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Modified atmosphere storage containers with gas-permeable membranes are effective in reducing post-harvest losses, mainly at low temperatures but become ineffective in controlling O 2 and CO 2 at temperatures above 3 °C. This study aimed to develop a method to control O 2 in a storage box. This method uses a diffusion tube, that blocks air from entering the box but allows for air exchange when an air blower is activated. The blower ON frequency (BOF) was modelled as a function of temperature, considering the type and amount of produce, blower properties, tube dimension, and the O 2 setpoint. The effects of temperature and gas composition on respiration rate kinetics were analysed with Michaelis-Menten and Arrhenius equations. The model was used to program the microcontroller to control the blower. Validation was performed using a 190 L box containing 25 kg of broccoli. The developed model maintained the desired O 2 concentration under fluctuating temperatures. The BOF values ranged from 25.6 to 549.2 s h−1, for temperature fluctuations between 4 and 23 °C. The time required to reach the 3% O 2 in the box differed depending on the blower's state. With the blower turned ON from the beginning, it took 24.9 h. However, when the blower was initially turned OFF, it took 11.1 h. Despite temperature changes, the system maintained O 2 at 3.8 ± 0.29% and CO 2 at 14.4 ± 0.66%. The system is promising for commercial use and best suited for CO 2 tolerant produce because it requires a separate mathematical model and control mechanism. • Simple method to control gas concentrations under fluctuating temperature. • Real-time O 2 control in storage based on temperature sensor and predictive model. • Mathematical model guides real-time air-blower activation for maintaining O 2. • Suitable for CO 2 -tolerant produce or when combined with CO 2 removal tools. [ABSTRACT FROM AUTHOR]

Desiccation is a severe survival problem for organisms. We have been studying the desiccation tolerance mechanisms in the true slime mold Physarum polycephalum. We measured the trehalose content of P. polycephalum vegetative cells (plasmodia) and drought cells (sclerotia). Surprisingly, we found that the content in sclerotia was about 473-fold greater than in the plasmodia. We then examined trehalose metabolism-related genes via RNAseq, and consequently found that trehalose 6-phosphate phosphorylase (T6pp) expression levels increased following desiccation. Next, we cloned and expressed the genes for T6pp, trehalose 6-phosphate synthase/phosphatase (Tps/Tpp), maltooligosyltrehalose trehalohydrolase (TreZ), and maltooligosyltrehalose synthase (TreY) in E. coli. Incidentally, TreY and TreZ clones have been reported in several prokaryotes, but not in eukaryotes. This report in P. polycephalum is the first evidence of their presence in a eukaryote species. Recombinant T6pp, TreY, and TreZ were purified and confirmed to be active. Our results showed that these enzymes catalyze reactions related to trehalose production, and their reaction kinetics follow the Michaelis-Menten equation. The t6pp mRNA levels of the sclerotia were about 15-fold higher than in the plasmodia. In contrast, the expression levels of TreZ and TreY showed no significant change between the sclerotia and plasmodia. Thus, T6pp is probably related to desiccation tolerance, whereas the contribution of TreY and TreZ is insufficient to account for the considerable accumulation of trehalose in sclerotia. • Study for the desiccation tolerance mechanisms in a true slime mold Physarum polycephalum. • A large amount of trehalose was accumulated in the drought cells (sclerotia). • The genes (t6pp , tps/tpp , treZ , treY) related to trehalose metabolism were cloned. • T6pp is probably related to desiccation tolerance. • This is the first report of treY and treZ genes from eukaryotic species. [ABSTRACT FROM AUTHOR]

