from Methylococcus capsulatus

1974 ◽  
Vol 144 (3) ◽  
pp. 477-486 ◽  
Author(s):  
T Ferenci ◽  
T Strøm ◽  
J R Quayle
Keyword(s):  
Equilibrium Constants ◽  
Gel Filtration ◽  
Reverse Reaction ◽  
Methylococcus Capsulatus ◽  
Forward Reaction ◽  
Bivalent Cation ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Michaelis Constants ◽  
Phosphate Synthase

3-Hexulose phosphate synthase and phospho-3-hexuloisomerase were purified 40- and 150-fold respectively from methane-grown Methylococcus capsulatus. The molecular weights of the enzymes were approximately 310000 and 67000 respectively, as determined by gel filtration. Dissociation of 3-hexulose phosphate synthase into subunits of molecular weight approx. 49000 under conditions of low pH or low ionic strength was observed. Within the range of compounds tested, 3-hexulose phosphate synthase is specific for formaldehyde and d-ribulose 5-phosphate (forward reaction) and d-arabino-3-hexulose 6-phosphate (reverse reaction), and phospho-3-hexuloisomerase is specific for d-arabino-3-hexulose 6-phosphate (forward reaction) and d-fructose 6-phosphate (reverse reaction). A bivalent cation is essential for activity and stability of 3-hexulose phosphate synthase; phospho-3-hexuloisomerase is inhibited by many bivalent cations. The pH optima of the two enzymes are 7.0 and 8.3 respectively and the equilibrium constants are 4.0×10-5m and 1.9×102m respectively. The apparent Michaelis constants for 3-hexulose phosphate synthase are: d-ribulose 5-phosphate, 8.3×10-5m; formaldehyde, 4.9×10-4m; d-arabino-3-hexulose 6-phosphate, 7.5×10-5m. The apparent Michaelis constants for phospho-3-hexuloisomerase are: d-arabino-3-hexulose 6-phosphate, 1.0×10-4m; d-fructose 6-phosphate, 1.1×10-3m.

Purification and Properties of the Soluble 17β-Hydroxysteroid Dehydrogenase of Rabbit Uterus

1979 ◽  
Vol 34 (9-10) ◽  
pp. 726-737 ◽  
Author(s):  
Kunhard Pollow ◽  
Walter Eiger ◽  
Herrmann Heßlinger ◽  
Barbara Pollow
Keyword(s):  
Enzyme Activity ◽  
Dehydrogenase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Hydroxysteroid Dehydrogenase ◽  
Maximal Velocity ◽  
Ammonium Sulfate Precipitation ◽  
Ph Optimum ◽  
Mobility Data ◽  
Purification And Properties

Abstract 17 β-Hydroxysteroid dehydrogenase activity towards estradiol-17 β has been demonstrated in the 105,000 X g supernatant of rabbit uterus. Hydroxylapatite chromatography of the enzyme activity isolated by ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromato­graphy yielded a single 17 β-hydroxysteroid dehydrogenase activity. Further purification of the enzyme preparation by isoelectric focusing resulted in multiple peaks of activity. The molecular weight or the enzyme, calculated from mobility data on Sephadex gel, is approximately 64,000. Some properties of partially purified 17 β-hydroxysteroid dehydrogenase activity have been studied. Estradiol-17 β reacts at a faster rate than testosterone. The Km for estradiol is 4.16X 10-5 mol/1 for the NAD-linked enzyme activity and 4.37 X 10-5 mol/1 when NADP as cofactor was used. The ratio of the maximal velocity for NADP to that for NAD was 1.42. The pH-optimum for estradiol appears between 9.5 and 10.5 and for estrone between 5.5 and 6.5. The enzyme appears to be of the sulfhydryl type.

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