scholarly journals Purification and regulatory properties of phosphofructokinase from Trypanosoma (Trypanozoon) brucei brucei

1985 ◽  
Vol 227 (1) ◽  
pp. 113-124 ◽  
Author(s):  
C N Cronin ◽  
K F Tipton
Keyword(s):  
Enzyme Activity ◽  
Kinetic Studies ◽  
Cysteine Proteinases ◽  
Allosteric Inhibitor ◽  
Step Procedure ◽  
Low Concentrations ◽  
Tetrameric Structure ◽  
High Concentrations ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties

Phosphofructokinase (EC 2.7.1.11) from Trypanosoma (Trypanozoon) brucei brucei was purified to homogeneity by using a three-step procedure that may be performed within 1 day. Proteolysis, which removes a fragment of Mr approx. 2000, may occur during the purification, but this can be prevented by including antipain, an inhibitor of cysteine proteinases, in the buffers during the purification. The subunits of the enzyme appear to be identical in size, with an Mr of 49 000. The Mr of the native enzyme was estimated to be approx. 220 000, suggesting a tetrameric structure. Kinetic studies showed the activity to depend hyperbolically on the concentration of ATP but sigmoidally on the concentration of fructose 6-phosphate. Although cyclic AMP, AMP and ADP stimulated the enzyme activity at low concentrations of fructose 6-phosphate, the last two nucleotides were inhibitory at high concentrations of this substrate. Phosphoenolpyruvate behaved as an allosteric inhibitor of the phosphofructokinase. Citrate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and Pi did not influence significantly the activity of the enzyme.

Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

1971 ◽  
Vol 17 (3) ◽  
pp. 183-191 ◽  
Author(s):  
Philip J Garry
Keyword(s):  
Enzyme Activity ◽  
Sodium Fluoride ◽  
Phosphate Buffer ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Serum Cholinesterase ◽  
Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

scholarly journals The enzymology of short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata. Kinetic studies and a proposed reaction mechanism

1974 ◽  
Vol 137 (3) ◽  
pp. 435-442 ◽  
Author(s):  
Owen A. Young ◽  
John W. Anderson
Keyword(s):  
Fatty Acid ◽  
Pinus Radiata ◽  
Kinetic Studies ◽  
Single Species ◽  
Competitive Inhibitor ◽  
Fatty Acyl ◽  
Short Chain ◽  
Low Concentrations ◽  
High Concentrations ◽  
Acyl Coenzyme A

1. Short-chain fatty acyl-CoA synthetase from seeds of Pinus radiata was examined by acetate- and propionate-dependent PPi–ATP exchange. Reaction mixtures came to equilibrium almost instantly as judged by rates of exchange and analysis of an incubation mixture. 2. The activity of the enzyme was correlated with the concentration of MgP2O72- but not with the concentration of Mg2+, as judged by PPi–ATP exchange and fatty acyl AMP-dependent synthesis of ATP in the presence of PPi. In PPi–ATP exchange assays, no clear relationship between activity and any single species of ATP was apparent. 3. High concentrations of fatty acid inhibited PPi–ATP exchange. PPi–dATP exchange was less than PPi–ATP exchange at low concentrations of fatty acid, but at higher concentrations PPi–dATP exchange exceeded PPi–ATP exchange. The rate of synthesis of fatty acyl-CoA in the presence of dATP was less than with ATP. 4. ATP and propionate inhibited the synthesis of ATP from propionyl-AMP and PPi. The inhibition by ATP was competitive with respect to propionyl-AMP and non-competitive with respect to PPi. The inhibition by propionate was non-competitive with respect to propionyl-AMP and PPi. 5. AMP was a competitive inhibitor of propionyl-AMP-dependent synthesis of ATP and competitively inhibited propionate-dependent PPi–ATP exchange when ATP was the variable substrate. 6. It was concluded that the first partial reaction catalysed by the enzyme is ordered; ATP is the first substrate to react with the enzyme and PPi is probably the only product released.

scholarly journals The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides

1972 ◽  
Vol 126 (4) ◽  
pp. 975-984 ◽  
Author(s):  
K. Dalziel ◽  
R. R. Egan
Keyword(s):  
Glutamate Dehydrogenase ◽  
Dissociation Constants ◽  
Equilibrium Dialysis ◽  
Kinetic Studies ◽  
Binding Studies ◽  
Purine Nucleotides ◽  
Active Centre ◽  
Sodium Phosphate Buffer ◽  
Low Concentrations ◽  
High Concentrations