[Display omitted] • Bi 2 Fe 4 O 9 nanozyme as a simple and low-cost colorimetric sensor was developed for easy dopamine detection. • The sensor achieved good performance with a linear range from 0.15 to 50 μM and a detection limit of 51 nM. • The nanozyme was reused up to 4 cycles without a significant decrease in its performance. This work describes the preparation of ternary bismuth ferrite oxide nanoparticles (Bi 2 Fe 4 O 9 NPs) with an enzyme mimetic activity for dopamine (DA) qualitative and quantitative detection. Bi 2 Fe 4 O 9 NPs were prepared using a facile, low cost, and one-pot hydrothermal treatment. The chemical composition, morphology, and optical properties of Bi 2 Fe 4 O 9 nanozyme were characterized using different techniques such as Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction pattern (XRD), X-ray photoelectron spectroscopy (XPS), thermo-gravimetric analysis (TGA), dynamic light scattering (DLS), field-emission scanning electron microscopy (FESEM) imaging, FESEM-energy dispersive X-ray spectroscopy (EDS), UV–vis absorption, and fluorescence emission spectroscopy. Bi 2 Fe 4 O 9 NPs were utilized to catalyze the oxidation of a typical chromogenic peroxidase substrate, 3,3′,5,5′-tetramethylbenzidine (TMB), to form the blue-colored oxidized product (oxTMB), in the presence of hydrogen peroxide (H 2 O 2). All reactions occurred in acetate buffer solution (pH 3.5) to generate hydroxyl radicals (•OH) and the kinetics were followed by UV–vis absorbance at 654 nm. The steady-state kinetic parameters were obtained from the Michaelis-Menten equation and exhibited a good catalytic efficiency of Bi 2 Fe 4 O 9 NPs as enzyme mimetics. Michaelis–Menten constant (K m) values were estimated as 0.07 and 0.73 mM for TMB and H 2 O 2 , respectively. The presented method is efficient, rapid, cost-effective, and sensitive for the colorimetric detection of dopamine with a linear range (LR) from 0.15 to 50 μM and a detection limit (LOD) of 51 nM. The proposed colorimetric sensor was successfully applied for the detection of different concentrations of dopamine in spiked fetal bovine serum (FBS) and horse serum (HS) samples. It is anticipated that Bi 2 Fe 4 O 9 nanozyme holds great potential in biomedical analysis and diagnostic applications of dopamine-related diseases. [ABSTRACT FROM AUTHOR]

The general characteristics of the effect of surfactants on the activity of lysozyme were demonstrated. The kinetics of bacterial cell lysis is consistent with the Michaelis-Menten equation and the presence of surfactants does not shift the pH-optimum of activity. Surfactants do not change the Km value but instead, affect the Vmax value. The experimental dependencies are well described by theoretical equations, which assume three surfactant binding sites on the lysozyme molecule. The dependencies of the activity of lysozyme on the surfactant concentration are either a step type (i.e., a higher plateau becomes a lower plateau), or a dependency with a maximum and continuation of the curve in the form of a plateau but with an increase in the surfactant concentration. It can be assumed that there is a mechanism for the regulation of lysozyme activity by an unknown natural factor that has a suitable hydrophobic radical capable of binding to the surface of lysozyme. [Display omitted] • The dependencies of the activity of lysozyme on various surfactants have common features. • The experimental data are well described by the theoretical curve, assuming three ligand binding sites by lysozyme. • It is possible that the activity of lysozyme can be regulated by natural factors that have suitable hydrophobic radicals. [ABSTRACT FROM AUTHOR]

Partial inhibition occurs when an enzyme-inhibitor complex is capable of producing a product, as opposed to complete inhibition where the enzyme-inhibitor complex is completely inactive and cannot produce a product. While not frequently encountered, partial inhibition is also not a rare phenomenon and can potentially have significant repercussions later on in a research project. It is therefore important to recognize partial inhibition when it occurs. However, it cannot be recognized from the standard velocity versus substrate concentration plots or even from other primary plots such as Lineweaver-Burk plots. Fortunately, partial inhibition is easily identified by using replots which can be generated from the data already gathered to produce the primary plots. Partial inhibition phenomena are also not usually addressed in introductory textbooks and are often given only brief mention in some articles or books dealing with enzyme kinetics. The same types of approaches can be used to study enzyme activation phenomena, which can be considered the flip-side of inhibition. This review presents a graphical approach to identifying partial inhibition in an attempt to provide a comprehensive treatment for those wishing for or needing additional information in carrying out steady-state kinetic inhibition analyses. [ABSTRACT FROM AUTHOR]

Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on many different cellular proteins. Despite extensive biochemical and structural studies, focusing on PKMT active site-peptide interactions, little is known regarding how PKMTs recognize globular substrates. To examine whether these enzymes recognize protein substrates through interactions that take place outside of the active site, we have measured SETD6 and SETD7 activity with both protein and peptide RelA substrate. We have utilized the MTase-Glo™ methyltransferase assay to measure the activity of SETD6 and SETD7 with the different RelA substrates and calculated the Michaelis-Menten (MM) parameters. We found an up to ∼12-fold increase in K M of the PKMTs activity with RelA peptide relative to the respective full-length protein, emphasizing the significantly higher PKMT-protein interaction affinity. Examination of SETD6 and SETD7 activity toward the same RelA substrates highlight the similarity in substrate recognition for both PKMTs. Our results show that the interaction affinity of SETD6 and SETD7 with RelA is enhanced through interactions that occur outside of the active site leading to higher catalytic efficiency and specificity. These interactions can significantly vary depending on the PKMT and the specific methylation site on RelA. Overall, our results underline that PKMTs can recognize their substrates through docking interactions that occur out of the active site-peptide region for enhancing their activity and specificity in the cellular environment. [ABSTRACT FROM AUTHOR]

Bioluminescence (BL) imaging based on d -luciferin ( d -luc)–luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d -luc–luciferase reaction, an approach to increase intracellular levels of d -luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d -luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d -luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d -luc was higher than that in control cells, and the specific d -luc uptake mediated by OAT1 was saturable with a Michaelis constant (K m ) of 0.23 μM. The interaction between OAT1 and d -luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d -luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis–Menten kinetics with an apparent K m of 0.36 μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d -luc could be a rate-limiting step in the d -luc–luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d -luc. Our findings suggest that because OAT1 is capable of transporting d -luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells. [ABSTRACT FROM AUTHOR]

Microscopic randomness and the small volumes of living cells combine to generate random fluctuations in molecule concentrations called “noise”. Here I investigate the effect of noise on biochemical reactions obeying Michaelis–Menten kinetics, concluding that substrate noise causes these reactions to slow. I derive a general expression for the time evolution of the joint probability density of chemical species in arbitrarily connected networks of non-linear chemical reactions in small volumes. This equation is a generalization of the chemical master equation (CME), a common tool for investigating stochastic chemical kinetics, extended to reaction networks occurring in small volumes, such as living cells. I apply this equation to a generalized Michaelis-Menten reaction in an open system, deriving the following general result: 〈 p 〉 ≤ p ¯ and 〈 s 〉 ≥ s ¯ , where s ¯ and p ¯ denote the deterministic steady-state concentration of reactant and product species, respectively, and 〈 s 〉 and 〈 p 〉 denote the steady-state ensemble average over independent realizations of a stochastic reaction. Under biologically realistic conditions, namely when substrate is degraded or diluted by cell division, 〈 p 〉 ≤ p ¯ . Consequently, noise slows the rate of in vivo Michaelis–Menten reactions. These predictions are validated by extensive stochastic simulations using Gillespie's exact stochastic simulation algorithm. I specify the conditions under which these effects occur and when they vanish, therefore reconciling discrepancies among previous theoretical investigations of stochastic biochemical reactions. Stochastic slowdown of reaction flux caused by molecular noise in living cells may have functional consequences, which the present theory may be used to quantify. [ABSTRACT FROM AUTHOR]

