scholarly journals Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans

1977 ◽  
Vol 161 (2) ◽  
pp. 357-370 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Cytochrome C ◽  
Electron Acceptor ◽  
Specific Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Transport Chain ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Triton X ◽  
Sepharose 4B

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.

scholarly journals Acetylcholinesterase aus Rindererythrozyten. Reinigung und Eigenschaften des mit und ohne Triton X-100 solubilisierten Enzyms / Acetylcholinesterase from Bovine Erythrocytes. Purification and Properties of the En­zyme Solubilized in the Presence and the Absence of Triton X-100

1979 ◽  
Vol 34 (9-10) ◽  
pp. 721-725 ◽  
Author(s):  
Heinz Großmann ◽  
Manfred Liefländer
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Multiple Forms ◽  
Terminal Amino Acid ◽  
Enzyme Preparations ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Terminal Amino ◽  
Triton X ◽  
Bovine Erythrocytes

Abstract Acetylcholinesterase was released from bovine erythrocytes by Triton X-100 treatment and pu­rified by twofold affinity chromatography. The detergentfree enzyme was obtained with a specific activity of 4130 U /mg (303 000-fold purification) and a 25% yield. Alternatively, the commercial available crude enzyme was purified. The latter preparation has an uniform molecular weight (Mr 175 000). The Triton-solubilized enzyme, however, can be resolved after removal of the detergent in eight multiple forms (Mr 175 000 and multiple values), in the presence of Triton there exists only one form (Mr 338 000). The amino acid composition of the two enzyme preparations differs significantly. No differences were observed with respect to other properties: SDS gel electrophore­sis revealed two protein bands (Mr 166 000 and 86 000) with both preparations. The enzyme is a glycoprotein with a pI value of 4.3 and contains strongly bound phosphatidylethanolamine. The N-terminal amino acid has been found to be Glu (or Gin).

Properties of formate dehydrogenase from Desulfovibrio gigas

1986 ◽  
Vol 32 (5) ◽  
pp. 430-435 ◽  
Author(s):  
Mary Ann Riederer-Henderson ◽  
Harry D. Peck Jr.
Keyword(s):  
Cytochrome C ◽  
Electron Acceptor ◽  
Formate Dehydrogenase ◽  
Specific Activity ◽  
Ph Optimum ◽  
Benzyl Viologen ◽  
Desulfovibrio Gigas ◽  
Diaphorase Activity ◽  
Lag Period ◽  
Molecular Radius

The formate dehydrogenase from extracts of Desulfovibrio gigas was partially purified to a specific activity of 5600 nmol CO2 ∙ min−1 ∙ mg protein−1. Uniquely for a formate dehydrogenase from anaerobes, the enzyme was stable when stored aerobically. Nevertheless, thiols were required in the assay mixture for enzymatic activity. If the enzyme first catalyzed the transfer of electrons from thiols to benzyl viologen (a diaphorase activity), then formate was oxidized rapidly without a lag period. The enzyme had a molecular weight of approximately 240 000, a pH optimum of 7.5–8.0, and a temperature optimum of 56 °C. Activity with cytochrome c3 (molecular radius (Mr) = 13 000) was about twice that with ferredoxin or flavodoxin as the electron acceptor. These results suggest that the formate dehydrogenase from D. gigas can be activated by transferring electrons from thiols to an electron acceptor and uses cytochrome c3 as the natural electron carrier for the oxidation of formate.

Isolation of a b-Type Cytochrome Oxidase from Membranes of the Phototrophic Bacterium Rhodopseudomonas capsulata

1982 ◽  
Vol 37 (3-4) ◽  
pp. 193-198 ◽  
Author(s):  
Hendrik Hüdig ◽  
Gerhart Drews
Keyword(s):  
Cytochrome C ◽  
Affinity Chromatography ◽  
Cytochrome Oxidase ◽  
Membrane Fraction ◽  
Rhodopseudomonas Capsulata ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Triton X ◽  
Sepharose 4B ◽  
Horse Cytochrome

