scholarly journals Chemical structure and biodegradability of halogenated aromatic compounds. Two catechol 1,2-dioxygenases from a 3-chlorobenzoate-grown pseudomonad

1978 ◽  
Vol 174 (1) ◽  
pp. 73-84 ◽  
Author(s):  
E Dorn ◽  
H J Knackmuss
Keyword(s):  
Heavy Metals ◽  
Aromatic Compounds ◽  
Chemical Structure ◽  
Specific Activity ◽  
Pseudomonas Sp ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Halogenated Aromatic Compounds

1. Two catechol 1,2-dioxygenases, pyrocatechase I and pyrocatechase II, were found in 3-chlorobenzoate-grown cells of Pseudomonas sp. B 13. The latter enzyme showed high relative activities with 3- and 4-chlorocatechol compared with catechol. 2. In benzoate-grown cells, only pyrocatechase I was induced. It was purified 29-fold with a final specific activity of 20 mumol of catechol oxygenated/min per mg of protein and an overall yield of 22%. Because of the instability of pyrocatechase II on chromatography and dialysis, no increase of specific activity was obtained during the purification experiments. 3. Molecular weights of pyrocatechase I and pyrocatechase II were 82000 and 67000 respectively. 4. For both pyrocatechases the pH optimum was found to be at 8.0.5. Inhibitions of the two pyrocatechases by Cu2+ and Hg2+ ions and p-chloromercuribenzoate were different. The effect on pyrocatechase I after incubation for 20 h with the heavy metals was decreased by addition of 1 mM-2-mercaptoethanol to the reaction mixture. The inhibition of pyrocatechase II was even enhanced under these conditions. 6. Extradiol cleavage of 3-methylcatechol in addition to intradiol fission at a ratio of 1:14 was observed only with pyrocatechase I.

scholarly journals Chemical structure and biodegradability of halogenated aromatic compounds. Conversion of chlorinated muconic acids into maleoylacetic acid

1980 ◽  
Vol 192 (1) ◽  
pp. 339-347 ◽  
Author(s):  
E Schmidt ◽  
H J Knackmuss
Keyword(s):  
Aromatic Compounds ◽  
Chemical Structure ◽  
Pseudomonas Sp ◽  
Muconic Acid ◽  
Low Activity ◽  
Muconate Cycloisomerase ◽  
Halogenated Aromatic Compounds

1. An enzyme for the cycloisomerization of 2- and 3-chloro-cis, cis-muconic acid was isolated from 3-chlorobenzoate-grown cells of Pseudomonas sp. B13. It was named muconate cycloisomerase II, because it could it clearly be differentiated by its Km and Vmax. values from an ordinary muconate cycloisomerase, which functioned in benzoate catabolism and exhibited low activity with the chlorinated substrates. 2-Chloro-cis, cis-muconic acid was converted into trans- and 3-chloro-cis, cis–muconic acid into cis-4-carboxymethylenebut-2-en-4-olide together with dehalogenation. 2. An enzyme was isolated from chlorobenzoate-grown cells, which converted the 4-carboxymethylenebut-2-en-4-olides into maleoylacetic acid.

Purine catabolism in man: characterization of placental microsomal 5′-nucleotidase

1976 ◽  
Vol 54 (5) ◽  
pp. 462-469 ◽  
Author(s):  
Irving H. Fox ◽  
Pamela J. Marchant
Keyword(s):  
Specific Activity ◽  
Ph Optimum ◽  
Electrophoretic Variants ◽  
Molecular Weights ◽  
Wide Range ◽  
Stokes Radius ◽  
Michaelis Constants ◽  
Light Form ◽  
Molecular Weight Form

Human placental microsomal 5′-nucleotidase (EC 3.1.3.5) was prepared free of alkaline phosphatase by isoelectric focusing. A total of seven electrophoretic variants were isolated during the preparation of six placentas. Only three to six variants were found in a single placenta. The isoelectric pH's were 6.70, 6.44, 6.23, 6.02, 5.76, 5.63 and 5.44. These were found to be composed of variable quantities of a large, medium and low molecular weight form. The apparent molecular weights of the medium and light form of the enzyme were 86 500 and 43 500, respectively, as estimated from Stokes radius and sedimentation velocity determinations. The electrophoretic variants were not distinguishable with respect to specific activity and Michaelis constants for AMP, GMP or CMP or inhibition by ATP, CTP or adenosine. These electrophoretic variants appeared to be pseudoisozymes based upon different states of aggregation of a common primary sequence.There was a wide range of substrate specificity among nucleoside 5′-monophosphates which included 2-deoxyribose compounds. With AMP as 100, substrate activity was: CMP, 122; NMN, 74; GMP, 68; IMP, 63; XMP, 28 and UDP–glucose, 68. The Michaelis constants for AMP, GMP and CMP ranged from 12–18 μM, from 33–67 μM and from 170–250 μM, respectively. Although 5′-nucleotidase was active in the absence of divalent cation, 5 mM MgCl2 stimulated the enzyme activity to 234% of control and shifted the pH optimum of 9.8 to a plateau from pH 7.4–9.8.

scholarly journals Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effects on 1,2-dioxygenation of catechol

