Simple Michaelis-Menten Equation Research Articles

The Drug of Abuse γ-Hydroxybutyrate Is a Substrate for Sodium-Coupled Monocarboxylate Transporter (SMCT) 1 (SLC5A8): Characterization of SMCT-Mediated Uptake and Inhibition

Gamma-hydroxybutyric acid (GHB), a drug of abuse, is a substrate of monocarboxylate transporters (MCTs). Sodium-coupled monocarboxylate transporter 1 (SMCT1; SLC5A8) is expressed in kidney, thyroid gland, neurons, and intestinal tract and exhibits substrate specificity similar to that of the proton-dependent MCT (SLC16A) family. The role of SMCT1 in GHB disposition has not been determined. In this study we characterized the driving force, transport kinetics, and inhibitors of GHB uptake, as well as expression of SMCT and MCT isoforms, in rat thyroid follicular (FRTL-5) cells. GHB, as well as the monocarboxylates butyrate and d-lactate, exhibited sodium-dependent uptake at pH 7.4, which could be described with a simple Michaelis-Menten equation plus a diffusional component [K(m) 0.68 +/- 0.30 mM, V(max) 3.50 +/- 1.58 nmol . mg(-1) . min(-1), and diffusional clearance (P) 0.25 +/- 0.08 microl . mg(-1) . min(-1)]. In the absence of sodium, GHB uptake was significantly increased at lower pH, suggesting proton-gradient dependent transport. Reverse transcriptase-polymerase chain reaction and Western analyses demonstrated the expression of SMCT1, MCT1, and MCT2 in FRTL-5 cells, supporting the activity results. Sodium-dependent GHB uptake in FRTL-5 cells was inhibited by MCT substrates (d-lactate, l-lactate, pyruvate, and butyrate), nonsteroidal anti-inflammatory drugs (ibuprofen, ketoprofen, and naproxen), and probenecid. IC(50) values for l-lactate, ibuprofen, ketoprofen, and probenecid were 101, 31.6, 64.4, and 380 muM, respectively. All four inhibitors also significantly inhibited GHB uptake in rat MCT1 gene-transfected MDA/MB231 cells, suggesting they are not specific for SMCT1. Luteolin and alpha-cyano-4-hydroxycinnimate represent specific proton-dependent MCT inhibitors. Our findings indicate that GHB is a substrate for both sodium- and proton-dependent MCTs and identified specific inhibitors of MCTs.

Um método de investigação da assimilação heterotrófica do amônio e nitrato por organismos planctônicos

The kinetics of assimilation of ammonium and nitrate is investigated for various predetermined concentrations of water samples extracted from the upper subsurface of the littoral zone of a small reservoir (Lake IAG) in Sao Paulo, Brazil ( 23o39’ S and 46o37’ W). Five concentrations were predetermined for added ammonium: 300; 370; 440; 650; 1280 µg.N.L -1 and nitrate: 340; 590; 890; 1090; 1590 µg.N.L -1 . The samples were maintained under dark and oxygenated at a controlled temperature of 20 o C ± 1 o C, for the period of 9 days for ammonium and 11 days for nitrate. Once a day the concentrations of ammonium and nitrate in the flasks were analyzed colorimetric method. The highest uptake rate occurred at the initial concentrations of 300 µg.N.L -1 (k = 0,23 day -1 ) and 340 µg.N.L -1 (k = 0,25 day -1 ) for ammonium and nitrate respectively. These results indicate that the latter concentrations are close to the ideal conditions for assimilation of ammonium and nitrate by the microorganisms in the system. It is suggested therefore that these are the concentrations at which incubation processes should be started. In addition, concentrations above 500 µg.N.L -1 for ammonium and 600 µg.N.L -1 for nitrate are likely to cause inhibition of the assimilation process. The data do not follow a simple Michaelis-Menten equation, probably because some inhibition in the assimilation occurred.