Abstract A cytochrome oxidase (EC 1.9.3.1) was solubilized from the membrane fraction of aerobically grown cells of Rhodopseudomonas capsulata by treatment with Triton X-100. The enzyme was purified 160 fold by chromatography on DEAE-Sepharose CL-6B and affinity chromatography on cytochrome c-thiol activated Sepharose 4B.The purified enzyme has a pH-optimum at 8.5 and a temperature optimum at 35 °C. The ap­ parent Km for reduced horse cytochrome c is 24 μм (at pH 8 and 30 °C). The purified cytochrome oxidase was 50% inhibited by 1.5 μм KCN and 10 μм NaN3. The purified enzyme contained one polypeptide of mr 65,000 and 6-type cytochrome.

scholarly journals Purification and properties of neutral maltase from human granulocytes

1989 ◽  
Vol 263 (3) ◽  
pp. 647-652 ◽  
Author(s):  
P Delqué Bayer ◽  
C Vittori ◽  
P Sudaka ◽  
J Giudicelli
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Human Kidney ◽  
Ph Optimum ◽  
Major Band ◽  
Purification And Properties ◽  
A Minor ◽  
Triton X ◽  
Human Polymorphonuclear Leukocytes

A procedure for the purification of neutral maltase from human polymorphonuclear leukocytes is described, involving solubilization with Triton X-100, proteolytic attack and three chromatographic steps: DEAE ion exchange, AcA 22 gel filtration and a second DEAE chromatography. The enzyme was obtained with a final specific activity of 30 units/mg of protein, comparable with that of other neutral maltases previously purified. The Mr of the enzyme was 550,000 as determined by gel filtration. SDS/polyacrylamide-gel electrophoresis, under non-denaturing conditions, led to a major band of 500,000 and a minor one of 260,000, both active, suggesting a polymeric or aggregated form of the protein. The catalytic properties of the human granulocytic neutral maltase were investigated. The pH optimum was around 6. The enzyme exhibited a broad range of substrate specificity, hydrolysing di- and oligosaccharides with alpha (1→2), alpha (1→3) and alpha (1→4) glucosidic linkages. The highest activities were observed for alpha (1→4) glucose oligomers of three to five residues. It was also found to hydrolyse polysaccharides such as starch and glycogen. The results of the inhibition studies are interpreted in terms of the existence of a large site including several subsites. The enzyme properties are broadly similar to those observed for other purified neutral alpha-glucosidases, in particular that of human kidney origin.

scholarly journals Isolation of a detergent-solubilized maltase/glucoamylase from rat intestine and its comparison with a maltase/glucoamylase solubilized by papain

1980 ◽  
Vol 187 (2) ◽  
pp. 437-446 ◽  
Author(s):  
Lilian M. Y. Lee ◽  
Antonieta K. Salvatore ◽  
Peter R. Flanagan ◽  
Gordon G. Forstner
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Solubilized Enzyme ◽  
Triton X ◽  
Sepharose 4B

Maltase/glucoamylase from the rat intestinal brush-border membrane was solubilized by homogenization of the intestinal mucosa in buffer containing 0.5% Triton X-100. After removal of the detergent with butan-1-ol, the enzyme was purified by chromatography on Sepharose 4B and DEAE-cellulose. The final specific activity was 70.3 units/mg of protein in six preparations, comparing favourably with the specific activity of 65.0 units/mg of protein of a pure papain-solubilized maltase/glucoamylase previously isolated and characterized by us [Flanagan & Forstner (1978) Biochem. J.173, 553–563]. The two enzymes were compared. Both migrated as single bands with the same mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were eluted at the same volume from Sepharose 4B, and had the same sedimentation pattern in mannitol gradients. The amino acid composition was similar; content of total apolar residues differed by 1.0mol%. Antibodies prepared against either enzyme gave identical precipitin lines with each. Neither enzyme bound tritiated Triton X-100. The only difference noted was the tendency of the detergent-solubilized enzyme to aggregate on storage, whereas the papain-solubilized enzyme remained unchanged. Both enzymes had two N-termini, glycine and arginine. When the two enzymes were dissociated by boiling in sodium dodecyl sulphate, each exhibited the same five species on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Single N-termini were found in the two smaller species, 1 (glycine) and 2 (arginine), whereas larger species (3–5) had both N-terminal amino acids. Both the Triton- and papain-solubilized enzymes appear to be oligomers of species 1 and 2, indicating that the native enzyme contains two subunit types. Aggregation in aqueous solutions does not depend on a proteolytically susceptible peptide fragment at the N-terminus of either subunit.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

