scholarly journals Reexamining Michaelis-Menten enzyme kinetics for xanthine oxidase

2013 ◽  
Vol 37 (1) ◽  
pp. 37-48 ◽  
Author(s):  
James B. Bassingthwaighte ◽  
Tamara M. Chinn
Keyword(s):  
Xanthine Oxidase ◽  
Enzyme Concentration ◽  
Product Inhibition ◽  
Parameter Estimates ◽  
Michaelis Constant ◽  
Binding Model ◽  
Initial Substrate ◽  
In Series ◽  
Substrate Concentrations

Abbreviated expressions for enzyme kinetic expressions, such as the Michaelis-Menten (M-M) equations, are based on the premise that enzyme concentrations are low compared with those of the substrate and product. When one does progress experiments, where the solute is consumed during conversion to form a series of products, the idealized conditions are violated. Here, we analyzed data of xanthine oxidase in vitro from Escribano et al. ( Biochem J 254: 829, 1988) on two conversions in series, hypoxanthine to xanthine to uric acid. Analyses were done using four models: standard irreversible M-M reactions ( model 1), Escribano et al.'s M-M forward reaction expressions with product inhibition ( model 2), fully reversible M-M equations ( model 3), and standard differential equations allowing forward and backward reactions with mass balance accounting for binding ( model 4). The results showed that the need for invoking product inhibition vanishes with more complete analyses. The reactions were not quite irreversible, so the backward reaction had a small effect. Even though the enzyme concentration was only 1–2% of the initial substrate concentrations, accounting for the fraction of solutes bound to the enzyme did influence the parameter estimates, but in this case, the M-M model overestimated Michaelis constant values by only about one-third. This article also presents the research and models in a reproducible and publicly available form.

Intestinal transport of amino acids studied with l-valine

1965 ◽  
Vol 208 (5) ◽  
pp. 914-921 ◽  
Author(s):  
Sheldon Reiser ◽  
Philip A. Christiansen
Keyword(s):  
Amino Acids ◽  
Competitive Inhibition ◽  
Intestinal Transport ◽  
Acceptor Site ◽  
Maximal Velocity ◽  
Tissue Uptake ◽  
Michaelis Constant ◽  
Tissue Water ◽  
Initial Substrate ◽  
Substrate Concentrations

l-Valine (0.003 m) was transported against a concentration gradient by everted rat intestinal sacs. After 5 min a mucosa-rich fraction had near-maximum valine concentration. There was a 45-min lag before the valine concentration in the serosal medium became equal to that in the tissue water. Placing valine either in the mucosal or serosal medium resulted in the same final ratio of valine between them (8/1, serosal/mucosal after 2 hr). Net transport was inhibited by substrate concentrations greater than 0.01 m, whereas tissue uptake was proportional to the initial substrate concentration through the range 0–0.05 m. The apparent Michaelis constant for valine net transport was 2.89 x 10–3 m and the maximal velocity of the system was 16.3 µmoles valine/500 mg tissue per hr. Potent inhibitors of tissue uptake and net transport were anaerobiosis, cyanide (0.002 m), p-chloromercuribenzoate (0.0004 m), 2,4-dinitrophenol (0.0002 m) (noncompetitively), and l-leucine (0.005 m) (competitively). Neutral l-amino acids having lipophilic side chains inhibited valine translocation in a predictable manner on the basis of competitive inhibition for an acceptor site on the tissue.

Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

1981 ◽  
Vol 45 (03) ◽  
pp. 290-293 ◽  
Author(s):  
Peter H Levine ◽  
Danielle G Sladdin ◽  
Norman I Krinsky
Keyword(s):  
Superoxide Dismutase ◽  
Cytochrome C ◽  
Xanthine Oxidase ◽  
Direct Effect ◽  
Platelet Function ◽  
Experimental Conditions ◽  
Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

