Activation of phosphoenolpyruvate carboxykinase isolated from Veillonella parvula

1986 ◽  
Vol 64 (9) ◽  
pp. 898-905
Author(s):  
Helen S. Chau ◽  
Stephen K. Ng
Keyword(s):  
Ammonium Sulphate ◽  
Column Chromatography ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Maximum Activity ◽  
Acetyl Phosphate ◽  
Acetyl Coa ◽  
Protamine Sulphate ◽  
Ammonium Sulphate Precipitation ◽  
Fructose 1,6 Bisphosphate

Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) has been purified 940-fold from Veillonella parvula using protamine sulphate treatment, ammonium sulphate precipitation, and column chromatography. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity in the direction of oxaloacetate (OAA) decarboxylation was exhibited at pH 9.0. At this pH, the V. parvula enzyme catalysed phosphenolpyruvate formation in the presence of Mn2+ ions. In the presence of varying concentrations of OAA and ATP, the PEPCK from V. parvula exhibited hyperbolic kinetics with Kms of 0.16 and 0.46 mM, respectively. PEPCK from the anaerobe was not inhibited by NADH, succinate, glutamate, D-glucose 6-phosphate, acetyl phosphate, D-fructose 1,6-bisphosphate, pyruvate, ribose 5-phosphate, and aspartate. However, acetyl CoA, glyceraldehyde 3-phosphate, 3-phospho-D-glycerate, CTP, and GTP activated the enzyme. The activation of acetyl CoA was uncompetitive and noncooperative.

Anreicherung und Charakterisierung einer „Transhydrogenase-Aktivität“ und der 17 β-Hydroxysteroid-Oxidoreduktase aus der Cytoplasma-Fraktion der Leber von weiblichen Ratten / The Enrichment and Characterisation of Cytoplasmatic “Transhydrogenase-activity” and 17 β-hydroxysteroid-oxidoreductase from Rat Liver

1972 ◽  
Vol 27 (8) ◽  
pp. 981-988
Author(s):  
Kunhard Pollow ◽  
Barbara Pollow
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Isoelectric Focusing ◽  
Isoelectric Point ◽  
Column Chromatography ◽  
Enzymatic Activities ◽  
Kinetic Constants ◽  
Gel Chromatography ◽  
Ammonium Sulphate Precipitation

The cytoplasmatic fraction of rat liver contains both 17 β-hydroxysteroid-oxidoreductase and a “transhydrogenase-activity”, which catalyses the transfer of hydrogen from the 17 β-position of estradiol-17 β to the 17-position of 4-androstene-3,17-dione. The 17 β-hydroxysteroid-oxidoreductase was purified 718-fold and the “transhydrogenase-activity” 264-fold by ammonium sulphate precipitation, gel chromatography with Sephadex G-200, column chromatography on DEAE-Sephadex and isoelectric focusing. The two enzymic activities could not be separated. The characteristics of the two enzymatic activities give some evidence that the “transhydrogenase-activity” is identical with the already known 17 β-hydroxysteroid-oxidoreductase.Isoelectric focusing of the chromatographycally enriched 17 β-enzyme gave an isoelectric point at 5,2. The 17 β-enzyme has a molecular weight of 62 — 65 000 as determined by mobility on Sephadex G-200 superfine.The kinetic constants for both the 17 β-enzyme and the “transhydrogenase-activity” were determined.

scholarly journals Studies on tryptophan synthetases from various strains of Bacillus subtilis

1969 ◽  
Vol 112 (3) ◽  
pp. 285-292
Author(s):  
Jerzy W. Meduski ◽  
Stephen Zamenhof
Keyword(s):  
Bacillus Subtilis ◽  
Ammonium Sulphate ◽  
Column Chromatography ◽  
Proteolytic Enzymes ◽  
Deae Cellulose ◽  
Deae Cellulose Column ◽  
Ammonium Sulphate Precipitation ◽  
Tryptophan Synthetase ◽  
Cellulose Column

1. Tryptophan synthetase B of three strains of Bacillus subtilis was prepared from ‘exo-protoplastic’ and ‘endo-protoplastic’ fractions; the enzyme from ‘exo-protoplastic’ fraction was purified 30- to 120-fold by ammonium sulphate precipitation and DEAE-cellulose column chromatography; the latter step separated this enzyme from tryptophan synthetase A, tryptophanase and proteolytic enzymes, but the purified preparations were not stable. 2. The activity of tryptophan synthetase B did not depend on the presence of tryptophan synthetase A. 3. Tryptophan synthetases B of the strains tested differed in their utilization of 2- and 7-methylindole as compared with indole; this suggests that these tryptophan synthetases B are not identical.