We propose that the time course of an enzyme reaction following the Michaelis-Menten reaction mechanism can be conveniently described by a newly derived algebraic equation, which includes the Lambert Omega function. Following Northrop's ideas [ Anal. Biochem. 321 , 457–461, 1983], the integrated rate equation contains the Michaelis constant ( K M ) and the specificity number ( k S ≡ k cat / K M ) as adjustable parameters, but not the turnover number k cat . A modification of the usual global-fit approach involves a combinatorial treatment of nominal substrate concentrations being treated as fixed or alternately optimized model parameters. The newly proposed method is compared with the standard approach based on the “initial linear region” of the reaction progress curves, followed by nonlinear fit of initial rates to the hyperbolic Michaelis-Menten equation. A representative set of three chelation-enhanced fluorescence EGFR kinase substrates is used for experimental illustration. In one case, both data analysis methods (linear and nonlinear) produced identical results. However, in another test case, the standard method incorrectly reported a finite (50–70 μM) K M value, whereas the more rigorous global nonlinear fit shows that the K M is immeasurably high. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is an environmentally prevalent toxicant posing increasing risk to human health worldwide. As compared to the extensive research in Cd tissue accumulation, little was known about the elimination of Cd, particularly its toxic form, Cd ion (Cd 2 + ). In this study, we aimed to examine whether Cd 2 + is a substrate of multidrug and toxin extrusion proteins (MATEs) that are important in renal xenobiotic elimination. HEK-293 cells overexpressing the human MATE1 (HEK-hMATE1), human MATE2-K (HEK-hMATE2-K) and mouse Mate1 (HEK-mMate1) were used to study the cellular transport and toxicity of Cd 2 + . The cells overexpressing MATEs showed a 2–4 fold increase of Cd 2 + uptake that could be blocked by the MATE inhibitor cimetidine. A saturable transport profile was observed with the Michaelis-Menten constant ( K m ) of 130 ± 15.8 μM for HEK-hMATE1; 139 ± 21.3 μM for HEK-hMATE2-K; and 88.7 ± 13.5 μM for HEK-mMate1, respectively. Cd 2 + could inhibit the uptake of metformin, a substrate of MATE transporters, with the half maximal inhibitory concentration (IC 50 ) of 97.5 ± 6.0 μM, 20.2 ± 2.6 μM, and 49.9 ± 6.9 μM in HEK-hMATE1, HEK-hMATE2-K, and HEK-mMate1 cells, respectively. In addition, hMATE1 could transport preloaded Cd 2 + out of the HEK-hMATE1 cells, thus resulting in a significant decrease of Cd 2 + -induced cytotoxicity. The present study has provided the first evidence supporting that MATEs transport Cd 2 + and may function as cellular elimination machinery in Cd intoxication. [ABSTRACT FROM AUTHOR]

[Display omitted] • Investigation of A. elasticus lead to new dihydrobenzoxanthones inhibiting BNA. • Eight compounds, including two new, were characterized by spectroscopic methods. • Kinetic parameters determined by Michaelis-Menten equation double reciprocal plots. • Binding affinities between the BNA and compounds were measured by FS quenching. • The binding site was found by molecular docking experiments. Artocarpus elasticus is a popular fruit tree in the tropical regions. Primary screenings of methanol extracts of the root bark confirmed its potent inhibition of bacterial neuraminidase (BNA), which plays an essential role in the pathogenesis of many microbial diseases. Assessments of the responsible phytochemicals were conducted by isolating eight compounds (1 – 8) and two of them (6 and 8) were identified as new compounds. Among the isolates, the dihydrobenzoxanthones attained the highest BNA inhibition with IC 50 values of 0.5 ∼ 3.9 µM. Further investigation of the inhibitory mechanism by Lineweaver-Burk plots revealed the phytochemicals to function as reversible noncompetitive inhibitors. Fluorescence quenching showed their binding affinities were highly correlated with their inhibitory potential dose-dependently. Molecular docking experiments suggested the dihydrobenzoxanthones (4 and 6) as noncompetitive inhibitors of BNA with unique interaction with Tyr435 of BNA in comparison with the mother flavonoid (7). [ABSTRACT FROM AUTHOR]