Phosphorus Release Kinetics in Biofilm Reactors

1992 ◽  
Vol 26 (3-4) ◽  
pp. 567-576 ◽  
Author(s):  
F. A. Ruiz-Treviño ◽  
S. González-Martínez ◽  
C. Doria-Serrano ◽  
M. Hernández-Esparza
Keyword(s):  
Phosphorus Removal ◽  
Release Kinetics ◽  
Phosphorus Release ◽  
Organic Substrate ◽  
Biofilm Reactor ◽  
Substrate Uptake ◽  
Biofilm Reactors ◽  
Initial Substrate ◽  
Linear Behaviour ◽  
Substrate Concentrations

This paper presents the kinetic analysis, using Generalized Power-Law equations to describe the results of an experimental investigation conducted on a batch submerged biofilm reactor for phosphorus removal under an anaerobic/aerobic cycle. The observed rates and amounts of phosphorus release and organic substrate uptake in the anaerobic phase leads to a kinetic model in which these two variables are dependent on each other with a non-linear behaviour and reach equilibrium values in both cases, at different times and are function of rate constants ratio. The model has a good fit with experimental data except for C uptake at anaerobic contact times longer than four hours, where other kinetics are implied. Kinetic parameters were obtained with different initial substrate concentrations, anaerobic contact cycles, and type of substrates.

Peroxidases from Tulip Bulbs (Tulipa fosteriana, L.) Oxidize Xenobiotics NNitrosodimethylamine and N-Nitroso-N-methylaniline in vitro

1997 ◽  
Vol 62 (11) ◽  
pp. 1804-1814 ◽  
Author(s):  
Marie Stiborová ◽  
Hana Hansíková
Keyword(s):  
Cytochromes P450 ◽  
Kinetic Studies ◽  
The Other ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
Other Hand ◽  
Plant Peroxidases ◽  
Tulip Bulbs

Tulip bulbs (Tulipa fosteriana, L.) contain peroxidases catalyzing the oxidation of the xenobiotics N-nitrosodimethylamine (NDMA) and N-nitroso-N-methylaniline (NMA). Three anionic (A1, A2, A3) and four cationic (B, C, D, E) peroxidases were purified from this tissue, partially characterized and used for kinetic studies. Demethylation of NDMA and NMA producing formaldehyde is catalyzed by one anionic (A1) and three cationic (C, D, E) peroxidases. The oxidation of NDMA by tulip peroxidases exhibits the Michaelis-Menten kinetics. The apparent Michaelis constant and the maximal velocity values for this substrate were determined. On the other hand, non-Michaelian kinetics for the NMA oxidation were observed with tulip peroxidases. The most abundant cationic peroxidase (peroxidase C) was used for detailed enzymatic studies. In addition to formation of formaldehyde, methylaniline, aniline, 4-aminophenol and phenol were found to be metabolites formed from NMA. Phenol was formed presumably by N-demethylation via a benzenediazonium ion, while methylaniline, aniline and 4-aminophenol were products of denitrosation of the substrate. The efficiencies of plant peroxidases to oxidize NDMA and NMA in vitro are compared with those of cytochromes P450 and discussed.

scholarly journals Cellulase Activity as a Mechanism for Suppression of Phytophthora Root Rot in Mulches

2011 ◽  
Vol 101 (2) ◽  
pp. 223-230 ◽  
Author(s):  
Brantlee Spakes Richter ◽  
Kelly Ivors ◽  
Wei Shi ◽  
D. M. Benson
Keyword(s):  
Root Rot ◽  
Phytophthora Cinnamomi ◽  
Cellulase Activity ◽  
Enzyme Concentration ◽  
Disease Suppression ◽  
In Vitro Assays ◽  
Infested Soil ◽  
Phytophthora Root Rot ◽  
Standard Curve