Reaktionen der Hydrogenase aus Rhodopseudomonas capsulata im partikelgebundenen und gelösten Zustand

1969 ◽  
Vol 24 (5) ◽  
pp. 603-612 ◽  
Author(s):  
J.-H. Klemme
Keyword(s):  
Cytochrome C ◽  
Ammonium Sulfate ◽  
Deae Cellulose ◽  
Reaction Rates ◽  
Rhodopseudomonas Capsulata ◽  
Hydrogenase Activity ◽  
Original Activity ◽  
Dispersing Agent ◽  
Solubilized Enzyme ◽  
Triton X

In cell free extracts of Rps. capsulata obtained by exposure of cells to ultrasonic oscillation, about 90% of the hydrogenase is associated with the particulate chromatophore fraction. The particulate enzyme reacts with methylene blue (MB), menadione, phenazonium methosulfate (PMS), dichlorophenolindophenol (DCPIP), cytochrome c, p-benzoquinone (BQ), ferricyanide and O2,, but does not react with benzylviologen (BV), pyridinnucleotides and flavinnucleotides. Treatment of chromatophores with sodiumlaurylsulfate inactivates the hydrogenase reaction with PMS, DCPIP, BQ and ferricyanide. The MB-linked or menadione-linked hydrogenase is not destroyed by the detergent. The hydrogenase reaction with BV is increased more than 20-fold after incubation of the chromatophores with the lipid-dispersing agent. Treatment of chromatophores with acetone and petroleum ether almost completely inactivates the hydrogenase reaction with PMS and BQ. The reaction rate of the DCPIP-linked and the ferricyanide-linked hydrogenase is somewhat decreased, whereas the MB-linked, the menadione-linked and the BV-linked hydrogenase reactions still exhibit about 100% of the original activity. By extraction of the acetone-treated chromatophores with glycine-NaOH-buffer (pH 9), about 10 — 15% of the particulate hydrogenase is solubilized. The enzyme was 9-fold purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose. The purified enzyme contains no cytochrome. The relative reaction rates of the solubilized enzyme with different electron acceptors are similar to the corresponding reaction rates of the acetonetreated chromatophores. Extraction of chromatophores with n-butanol results in the solubilization of 5 — 10% of the particulate enzyme. By extraction of acetone-treated chromatophores with 0,5% Triton X-100, 40% of the particulate hydrogenase is solubilized. The fractionation of the extract with ammonium sulfate results in the isolation of a cytochrome c-containing particle which exhibits a 3-fold increased hydrogenase activity.

Enzymes involved in the metabolism of thiosulfate by Thiobacillus thioparus. II. Properties of adenosine-5′-phosphosulfate reductase

1970 ◽  
Vol 48 (3) ◽  
pp. 344-354 ◽  
Author(s):  
Ronald M. Lyric ◽  
Isamu Suzuki
Keyword(s):  
Molecular Weight ◽  
Cytochrome C ◽  
Electron Acceptor ◽  
Desulfovibrio Desulfuricans ◽  
Thiobacillus Denitrificans ◽  
Ph Optimum ◽  
Thiobacillus Thioparus ◽  
Aps Reductase

Adenosine-5′-phosphosulfate (APS) reductase was purified from Thiobacillus thioparus extracts 25- to 46-fold and the properties were studied. The molecular weight was 170 000 and the enzyme had 1 mole of FAD, 8–10 moles of iron, and 4–5 moles of labile sulfide. Cytochrome c as well as ferricyanide served as the electron acceptor. The pH optimum shifted from 7.4 to 9.5 when cytochrome c was used instead of ferricyanide. The Km values for sulfite and AMP were reduced from 2.5 mM and 100 μM to 17 μM and 2.5 μM, respectively, with cytochrome c as electron acceptor. Properties of the T. thioparus enzyme were compared to those of APS reductase isolated from Thiobacillus denitrificans and Desulfovibrio desulfuricans.

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 Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

1980 ◽  
Vol 191 (1) ◽  
pp. 117-124 ◽  
Author(s):  
R Zecher ◽  
H U Wolf
Keyword(s):  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Half Maximum ◽  
Purification And Characterization ◽  
Dimeric Molecule ◽  
Maximum Inhibition ◽  
Optimum Ph ◽  
Triton X

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

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