In this article, a new approach—namely, the extended Parker–Sochacki method (EPSM)—is presented for solving the Michaelis–Menten nonlinear enzymatic reaction model. The Parker–Sochacki method (PSM) is combined with a new resummation method called the Sumudu–Padé resummation method to obtain approximate analytical solutions for the model. The obtained solutions by the proposed approach are compared with the solutions of PSM and the Runge–Kutta numerical method (RKM). The comparison proves the practicality, efficiency, and correctness of the presented approach. It serves as a basis for solving other nonlinear biochemical reaction models in the future. [ABSTRACT FROM AUTHOR]

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. [ABSTRACT FROM AUTHOR]

We quantify the strength of miRNA–target interactions with MIRZA, a recently introduced biophysical model. We show that computationally predicted energies of interaction correlate strongly with the energies of interaction estimated from biochemical measurements of Michaelis–Menten constants. We further show that the accuracy of the MIRZA model can be improved taking into account recently emerged experimental data types. In particular, we use chimeric miRNA–mRNA sequences to infer a MIRZA–CHIMERA model and we provide a framework for inferring a similar model from measurements of rate constants of miRNA–mRNA interaction in the context of Argonaute proteins. Finally, based on a simple model of miRNA-based regulation, we discuss the importance of interaction energy and its variability between targets for the modulation of miRNA target expression in vivo . [ABSTRACT FROM AUTHOR]

Determination of individual rate constants for enzyme-catalyzed reactions is central to the understanding of their mechanism of action and is commonly obtained by stopped-flow kinetic experiments. However, most natural substrates either do not fluoresce/absorb or lack a significant change in their spectra while reacting and, therefore, are frequently chemically modified to render adequate molecules for their spectroscopic detection. Here, isothermal titration calorimetry (ITC) was used to obtain Michaelis–Menten plots for the trypsin-catalyzed hydrolysis of several substrates at different temperatures (278–318 K): four spectrophotometrically blind lysine and arginine N-free esters, one N-substituted arginine ester, and one amide. A global fitting of these data provided the individual rate constants and activation energies for the acylation and deacylation reactions, and the ratio of the formation and dissociation rates of the enzyme–substrate complex, leading also to the corresponding free energies of activation. The results indicate that for lysine and arginine N-free esters deacylation is the rate-limiting step, but for the N-substituted ester and the amide acylation is the slowest step. It is shown that ITC is able to produce quality kinetic data and is particularly well suited for those enzymatic reactions that cannot be measured by absorption or fluorescence spectroscopy. [ABSTRACT FROM AUTHOR]

Signaling cascades proliferate signals received on the cell membrane to the nucleus. While noise filtering, ultra-sensitive switches, and signal amplification have all been shown to be features of such signaling cascades, it is not understood why cascades typically show three or four layers. Using singular perturbation theory, Michaelis–Menten type equations are derived for open enzymatic systems. Cascading these equations we demonstrate that the output signal as a function of time becomes sigmoidal with the addition of more layers. Furthermore, it is shown that the activation time will speed up to a point, after which more layers become superfluous. It is shown that three layers create a reliable sigmoidal response progress curve from a wide variety of time-dependent signaling inputs arriving at the cell membrane, suggesting the evolutionary benefit of the observed cascades. [ABSTRACT FROM AUTHOR]

Bone is a hard-soft biomaterial built through a self-assembly process under genetic regulatory network (GRN) monitoring. This paper aims to capture the behavior of the bone GRN part that controls mineralization by using a mathematical model. Here, we provide an advanced review of empirical evidence about interactions between gene coding (i) transcription factors and (ii) bone proteins. These interactions are modeled with nonlinear differential equations using Michaelis-Menten and Hill functions. Compared to empirical evidence - coming from osteoblasts culture -, the two best systems (among 12 6 = 2 , 985 , 984 possibilities) use factors of inhibition from the start of the activation of each gene. It reveals negative indirect interactions coming from either negative feedback loops or the recently depicted micro-RNAs. The difference between the two systems also lies in the BSP equation and two ways for activating and reducing its production. Thus, it highlights the critical role of BSP in the bone GRN that acts on bone mineralization. Our study provides the first theoretical evidence of osteoblast self-inhibition after activation of the genetic regulatory network controlling mineralization with this work. [ABSTRACT FROM AUTHOR]

