scholarly journals Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis

1978 ◽  
Vol 172 (1) ◽  
pp. 69-76 ◽  
Author(s):  
A Akrigg
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Duplex Dna ◽  
Ph Optimum ◽  
Endonucleolytic Cleavage ◽  
Single Strand Breaks ◽  
Purification And Properties

A DNAase (deoxyribonuclease) was isolated from culture supernatants of sporulating Bacillus subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel electrophoresis. The enzyme differs from other DNAases of B. subtilis in molecular weight, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNAase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The molecular weight of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12000.

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.

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.

Properties and characteristics of partly purified glutamate dehydrogenase from sheep rumen mucosa

1981 ◽  
Vol 46 (11) ◽  
pp. 2766-2773
Author(s):  
Katarína Holovská ◽  
Viera Lenártová ◽  
Ivan Havassy
Keyword(s):  
Molecular Weight ◽  
Glutamate Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Enzymatic Reaction ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ph Optimum ◽  
Relative Molecular Weight ◽  
Sheep Rumen ◽  
The Individual

The purification of glutamate dehydrogenase from sheep rumen mucosa on DEAE-cellulose afforded two enzyme fractions with glutamate dehydrogenase activity. The enzyme fraction II (tissue glutamate dehydrogenase) was freed of contaminating proteins in the subsequent purification step on Sephadex G-200. The approximate relative molecular weight (260 000) of tissue glutamate dehydrogenase (fraction II) was determined by gel filtration on Sephadex G-200 and the approximate relative molecular weight of its polypeptide chain (48 000) was established by polyacrylamide gel electrophoresis in SDS. The pH-optimum of fraction II was 7.9. The effect of substrate concentration on the rate of the enzymatic reaction was examined and the following apparent Michaelis' constants were found for the individual substrates: NADH 6.25 . 10-5 mol/l, 2-oxoglutarate 4.5 . 10-3 mol/l, and NH4+ 77 . 10-3 mol/l.

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 Purification and properties of a 1,3-1,4-β-d-glucanase (lichenase, 1,3-1,4-β-d-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli

1988 ◽  
Vol 255 (3) ◽  
pp. 833-841 ◽  
Author(s):  
J D Erfle ◽  
R M Teather ◽  
P J Wood ◽  
J E Irvin
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Glucanase Activity ◽  
Ph Optimum ◽  
Beta Glucanase ◽  
Purification And Properties ◽  
Hydrolysis Of

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.

scholarly journals Purification and some properties of a novel heat-stable cis-toluene dihydrodiol dehydrogenase

1987 ◽  
Vol 244 (3) ◽  
pp. 585-590 ◽  
Author(s):  
H D Simpson ◽  
J Green ◽  
H Dalton
Keyword(s):  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sole Source ◽  
Product Inhibition ◽  
Enzyme Mechanism ◽  
Bacillus Species ◽  
Ph Optimum ◽  
Heat Stable ◽  
Dihydrodiol Dehydrogenase

cis-Toluene dihydrodiol dehydrogenase was purified 200-fold from cells of a thermotolerant Bacillus species grown with toluene as the sole source of carbon and energy. The purified enzyme preparation was remarkably heat-stable and exhibited a half-life of 100 min at 80 degrees C, the temperature optimum. The activation energy of the reaction was 36 kJ.mol-1. Isoelectric focusing indicated that the pI of the native enzyme was 6.4 and that of the denatured enzyme 6.5. Although the pH optimum was 9.8, the enzyme was most stable at pH 8. The Mr of the enzyme was approx. 172,000 as determined by gel filtration and 166,000 by polyacrylamide-gel electrophoresis. The enzyme was composed of six apparently identical subunits with Mr values of 29,500. Kinetic analysis revealed that the Km for cis-toluene dihydrodiol was 92 microM and for NAD+ was 80 microM. The apparent Km values for cis-benzene dihydrodiol and cis-naphthalene dihydrodiol were 330 microM and 51 microM respectively. The enzyme was inhibited by mercurials but was unaffected by metal-ion chelators. Steady-state kinetics and product-inhibition patterns suggested that the enzyme mechanism was ordered Bi Bi.