Wood-based mulches are used in avocado production and are being tested on Fraser fir for reduction of Phytophthora root rot, caused by Phytophthora cinnamomi. Research with avocado has suggested a role of microbial cellulase enzymes in pathogen suppression through effects on the cellulosic cell walls of Phytophthora. This work was conducted to determine whether cellulase activity could account for disease suppression in mulch systems. A standard curve was developed to correlate cellulase activity in mulches with concentrations of a cellulase product. Based on this curve, cellulase activity in mulch samples was equivalent to a cellulase enzyme concentration of 25 U ml–1 or greater of product. Sustained exposure of P. cinnamomi to cellulase at 10 to 50 U ml–1 significantly reduced sporangia production, but biomass was only reduced with concentrations over 100 U ml–1. In a lupine bioassay, cellulase was applied to infested soil at 100 or 1,000 U ml–1 with three timings. Cellulase activity diminished by 47% between 1 and 15 days after application. Cellulase applied at 100 U ml–1 2 weeks before planting yielded activity of 20.08 μmol glucose equivalents per gram of soil water (GE g–1 aq) at planting, a level equivalent to mulch samples. Cellulase activity at planting ranged from 3.35 to 48.67 μmol GE g–1 aq, but no treatment significantly affected disease progress. Based on in vitro assays, cellulase activity in mulch was sufficient to impair sporangia production of P. cinnamomi, but not always sufficient to impact vegetative biomass.

Notizen: Effect of Magnetic Field on Ascorbic Acid Oxidase Activity, I

1986 ◽  
Vol 41 (3) ◽  
pp. 355-358 ◽  
Author(s):  
V. S. Ghole ◽  
P. S. Damle ◽  
W. H.-P. Thiemann
Keyword(s):  
Magnetic Field ◽  
Ascorbic Acid ◽  
Homogeneous Magnetic Field ◽  
Acid Oxidase ◽  
High Substrate Concentration ◽  
Ascorbic Acid Oxidase ◽  
Rate Of Reaction ◽  
Substrate Concentrations ◽  
Burk Plot

A homogeneous magnetic field of 1.1 T strength exhibits a significant influence on the activity of the enzyme ascorbic acid oxidase in vitro. A Lineweaver-Burk plot of the reaction shows the typical pattern of a mixed-type inhibition, i.e. a larger rate of reaction at low substrate concentrations and a smaller rate of reaction at high substrate concentration than that of the control without magnetic field applied.

scholarly journals The determination of binding parameters when the total and free substrate concentrations are not approximately equal

1979 ◽  
Vol 179 (3) ◽  
pp. 697-700 ◽  
Author(s):  
N Gains
Keyword(s):  
Substrate Concentration ◽  
Graphical Method ◽  
Enzyme Concentration ◽  
Binding Parameters ◽  
Free Concentration ◽  
Equilibrium Binding ◽  
Substrate Concentrations

By using a standard graphical method values of Km and V may be found that are independent of the conditions and assumptions that the total substrate concentration approximates to its free concentration and that Km is much larger than the enzyme concentration. The procedure is also applicable to the determination of equilibrium binding parameters of a ligand to a macromolecule.

The Influence of Carbohydrate Gelling Agents on Rat Intestinal Transport of Monosaccharides and Neutral Amino Acids in Vitro

1980 ◽  
Vol 59 (5) ◽  
pp. 373-380 ◽  
Author(s):  
B. Elsenhans ◽  
U. Süfke ◽  
R. Blume ◽  
W. F. Caspary
Keyword(s):  
Intestinal Absorption ◽  
Membrane Filtration ◽  
Intestinal Transport ◽  
Michaelis Constant ◽  
Experimental Conditions ◽  
Plant Polysaccharides ◽  
Gelling Agents ◽  
Layer Resistance ◽  
Filtration Technique

1. In the present investigation with rings of everted rat small intestine, carbohydrate gelling agents (plant polysaccharides) such as guaran, pectin, tragacanth, carubin and carrageenan were employed to study their direct effect on intestinal absorption of α-methyl-d-glucoside, d-galactose, l-leucine and l-phenylalanine. 2. Inhibition was found to correlate with the viscosity of the incubation medium, a function only of the polysaccharide concentration, and was independent of other properties of the carbohydrate gelling agents. 3. Reversal of this inhibition was achieved either by washing the tissue free of polysaccharide or by raising tissue agitation. 4. Uptake kinetics in polysaccharide-containing solutions revealed a marked increase of the apparent Michaelis constant although the maximal transport capacity remained essentially unaltered. 5. Since there was no binding of the substrate by the polysaccharides under experimental conditions as judged by a membrane filtration technique, it is concluded that carbohydrate gelling agents may impair intestinal absorption by means of an increased unstirred layer resistance.

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