Transport of γ-Hydroxybutyrate in Rat Kidney Membrane Vesicles: Role of Monocarboxylate Transporters

Intoxication with gamma-hydroxybutyrate (GHB) is associated with coma, seizure, and death; treatment of overdoses is symptomatic. Previous studies in our laboratory have demonstrated that L-lactate and pyruvate treatment can increase the renal clearance of GHB and increase its elimination in rats, suggesting that GHB may undergo renal reabsorption mediated by monocarboxylic acid transporters (MCTs). The goals of this study were to characterize the renal transport of GHB in rats and to determine the role of MCT in its renal transport. Brush-border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from rat kidney cortex, and the uptake of L-lactate and GHB was characterized. L-Lactate and GHB undergo both pH- and sodium-dependent transport in BBM vesicles and pH-dependent transport in BLM vesicles. A simple Michaelis-Menten equation best described the pH-dependent uptake of GHB in BBM (Km, 8.0 +/- 1.8 mM; Vmax, 838 +/- 45 pmol/mg/s) and in BLM vesicles (Km, 10.5 +/- 2.6 mM; Vmax, 806 +/- 253 pmol/mg/s). mRNA of MCT1 and MCT2 was determined in rat kidney cortex using reverse transcriptase-polymerase chain reaction; using Western blot, the protein expression of MCT1 was present mainly in BLM vesicles, with weak expression in BBM vesicles, whereas that of MCT2 was exclusively in BLM vesicles. Studies with rat MCT1 gene-transfected MDA-MB231 cells demonstrated that GHB was a substrate of MCT1. The data suggest that rat MCT1 may represent an important transporter for GHB in renal tubule cells. This investigation provides evidence for the importance of MCTs in the reabsorption of the monocarboxylic acids l-lactate and GHB in the kidney.

Precise and continuous observation of cellulase-catalyzed hydrolysis of cello-oligosaccharides using isothermal titration calorimetry

Heat is a characteristic of all chemical reactions, and isothermal titration calorimetry (ITC) provides a possible way to continuously detect the heat from catalytic reactions with high sensitivity and reproducibility. Cellulase, the enzyme of glycosyl hydrolase, catalyzes the cleavage of β-1,4 glycosidic bonds in cellulose. In this paper, ITC was applied to evaluate cellulase activity using cello-oligosaccharides as substrates. The hydrolysis heat of a single glycosidic bond of the substrate was successfully detected by combining ITC and normal-phase HPLC, and the time course of the enzymatic reaction was monitored continuously by ITC. The enzymatic parameters k cat and K M, obtained from calorimetric observables, clearly indicated that the reaction was well approximated by a simple Michaelis–Menten equation under the experimental conditions of this study. The normal-phase HPLC analysis was combined with the ITC approach to observe hydrolysis patterns and was found to be an effective and precise way to evaluate the activity of cellulase against cello-oligosaccharides.

Effect of non-ionic detergents on apparent enzyme mechanism: V121A mutant of Streptomyces cholesterol oxidase endowed with enhanced sensitivity towards detergents.

One of the mutants of Streptomyces cholesterol oxidase with the Val121Ala mutation (V121A) was kinetically analysed. Although the reaction rate-substrate concentration curve of wild type follows a simple Michaelis-Menten equation, that of V121A is sigmoidal. The cooperativity was apparent and caused by non-ionic detergents that were used as a solvent of cholesterol. The concentration dependence of V121A on detergents was more significant than that of wild type, although the reaction rates of both enzymes decrease as the concentrations of detergents increase. Further experiments suggested that less hydrophobic interactions between V121A and detergents should be responsible for the apparent cooperativity. Since Val121 is in a hydrophobic loop located near the active site, the mutational effect is structurally discussed.