Abstract: Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-l-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis–Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65±0.02 and 0.72±0.03U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP’s role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome. [Copyright &y& Elsevier]

Abstract: The O-acetylation of peptidoglycan occurs at the C-6 hydroxyl group of muramoyl residues in many human pathogens, both gram positive and gram negative, such as Staphylococcus aureus and species of Campylobacter, Helicobacter, Neisseria, and Bacillus, including Bacillus anthracis. The process is a maturation event being catalyzed either by integral membrane O-acetylpeptidoglycan transferase (Oat) of gram-positive bacteria or by a two-component peptidoglycan O-acetyltransferase system (PatA/PatB) in gram-negative cells. Here, we describe the development of the first in vitro assay for any peptidoglycan O-acetyltransferase using PatB from Neisseria gonorrhoeae as the model enzyme. This assay is based on the use of chromogenic p-nitrophenyl acetate as the donor substrate and chitooligosaccharides as model acceptor substrates in place of peptidoglycan. The identity of the O-acetylated chitooligosaccharides was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rates of transacetylations were determined spectrophotometrically by monitoring p-nitrophenol release after accounting for both spontaneous and enzyme-catalyzed hydrolysis of the acetate donor. Conditions were established for use of the assay in microtiter plate format, and its applicability was demonstrated by determining the first Michaelis–Menten kinetic parameters for PatB. The assay is readily amenable for application in the high-throughput screening for potential inhibitors of peptidoglycan O-acetyltransferases that may prove to be leads for novel classes of antibiotics. [Copyright &y& Elsevier]