1. The binding of NAD+ and NADP+ to glutamate dehydrogenase has been studied in sodium phosphate buffer, pH7.0, by equilibrium dialysis. Approximate values for the dissociation constants are 0.47 and 2.5mm respectively. For NAD+ the value agrees with that estimated from initial-rate results. 2. In the presence of the substrate analogue glutarate both coenzymes are bound more firmly, and there is one active centre per enzyme subunit. The binding results cannot be described in terms of independent and identical active centres, and binding is stronger at low coenzyme concentrations than at high concentrations. Either the six subunits of the oligomer are not identical or there are negative interactions between them in the binding of coenzymes in ternary complexes with glutarate. The latter explanation is favoured. 3. The binding studies support the conclusions drawn from earlier kinetic studies of the glutamate reaction. 4. ADP and GTP respectively decrease and increase the affinity of the enzyme for NAD+ and NADP+, in both the presence and absence of glutarate. The negative binding interactions in the presence of glutarate are abolished by ADP, which decreases the affinity for the coenzymes at low concentrations of the latter. 5. In the presence of glutarate, GTP and NAD+ or NADP+, the association of enzyme oligomers is prevented, and the solubility of the enzyme is decreased; the complex of enzyme and ligands readily crystallizes. 6. The results are discussed in relation to earlier kinetic studies.

scholarly journals Solubilization of a mannose-polymerizing enzyme from Phaseolus aureus

1972 ◽  
Vol 128 (2) ◽  
pp. 243-252 ◽  
Author(s):  
J. S. Heller ◽  
C. L. Villemez
Keyword(s):  
Enzyme Activity ◽  
Enzyme Preparation ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Soluble Enzyme ◽  
Phaseolus Aureus ◽  
Original Activity ◽  
Solubilized Enzyme ◽  
High Concentrations

A soluble enzyme preparation, which catalyses the polymerization of mannose, was obtained by Triton X-100 extraction of a particulate fraction derived from Phaseolus aureus hypocotyls. The product that resulted when GDP-α-d-mannose was used as a substrate was a β-(1→4)-linked mannan, about three-quarters of which was alkali-insoluble. The mannose-polymerizing enzyme activity was at least as great in the soluble preparation as in the particulate preparation, and the specific activity of the solubilized enzyme was greater by a factor of at least 3.5. Kinetic studies of the soluble enzyme indicate that the apparent Km is 55–62μm, and a disproportionate increase in rate is observed at high concentrations. GDP-α-d-glucose is a strong competitive inhibitor of the mannose-polymerizing reaction, with an apparent Ki of 6.2μm. The soluble enzyme is relatively unstable, losing about two-thirds of its original activity in 5h at 0°C or in 24h at −20°C. A solvent (acetone, butanol, diethyl ether)-extracted particulate preparation, which also exhibits the same enzyme activity, is more stable, retaining full activity for at least 5 days at −20°C. There was no polymerizing-enzyme activity in the soluble enzyme preparation when UDP-d-glucose, UDP-d-galactose, UDP-d-xylose, UDP-l-arabinose or UDP-d-glucuronic acid were used as substrates. However, the soluble enzyme preparation would catalyse the polymerization of glucose, with GDP-d-glucose as substrate.

scholarly journals Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow-trout fructose diphosphatase

1969 ◽  
Vol 111 (3) ◽  
pp. 287-295 ◽  
Author(s):  
H. W. Behrisch ◽  
P. W. Hochachka
Keyword(s):  
Enzyme Activity ◽  
Rainbow Trout ◽  
Temperature Adaptation ◽  
Temperature Dependent ◽  
Ph Optimum ◽  
Physiological Temperature ◽  
Low Concentrations ◽  
High Concentrations ◽  
Regulation Of Enzyme Activity ◽  
Hill Plots