A kinetic study on the bioremediation of sodium cyanide and acetonitrile by free and immobilized cells ofPseudomonas putida

Pseudomonas putida capable of utilizing organic nitrile (acetonitrile) and inorganic cyanide (sodium cyanide) as the sole source of carbon and nitrogen was isolated from contaminated industrial sites and waste water. The bacterium possesses nitrile aminohydrolase (EC 3.5.5.1) and amidase (EC 3.5.1.4), which are involved in the transformation of cyanides and nitriles into ammonia and CO2 through the formation of amide as an intermediate. Both of the enzymes have a high selectivity and affinity toward the−Cn group. The rate of degradation of aceotnitrile and sodium cyanide to ammonia and CO2 by the calcium-alginate immobilized cells ofP. putida was studied. The rate of reaction during the biodegradation of acetonitrile and sodium cyanide, and the substrate- and product-dependent kinetics of these toxic compounds were studied using free and immobilized cells ofP. putida and modeled using a simple Michaelis-Menten equation.

Kinetics of enzymatic degradation of cyanide

Cyanidase is a new enzyme preparation capable of degrading cyanide in industrial wastewaters to ammonia and formate in an apparently one-step reaction, down to very low concentrations. This enzyme has both a high selectivity and affinity toward cyanide. A granular form of the biocatalyst was used in a recirculation fixed bed reactor in order to characterize the new biocatalyst with respect to pH, ionic strength, common ions normally present in wastewaters, mass transfer effects, and temperature. Long term stability was investigated. The kinetics of the enzymatic degradation of cyanide were studied in a batch reactor using the powdered immobilized enzyme preparation and modeled using a simple Michaelis-Menten equation.

Kinetics of serotonin uptake in isolated rabbit lungs.

The single-pass, bolus-injection method was used to study the effect of serotonin (5-HT) concentration on the extraction of 5-HT by isolated perfused rabbit lungs. The extraction pattern suggested that an uptake model, which includes multiple parallel uptake processes, provided a better representation of the data than the simple Michaelis-Menten equation, which has commonly been used to fit the saturable uptake data in previous studies. In particular, the rabbit lung data could be fit with two such parallel pathways. Since the 5-HT uptake could virtually be completely blocked by imipramine, both pathways can be considered to be carrier-mediated processes. The high-affinity pathway was saturable within the range of concentrations studied, with a Km and Vmax of approximately 0.84 microM and 0.21 nmol.s-1.g wet lung wt-1. The Km for the low-affinity pathway was larger than concentrations for which accurate uptake measurements are practical in the perfused organ. Thus, for the low-affinity pathway, only Vmax/Km was identifiable. Vmax/Km values for the high- and low-affinity pathways were approximately 2.87 and 0.35 ml/s, respectively. The results suggest that it will be worthwhile to investigate the behavior of these uptake parameters in response to changes in lung physiology and endothelial function in future studies.

Phosphate Uptake by Microorganisms in Lake Water: Deviations from Simple Michaelis–Menten Kinetics

Orthophosphate (31Pi) uptake rates by natural Lake Michigan microbial assemblages were measured to test a hypothesis that the instantaneous velocity of31Piuptake at low added substrate concentrations is higher than predicted by the simple Michaelis–Menten equation. Analysis of data from most experiments verified this prediction:31Piturnover times (Tcalc) obtained by back-extrapolating from "low" substrate regions in Woolf plots ranged from 25% to nearly 3000% of those calculated from "high" substrate regions. Simulation analysis demonstrated that deviations in Tcalccould be at least an order of magnitude higher than previously predicted. Large (>1000%) discrepancies from the simple Michaelis–Menten equation could be caused by "skewed" or "clumped" distributions, where the range in both species half-saturation constants (Kt) and relative abundances is very wide and species with the lowest Ktvalues are most abundant. A comparison of Ktvalues for mixed microbial assemblages in Lake Michigan (0.16–19.4 μg P∙L−1) with those from laboratory culture studies (11–364 μg P∙L−1) demonstrates that natural microbial populations have adapted to P-limited environments by synthesizing uptake systems that have Ktvalues at least an order of magnitude below those detected in culture studies.