1978 ◽  
Vol 174 (1) ◽  
pp. 85-94 ◽  
Author(s):  
E Dorn ◽  
H J Knackmuss
Keyword(s):  
Chemical Structure ◽  
Substituent Effects ◽  
Alcaligenes Eutrophus ◽  
Reaction Rates ◽  
Pseudomonas Sp ◽  
Substituent Constant ◽  
Binding Studies ◽  
Km Value ◽  
Michaelis Constants ◽  
Halogenated Aromatic Compounds

1. The influence of halogen substituents on the 1,2-dioxygenation of catechols was investigated. The results obtained with the two isoenzymes pyrocatechase I and pyrocatechase II from the haloarene-utilizing Pseudomonas sp. B 13 and the pyrocatechase from benzoate-induced cells of Alcaligenes eutrophus B.9 were compared. 2. Substituents on catechol were found to interfere with O2 binding by the two isoenzymes from Pseudomonas sp. B 13, whereas the Km value for catechol kept constant at different O2 concentrations. 3. Electron-attracting substituents decreased the Km values for catechols. 4. Results from binding studies with substituted catechols demonstrated narrow stereospecificities of pyrocatechase I from pseudomonas sp. B 13 and the pyrocatechase from alcaligenes eutrophus B.9. In contrast, a low steric hindrance by substituents in the binding of catechols with pyrocatechase II was observed. 5. Low pK′1 values of substituted catechols resulted in low Michaelis constants. 6. Electron-attracting substituents such as halogen decreased the reaction rates of catechol 1,2-dioxygenation. The correlation of the Vmax. values observed with pyrocatechase II from Pseudomonas sp. B 13 with the substituent constant sigma+ (Okamoto–Brown equation) was distinctly greater than with Hammett's sigma values. The corresponding logVmax. against sigma+ correlation for pyrocatechase I was considerably disturbed by steric influences of the substituents.

scholarly journals Chemical structure and biodegradability of halogenated aromatic compounds. Halogenated muconic acids as intermediates

1980 ◽  
Vol 192 (1) ◽  
pp. 331-337 ◽  
Author(s):  
E Schmidt ◽  
G Remberg ◽  
H J Knackmuss
Keyword(s):  
Chemical Structure ◽  
Acidic Solution ◽  
Pseudomonas Sp ◽  
Trans Form ◽  
Solution Ph ◽  
Muconic Acid ◽  
Acidic Solutions ◽  
Ph Conditions ◽  
Ylacetic Acid ◽  
Halogenated Aromatic Compounds

Substituted muconic acids were prepared from the corresponding catechols by pyrocatechase II from Pseudomonas sp. B13. The stabilities of substituted muconic acids were compared under different pH conditions. 3-Substituted cis, cis-muconic acids cycloisomerized readily in slightly acidic solutions, whereas 2-chloro- and 2-fluoro-cis, cis-muconic acids were stable under these conditions and could be isolated as crystalline compounds. They were isomerized to the cis, trans-form in highly acidic solution (pH 1), particularly when heated to 80 degrees C. Cycloisomerization of 2-chloro-cis, cis-muconic acid in 75% (v/v) H2SO4 yields 4-carboxymethyl-2-chloro-but-2-en-4-olide (4-chloro-2,5-dihydro-5-oxo-3H-furan-2-ylacetic acid). THe cis, cis-configuration of 2-chloromuconic acid was certified by 1H n.m.r. spectroscopy and by enzymic cycloisomerization. Although the cis, cis-configuration of 2-fluoromuconic acid was confirmed by corresponding spectroscopic data, it was not cycloisomerized by crude extracts or cycloisomerase II preparations from Pseudomonas sp. B13.

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 Isolation and properties of α-D-mannosidase from human kidney

1976 ◽  
Vol 155 (2) ◽  
pp. 217-223 ◽  
Author(s):  
D V. Marinkovic ◽  
J N. Marinkovic
Keyword(s):  
Heavy Metals ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Human Kidney ◽  
Human Albumin ◽  
Homogeneous State ◽  
Thiol Groups ◽  
Ph Optimum ◽  
Molecular Weights

α-D-Mannosidase activity exists in three forms that can be separated by DEAE-cellulose chromatography, α-D-Mannosidase was isolated from human kidney in a homogeneous state, and was purified 2100-fold, with p-nitrophenyl α-D-mannoside as substrate. The purified α-D-mannosidase was practically free from all other glycosidases tested. The Km of the synthetic substrate with the enzyme was 1 × 10(-3) M and the pH optimum 4.5. It was inhibited by heavy metals, sodium dodecyl sulphate, urea and compounds that react with the thiol groups, and was activated by Zn2+, Na+, 2-mercaptoethanol, human albumin and γ-globulin. The mol. wt. of the enzyme was estimated to be 180 000 +/- 4500. After pretreatment with 2-mercaptoethanol and sodium dodecyl sulphate, α-D-mannosidase dissociated into subunits of mol. wts. of 58 000 +/- 600 and 30 000 +/- 380 respectively. Subunits of the same molecular weights were also obtained after the enzyme was heated at 100 degrees C.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

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