Abstract: Nitrogen (N) is the most important, essential nutrient for all living organisms on earth; it is present in a number of complex organic molecules and plays extremely important roles in their activities. Ammonium N (NH4 +–N) and nitrate N (NO3 −–N) are the main forms taken up by plants in addition to some organic N compounds. More than 90% soil N is in organic form. The intermediate products of complicated organic N substances can be absorbed by plants. Organic N nutrition affects plant product quality and plant metabolism. Organic N passes through the cell wall and arrives at the plasma membrane through the apoplast and cytoplast systems and, in addition to endocytosis, may get transported across the plasma membrane by an active (sugar/proton cotransport) or passive process. After uptake by plants, simple organic N compounds such as amino acids can be rapidly assimilated and transformed into other amino acids by transamination and deamination. The uptake of NH4 +–N and NO3 −–N can be described by the Michaelis–Menten equation, and two parameters, the maximum absorption velocity (Vmax) and affinity constant or Michaelis constant (Km), have been used to measure the ability and efficiency of roots absorbing the two ions of crop plants. The uptake amounts of both NH4 +–N and NO3 −–N at the seedling stage are well in agreement with their absorption kinetic parameters, particularly at low concentrations, but are not fully in agreement with the entire growing periods of crops. In addition to root interception, NH4 + and NO3 − can move from bulk soil to the root surface by mass flow and diffusion. Diffusion is more important to NH4 +–N than NO3 −–N, while NO3 −–N movement mainly depends on mass flow. Roots are the major organs for the uptake of NH4 + and NO3 − ions. On arriving at the root surface, the two N forms can passively enter the root epidermis cell wall through the symplast and apoplast and then radically and vertically move across the cortex where the two ions in the apoplast enter the cortex symplast for passing through the endodermal Casparian trip to the endodermis. From the endodermis, the two ions go to the stele and empty into the xylem, or flow to the apoplast or get stored in vacuoles in addition to reduction or direct assimilation. The movement occurs from cell to cell. On emptying into the xylem, NH4 + and NO3 − are transported to the shoots via the transpiration stream. NO3 −–N is the dominant form of the mineral N with high concentrations in soil solutions, and is usually taken up in great amounts by crops and is readily mobile in the xylem. Transport of NO3 − across the plasma membrane along the electrochemical gradient is thought to be by H+/NO3 − cotransport, or by transport proteins or carriers or by specific ion channels. NH4 + is in equilibrium with NH3, and in most soils, the pH is considerably low and NH3 concentrations are usually very low. NH4 + uptake through the plasma membrane has been assumed to occur in three ways: either active or passive, or both. Passive uptake may occur at the initial stage of uptake, while at the second stage, active uptake may be predominating. For passive uptake, NH4 + ions passing through the membrane are thought to be present in either NH4 + or NH3 form, and in this way may be related to the facilitated diffusion through channels. The NH4 + ion resembles the K+ ion in terms of the ionic radius and size of the hydration shell, and therefore, it may be able to permeate the plasma membrane through K+ channels. The active uptake includes the H+/NH4 + cotransport, and specific transporters. Nitrate N cannot be directly used by plants until it is reduced to ammonia. The reduction is catalyzed by enzymes in two steps: the first step takes place in the cytoplasm by nitrate reductase (NR) transforming NO3 − into nitrous acid (HNO2), and the second occurs in chloroplasts (shoots) or proplastids (roots) by nitrite reductase (NiR) converting HNO2 to NH3. The NO3 − reduction to NO2 − is the rate-limiting step for NO3 − assimilation. Both roots and shoots are capable of reducing NO3 −. The uptake and reduction of NO3 − and the reductive ratios in roots and shoots depend on plant species, carbohydrates in plants, and nitrate reductase activity (NRA) as well as environmental conditions such as NO3 − concentration, medium pH, complementary ions, light, and ambient CO2 concentration. Due mainly to NO3 − uptake exceeding its assimilation by plants, a large amount of NO3 − is accumulated in vacuoles of roots and shoots at vegetative stages. With the exception of vegetables, the storage of NO3 − has no harmful effects on plant product quality and benefits the supply of N nutrient for later growing stages of plants. Ammonia is a central intermediate in plant N metabolism. NH3 is assimilated by plants by the mediation of glutamine synthetase–glutamine (z-) oxoglutarate aminotransferase enzyme systems in two steps: the first step requires adenosine triphosphate to add NH3 to glutamate to form glutamine (Gln), and the second step transfers the NH3 from Gln to α-ketoglutarate to form two glutamates. Once NH3 has been incorporated into glutamate, it can be transferred to other carbon skeletons by various transminases to form additional amino acids. The glutamate and Gln formed can rapidly be used for the synthesis of low-molecular-weight organic N compounds (LMWONCs) (such as other amides, amino acids, ureides, amines, and peptides) that will be further synthesized into high-molecular-weight organic N compounds (HMWONCs) such as proteins and nucleic acids. NH4 +–N and NO3 −–N may exhibit different effects on plant nutrition, growth, and crop production of different plant species. The preferences are the demand nature of plant species together with environmental conditions. The preferences have