1. The properties of fructose diphosphatase from the liver of rainbow trout (Salmo gairdnerii) were examined over the physiological temperature range of the organism. 2. Saturation curves for substrate (fructose 1,6-diphosphate) and a cofactor (Mg2+) are sigmoidal, and Hill plots of the results suggest a minimum of two interacting fructose 1,6-diphosphate sites and two interacting Mg2+ sites per molecule of enzyme. 3. Mn2+-saturation curves are hyperbolic, and the Ka for Mn2+, which inhibits the enzyme at high concentrations, is 50–100-fold lower than the Ka for Mg2+. 4. Fructose diphosphatase is inhibited by low concentrations of AMP; this inhibition appears to be decreased and reversed by increasing the concentrations of Mg2+ and Mn2+. Higher concentrations of AMP are required to inhibit the trout fructose diphosphatase in the presence of Mn2+. 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn2+ appear to be temperature-independent, whereas the affinities for Mg2+ and AMP are highly temperature-dependent. 6. The pH optimum of the enzyme depends on the concentrations of Mg2+ and Mn2+. In addition, pH determines the Ka for Mg2+; at high pH, Ka for Mg2+ is lowered. 7. The enzyme is inhibited by Ca2+ and Zn2+, and the inhibition is competitive with respect to both cations. 8. The possible roles of these ions and AMP in the modulation of fructose diphosphatase and gluconeogenic activity are discussed in relation to temperature adaptation.

scholarly journals Thermodynamic and Kinetic Aspects on the Biosorption of Cadmium by Low Cost Materials: A Review

2006 ◽  
Vol 3 (6) ◽  
pp. 400 ◽  
Author(s):  
Pablo Lodeiro ◽  
Roberto Herrero ◽  
Manuel E. Sastre de Vicente
Keyword(s):  
Low Cost ◽  
Kinetic Studies ◽  
Chemical Precipitation ◽  
Point Of View ◽  
Biological Origin ◽  
Biosorption Process ◽  
Low Concentrations ◽  
Passive Adsorption ◽  
High Concentrations

Environmental Context. The toxicity of cadmium in waters can be decreased by using a wide variety of low-cost biomaterials. A number of such investigations are reviewed here and the models used to describe the process of biosorption discussed. Fundamental investigations that probe the thermodynamics and kinetics of the biosorption process are essential for a strong understanding of all biosorption processes. Areas that still need addressing are highlighted, in particular with regard to cadmium biosorption, some models for which are ready to be tested in pilot plants. Abstract. Cadmium is internationally recognized as an important pollutant in the environment, and different methods for its removal from wastewaters (chemical precipitation being the most commonly used) have been reported in the literature. Those methods are in most cases oriented to situations with high concentrations of the pollutant. Thus, alternative removal and recovery methods are being considered for removing very low concentrations of cadmium. These methods are all based on biosorption, the passive adsorption and sequestration of metals by several natural materials of biological origin. In this review we have considered the biosorption of cadmium onto biomaterials from a physicochemical, thermodynamic, and kinetic perspective. The thermodynamic perspective is based on the characterization of the interactions of the binding sites of the biosorbents with cadmium species in aqueous solution. Traditionally, this approach has been quantified using different kinds of isotherms. In addition, the description is completed by taking into account electrostatic effects, and the influence of pH and ionic strength, which are associated with the negative charge developed, in most cases, by the biomaterial. The other point of view in this review is the kinetic one, which is necessary for a full physicochemical description of the sorbate–biosorbent system. Consequently, an updated description of the various approaches commonly employed in kinetic studies in biosorption has been carried out.

Properties and regulation of pyruvate carboxylase from Bacillus stearothermophilus

1970 ◽  
Vol 176 (1042) ◽  
pp. 1-19 ◽  
Keyword(s):  
Enzyme Activity ◽  
Pyruvate Carboxylase ◽  
Bacillus Stearothermophilus ◽  
Kinetic Studies ◽  
Allosteric Effectors ◽  
Regulatory Properties

Pyruvate carboxylase has been purified 400-fold from the thermophile, Bacillus stearothermophilus; it resembles pyruvate carboxylases purified from mesophilic organisms in its general kinetic and regulatory properties. The enzyme is virtually inactive in the absence of acetylcoenzyme A ; this activating effect is antagonized by L-aspartate. Kinetic studies show that these two compounds act as allosteric effectors. ADP inhibits the enzyme activity competitively with ATP. Although the thermophile enzyme is appreciably more thermostable than similar mesophile enzymes, it is quite labile at the temperature at which the organism grows optimally, but can be stabilized by the two allosteric effectors and by some of the reactants.

scholarly journals The effects of the tylosin and Cd on soil enzyme activity

2020 ◽  
Vol 213 ◽  
pp. 01032
Author(s):  
Zhaohong Meng ◽  
Shuman Wang ◽  
Jia Zhou
Keyword(s):  
Heavy Metals ◽  
Enzyme Activity ◽  
Soil Enzyme ◽  
Soil Enzyme Activity ◽  
Germination Index ◽  
Soil Microbial ◽  
Low Concentrations ◽  
Long Time ◽  
High Concentrations ◽  
Microbial Environment