ENZYMES OF PYRUVATE AND PHOSPHOENOLPYRUVATE CARBOXYLATION IN RUMEN BACTERIA

1974 ◽  
Vol 54 (4) ◽  
pp. 595-603 ◽  
Author(s):  
A. S. ATWAL ◽  
F. D. SAUER
Keyword(s):  
Calcium Phosphate ◽  
Ammonium Sulphate ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Rumen Fluid ◽  
Maximum Activity ◽  
Rumen Bacteria ◽  
Carboxylase Activity ◽  
Mixed Bacteria ◽  
Pep Carboxykinase

Extracts of mixed bacteria collected from bovine rumen fluid contained enzymes that carboxylated pyruvate and phosphoenolpyruvate (PEP). Fresh extracts showed high pyruvate carboxylase activity (EC 6.4.1.1. pyruvate carboxylase) which, however, was cold-labile and lost activity on dialysis at 4 C. Enzyme(s) catalyzing the carboxylation of PEP were stable under these conditions. The carboxylation of PEP was maximally stimulated by ADP and to a lesser degree by GDP. The ADP–requiring PEP carboxykinase [EC 4.1.1.49 phosphoenolpyruvate carboxykinase (ATP)] was equally active with either Mg++ or Mn++ and showed maximum activity at pH 6.5. The GDP–requiring PEP carboxykinase [EC 4.1.1.32 phosphoenolpyruvate carboxykinase (GTP)] required Mn++ and was almost inactive if Mg++ was substituted. Maximum activity was at pH 7.0. These nucleotides were most effective at 2.5-mM concentration and were inhibitory at higher concentrations. In the absence of added ADP or GDP, the carboxylation of PEP continued at a low but persistent rate. Precipitation with ammonium sulphate and adsorption on calcium phosphate gel resulted in fractions containing different proportions of the three activities. These results suggest that in mixed rumen bacterial extracts there are four separate enzymes capable of synthesizing oxaloacetate: one that catalyzes the carboxylation of pyruvate and three that catalyze the carboxylation of PEP.

scholarly journals Contributions of Environmental Signals and Conserved Residues to the Functions of Carbon Storage Regulator A of Borrelia burgdorferi

2013 ◽  
Vol 81 (8) ◽  
pp. 2972-2985 ◽  
Author(s):  
S. L. Rajasekhar Karna ◽  
Rajesh G. Prabhu ◽  
Ying-Han Lin ◽  
Christine L. Miller ◽  
J. Seshu
Keyword(s):  
Borrelia Burgdorferi ◽  
Carbon Storage ◽  
Vertebrate Host ◽  
Acetyl Phosphate ◽  
Mobility Shift ◽  
Acetyl Coa ◽  
Site Specific ◽  
Content Type ◽  
Environmental Signals ◽  
Carbon Storage Regulator

ABSTRACTCarbon storage regulator A ofBorrelia burgdorferi(CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABbare dependent on environmental signals and on select residues. We analyzed the phenotype ofcsrABbdeletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABbin thecsrABbmutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in thecsrABbmutant to parental levels, indicating that both the levels of CsrABband the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb(8S) with alanines resulted in increased levels of CsrABband reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members ofrpoSregulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb(7D) resulted in reducedcsrABbtranscripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream ofptacompared to the parental and 7D truncated protein. Two CsrABbbinding sites were also identified upstream offliWwithin theflgKcoding sequence. These observations reveal conserved and unique functions of CsrABbthat regulate adaptive gene expression inB. burgdorferi.

scholarly journals Prospects of Biocatalyst Purification Enroute Fermentation Processes

2021 ◽  
Author(s):  
Michael Bamitale Osho ◽  
Sarafadeen Olateju Kareem
Keyword(s):  
Fermentation Process ◽  
Colloidal Particles ◽  
Methyl Cellulose ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Inorganic Materials ◽  
Organic Substrates ◽  
Ammonium Sulphate Precipitation ◽  
Sodom Apple ◽  
Microbial Agents

Biotransformation of broth through fermentation process suffers a major setback when it comes to disintegration of organic substrates by microbial agents for industrial applications. These biocatalysts are in crude/dilute form hence needs to be purified to remove colloidal particles and enzymatic impurities thus enhancing maximum activity. Several contractual procedures of concentrating dilute enzymes and proteins had been reported. Such inorganic materials include ammonium sulphate precipitation; salting, synthetic polyacrylic acid; carboxy-methyl cellulose, tannic acid, edible gum and some organic solvents as precipitants etc. The emergence of organic absorbents such as sodom apple (Calostropis procera) extract, activated charcoal and imarsil had resulted in making significant impact in industrial circle. Various concentrations of these organic extracts have been used as purifying agents on different types of enzyme vis: lipase, amylase, protease, cellulase etc. Purification fold and stability of the enzyme crude form attained unprecedented results.

scholarly journals Genome-Scale Metabolic Network Reconstruction and In Silico Analysis of Hexanoic acid Producing Megasphaera elsdenii

2020 ◽  
Vol 8 (4) ◽  
pp. 539 ◽  
Author(s):  
Na-Rae Lee ◽  
Choong Hwan Lee ◽  
Dong-Yup Lee ◽  
Jin-Byung Park
Keyword(s):  
Cell Growth ◽  
Acid Production ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Flux Ratio ◽  
Value Added ◽  
Tca Cycle ◽  
Hexanoic Acid ◽  
Acetyl Coa ◽  
Megasphaera Elsdenii ◽  
Genome Scale