AMP metabolism by the marine bacterium Vibrio (Beneckea) natriegens: purification and properties of adenylate kinase

1981 ◽  
Vol 27 (10) ◽  
pp. 1053-1059 ◽  
Author(s):  
Karamchand Ramotar ◽  
Michael A. Pickard
Keyword(s):  
Molecular Weight ◽  
Adenylate Kinase ◽  
Gel Electrophoresis ◽  
Marine Bacterium ◽  
Specific Activity ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Atp Formation ◽  
Purification And Properties ◽  
Beneckea Natriegens

Adenylate kinase (EC 2.7.4.3) has been purified 484-fold from extracts of Vibrio natriegens to a specific activity of 1350 μmol ADP formed∙min−1∙mg protein−1. The preparation was 97% pure as judged by gel electrophoresis and exhibited molecular weight values of 29 000 by gel filtration and 32 000 by SDS–gel electrophoresis. The isoelectric point was at pH 4.7. Only ATP (Km 0.067 mM), ADP (Km 0.45 mM), and AMP (Km 0.12 mM) exhibited high activity as substrates, though dATP or dAMP could serve as cosubstrates with AMP or ATP, respectively, at reduced rates. The equilibrium constant in the direction of ATP formation was 1.09, and the pH optimum in both directions was broad, from pH 7.2 to pH 7.6. Enzyme activity was sensitive to the thiolalkylating agents iodacetamide and p-chloromercuriphenyl sulfonate.

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.

The Purification and Properties of an Amidohydrolase from Soybean

1974 ◽  
Vol 52 (10) ◽  
pp. 903-910 ◽  
Author(s):  
Robert E. Hoagland ◽  
George Graf
Keyword(s):  
Arrhenius Plot ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hydrolysis Rate ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Hydrolysis Rates ◽  
Hydrolysis Of

An amidohydrolase (EC 3.5.1.13) was isolated from the roots of soybean (Glycine max Merril, var. Hawkeye) seedlings and purified 130-fold over the crude extract with 30% recovery. The purification steps entailed ammonium sulfate precipitation, gel filtration, cellulose ion-exchange chromatography, and polyacrylamide gel electrophoresis. The specific activity of the purified enzyme for the hydrolysis of Nα-benzoyl-DL-arginine p-nitroanilide (BAPA) was 810 mU/mg. The Km of the enzyme for this substrate was 5.78 × 10−6 M. The enzyme possessed a broad substrate specificity and catalyzed the hydrolysis of BAPA, glycine p-nitroanilide, L-leucine p-nitroanilide, and L-lysine p-nitroanilide. Specificity studies with a series of aminoacyl β-naphthylamides revealed a high hydrolysis rate on Nα-benzoyl-L-arginine β-naphthylamide, and lower hydrolysis rates on several other aminoacyl-substituted β-naphthylamides. The enzyme also displayed dipeptide hydrolase activity on several dipeptide substrates. The enzyme had a pH optimum of 8.0 in 0.05 M phosphate buffer with Nα-benzoyl-DL-arginine p-nitroanilide as substrate. The temperature optimum was 50 °C. The apparent activation energy determined from an Arrhenius plot was 6.3 kcal/mol (26 400 J/mol). The molecular weight estimated by gel filtration was approximately 63 000. Mercury (II) ion, silver (I) ion, p-benzoquinone, p-chloromercuribenzoate, and N-ethylmaleimide were effective inhibitors of the enzyme.

scholarly journals Purification and properties of hyaluronidase from human liver. Differences from and similarities to the testicular enzyme

1982 ◽  
Vol 205 (1) ◽  
pp. 69-74 ◽  
Author(s):  
E W Gold
Keyword(s):  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Human Umbilical Cord ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Significant Difference ◽  
High Concentrations ◽  
Different Response

Human liver hyaluronidase was purified to homogeneity by (NH4)2SO4 fractionation, chromatography on hydroxyapatite and DEAE-cellulose, and preparative disc polyacrylamide-gel electrophoresis. The enzyme had a pH optimum of 3.8-4.0, a molecular weight (determined by gel filtration) of 76000, and a Km of 0.05 mg/ml for purified human umbilical-cord hyaluronic acid. It generally resembled hyaluronidases studied in other tissues which are believed to be lysosomal, but shared a number of characteristics with a partially purified bovine testicular hyaluronidase. Neither enzyme exhibited inhibition by high concentrations of substrate, but both were competitively inhibited by dermatan sulphate and keratan sulphate. Both enzymes exhibited increased activity in the presence of albumin, probably owing to an increased susceptibility of substrate to enzyme action. The liver enzyme was inhibited by NaCl, but the testicular enzyme exhibited an increase in activity in the presence of the salt which was similar to the effect observed with albumin. The different response toward Cl- ion appeared to be the most significant difference between the two enzymes.

Sign in / Sign up

Export Citation Format

Share Document