Soil microbial environment have been affected by different concentration heavy metals Cd (HM) and tylosin (TYL) and combination of TYL and HM interactions. Degradation of TYL was caused certain inhibition due to the addition of HM. The germination index of seed had been inhibited owing to the toxic effects of HM and TYL, but we found that the low concentrations of HM (4 mg/kg), the germination index higher than the soil which unadded HM and TYL in it. The soil enzyme activity was significantly suppressed by the addition of HM and TYL. Actinomycete was inhibited by high concentrations of HM for a long time. The studies demonstrated that the pollution of the soil micro-environment has been serious than only add HM or TYL in the soil.

scholarly journals The kinetics of effector binding to phosphofructokinase. The influence of effectors on the allosteric conformational transition

1980 ◽  
Vol 189 (3) ◽  
pp. 569-579
Author(s):  
D Roberts ◽  
G L Kellett
Keyword(s):  
Cyclic Amp ◽  
Conformational Transition ◽  
Slow Phase ◽  
Protein Fluorescence ◽  
Allosteric Transition ◽  
Low Concentrations ◽  
Inhibitory Site ◽  
High Concentrations ◽  
Isomerization Pathway ◽  
Fructose 1,6 Bisphosphate

1. The extent of the allosteric transition from the R into the T conformation of rabbit skeletal muscle phosphofructokinase induced by Mg2+-1,N6-etheno-ATP was determined by stopped-flow fluorimetry from the amplitude of the slow phase of the Mg2+-1,N6-etheno-ATP fluorescence enhancement [Roberts & Kellet (1979) Biochem. J. 183, 349–360]. 2. The amplitude of the slow phase was decreased by low concentrations of the activators cyclic AMP and fructose 1,6-bisphosphate, but increased in a complex manner by the inhibitor citrate. 3. Mg2+-1,N6-etheno-ATP and Mg2+-ATP are unable to induce the T conformation to a detectable extent in the presence of saturating cyclic AMP, but can do so readily in the presence of saturating fructose 1,6-bisphosphate. 4. The conformational transitions induced in enzyme alone by different ligands were observed by changes in intrinsic protein fluorescence. In general, an R-type conformation has diminished protein fluorescence compared with a T-type conformation. 5. Mg2+-ATP exerts a complex effect on protein fluorescence; both the enhancement at low concentrations and the quenching at high concentrations of Mg2+-ATP result from the binding of Mg2+-ATP to the inhibitory site and the ensuing allosteric transition. Enhancement reflects the extent of the allosteric transition and involves both tyrosine and tryptophan, probably in the region of the active site; quenching reflects occupation of the inhibitory site and involves tyrosine at the inhibitory site. 6. The mechanism of the allosteric transition from the R into the T conformation induced by Mg2+-1,N6-etheno-ATP at low concentrations occurs predominantly by a ‘prior-isomerization’ pathway; at higher concentrations a limited contribution from a ‘substrate-guided’ pathway occurs. 7. The allosteric behaviour of phosphofructokinase with respect to Mg2+-ATP and Mg2+-1,N6-ethenol-ATP binding may be accounted for in terms of the simple, concerted model.

Radioenzymatic Assay of Catecholamines: Reversal of Aluminium Inhibition of Enzymatic O-Methylation by Desferrioxamine

1983 ◽  
Vol 20 (2) ◽  
pp. 105-111 ◽  
Author(s):  
L Mason ◽  
C Weinkove
Keyword(s):  
Complex Formation ◽  
Biological Samples ◽  
Chelating Agent ◽  
Kinetic Studies ◽  
Metal Chelating ◽  
Radioenzymatic Assay ◽  
Low Concentrations ◽  
High Concentrations

Alumina is commonly used for the purification and concentration of catecholamines in biological specimens before analysis. Acid eluates of alumina, found to contain high concentrations of A13+, interfered with O-methylation but not with N-methylation. The chemistry of the catecholamines, supported by kinetic studies, suggest that complex formation between aluminium and the substrate account for the observed inhibition of O-methylation. The addition of desferrioxamine, a metal-chelating agent, to the reaction mixture reversed this inhibition and, by allowing a preliminary alumina extraction, permits the measurement of low concentrations of catecholamines in biological samples.

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