Hexanoic acid and its derivatives have been recently recognized as value-added materials and can be synthesized by several microbes. Of them, Megasphaera elsdenii has been considered as an interesting hexanoic acid producer because of its capability to utilize a variety of carbons sources. However, the cellular metabolism and physiology of M. elsdenii still remain uncharacterized. Therefore, in order to better understand hexanoic acid synthetic metabolism in M. elsdenii, we newly reconstructed its genome-scale metabolic model, iME375, which accounts for 375 genes, 521 reactions, and 443 metabolites. A constraint-based analysis was then employed to evaluate cell growth under various conditions. Subsequently, a flux ratio analysis was conducted to understand the mechanism of bifurcated hexanoic acid synthetic pathways, including the typical fatty acid synthetic pathway via acetyl-CoA and the TCA cycle in a counterclockwise direction through succinate. The resultant metabolic states showed that the highest hexanoic acid production could be achieved when the balanced fractional contribution via acetyl-CoA and succinate in reductive TCA cycle was formed in various cell growth rates. The highest hexanoic acid production was maintained in the most perturbed flux ratio, as phosphoenolpyruvate carboxykinase (pck) enables the bifurcated pathway to form consistent fluxes. Finally, organic acid consuming simulations suggested that succinate can increase both biomass formation and hexanoic acid production.

scholarly journals Laundry Detergent Compatibility of Papain Like Protease Purified From Piper Betel Leaves

2018 ◽  
Vol 7 (3.3) ◽  
pp. 132
Author(s):  
A Mousami Shankar ◽  
Dr G.V.D. Sirisha ◽  
Dr K. Vijaya Rachel
Keyword(s):  
Laundry Detergent ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Native Page ◽  
Chromatographic Techniques ◽  
Detergent Compatibility ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
The Stability ◽  
Better Than

Enzymes have wide applications in detergent industry from early 1900’s. Mostly, clothes are soiled by protein based grime. Most of the detergents have either amylase / protease. Various sources were scrutinized for potent protease activity and Betel leaves were selected, the enzyme was then isolated, purified to homogeneity by ammonium sulphate precipitation, DEAE-Cellulose and gel permeation chromatographic techniques. The enzyme was monomeric in nature with a molecular mass of 38kDa as determined by native PAGE and SDS-PAGE. The enzyme shows maximum activity at 60oC and pH 4.0. The Km and Vmax of the enzyme were 4x10-3M and 54µmol/min/mg respectively. The enzyme was categorically inhibited by PCMB and iodo-acetamide suggesting it to have papain like nature. The stability of the enzyme is assessed over the stretch of alkaline pH and temperature. This evaluation validates the stability of the enzyme and its use in detergent formulations. It was evident that after adding the enzyme preparation the stains (tea, chocolate, blood) were removed much better than that of the controls, which affirms that papain like enzyme from betel leaves, enhances detergent activity.  

Tetanus toxoid purification: Chromatographic procedures as an alternative to ammonium-sulphate precipitation

2011 ◽  
Vol 879 (23) ◽  
pp. 2213-2219 ◽  
Author(s):  
Ivana Stojićević ◽  
Ljiljana Dimitrijević ◽  
Nebojša Dovezenski ◽  
Irena Živković ◽  
Vladimir Petrušić ◽  
...  
Keyword(s):  
Ammonium Sulphate ◽  
Tetanus Toxoid ◽  
Ammonium Sulphate Precipitation

Photosynthesis in Phosphoenolpyruvate Carboxykinase-Type C4 Species: Properties of Nad-Malic Enzyme From Urochloa panicoides

1987 ◽  
Vol 14 (5) ◽  
pp. 517 ◽  
Author(s):  
JN Burnell
Keyword(s):  
Malic Enzyme ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Bundle Sheath ◽  
Photosynthetic Pathway ◽  
C4 Plant ◽  
C4 Species ◽  
Absolute Requirement ◽  
Pep Carboxykinase ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties

NAD-malic enzyme (EC 1.1.1.39) was purified from bundle sheath strands of Urochloa panicoides (a phosphoenolpyruvate carboxykinase-type C4 plant) and its kinetic and regulatory properties were investigated. The native enzyme has a molecular weight of about 470 000 and is an octomer composed of two slightly different monomers which occur in a 1 : 1 ratio. The enzyme has an absolute requirement for Mn2+, is stimulated by CoA, acetyl CoA, fructose 1,6-bisphosphate and SO42- and is inhibited by HCO3, oxaloacetate, 2-oxoglutarate and pyruvate. The enzyme is shown to be localised in the mito- chondria. The purified NAD-malic enzyme is unable to catalyse the carboxylation of pyruvate according to the reverse reaction. These findings are discussed in relation to the C4 photosynthetic pathway and its possible role in PEP carboxykinase-type C4 plants.

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