been evaluated mainly by plant biomass, yield production, or N uptake amount by the application of NH4 +–N or NO3 −–N alone. Based on current results, crop plants may be classified into four types: preference to NH4 +–N; preferences to NO3 −–N; equal effect of NH4 +–N and NO3 −–N; combinative use of the two N sources being superior to either NH4 + or NO3 − alone. Based on the fact that when NH4 +–N and NO3 −–N coexist in media, crops have a higher dry matter production, N uptake, and N utilization efficiency compared to NH4 + or NO3 − alone, the concepts, enhancement of NH4 +–N nutrition to the majority of crops and enhancement of NO3 −–N nutrition to rice, have been proposed. Both measures have significantly improved plant physiological properties, N use efficiency, and crop yields. Plant preferences to NH4 +–N and NO3 −–N are determined by many plant internal factors such as plant species, cultivars, and growing stages. For example, because of a lack of nitrate reductive ability, some plant species cannot efficiently use NO3 − as their major N source. The preferences are also determined by the chemical properties of the two ions, NH4 + and NO3 −, which affect N loss and N efficiency in plants, the uptake effects on absorption of other anions and cations, the balance between cations and anions in plants by uptake of NO3 − or NH4 + alone, and the influence of Fe and Mn uptake or their bioavailability as well as plant tolerances to Mn and Al toxicities. External or environmental factors, such as media pH, cations and anions, NH4 + concentration in solution, temperature, illumination, and aeration, also have great effects on their preferences. The toxicity produced by NH4 +/NH3 nutrition is an important reason for plant preferences for the two N forms. By using NH4 +–N as the sole N source, the toxicity is often characterized by an immediate restriction in plant growth, stem lesions, leaf area decline, total biomass reduction, and finally plant death. Influence of root growth and production of short, thick, less branched and darkly colored roots is the particular feature of the toxicity. The toxicity to plants is the combinative result of internal and external factors. Different species may have different tolerances to NH4 + or NH3, and thus toxicity, can appear to be different among species and even among cultivars in the same species. Carbohydrate amounts in plants substantially affect toxicity. Strong acidification of rhizosphere soil is an important environmental factor and medium acidification associated with NH4 + absorption has been shown to be toxic to many crop plants. The degree of the toxicity is associated with crops’ sensitivity to pH. Depending on the plant species and particular growth conditions, each of these factors may contribute to toxicity. Two hypotheses have been put forward to explain the physiological source or the cause of the toxicity. Some scientists consider that toxicity is due mainly to free ammonia (NH3) that affects plant growth and metabolism at low concentration levels at which NH4 + is found to be not harmful. The NH3 molecules can directly penetrate the membrane and enter cells through diffusion resulting in an increase in cytoplasmic pH that inhibits Glu synthase activity as the primary cause. However, based on the fact that under conditions without the possibility to produce NH3, the toxicity effect still exists, and it is often enhanced by water stress, NH4 + toxicity has been proposed. Injury of membrane and cell wall structure as well as changes of membrane enzyme activities and metabolism have been regarded as the primary causes of NH4 + toxicity, and the retardation of nutrient absorption, decrease of the uptake of essential cations and plant growth, disorders of NH4 +-induced pH changes, excessive consumption of sugars as the secondary causes of the NH4 + toxicity. Crops growing in solution cultures with equal amounts of NH4 +–N and NO3 −–N in their entire life cycle show that they did not absorb equal amounts of each N form, varying with plant species and growing stages of each crop and solution pH. The media pH affects the uptake ratio of NH4 +–N to NO3 −–N of wheat, and the unequal uptake of the two N forms results in significant changes in the pH in the solution. The amounts and ratios of NH4 +–N to NO3 −–N taken up by rice vary with growing stages. Application of NH4 +–N at early stages and alternatively applying NH4 +–N and NO3 −–N at late stages may be a way for the promotion of rice growth and increasing its yield. NH4 +–N and NO3 −–N forms affect some morphological and physiological characteristics of crop plants. Supply of NO3 −–N can increase lateral root growth, but supply of NH4 +–N inhibits root growth and produces abnormal growth of lateral roots. In coexistence of NH4 +–N and NO3 −–N, chlorophyll contents and the net photosynthetic rate are the highest, followed by NO3 −–N, while NH4 +–N is the lowest. NH4 +–N, NO3 −–N, and their mixed N source exerted a greater effect on mesophyll conductance: the highest occurs for the mixed N source, followed by NO3 −–N and the smallest for NH4 +–N. Some results show that on supplying NH4 +–N, the total N, free amino acids, and amides are higher than NO3 −–N, but others show that the two N forms have no great influence on crop N accumulation and N-containing components. Application of NO3 −–N benefits the accumulation of sucrose and increases plant organic acids, total soluble sugar amounts, reductive sugar accumulation, structured polysaccharides contents (cellulose) in roots, stems and leaves of maize seedlings, while NH4 +–N benefits starch accumulation and consumes large amounts of organic acids and carbohydrates and reduces sugar content. The two N forms also affect the mineral ion uptake and nutrient accumulation in plants. The RN amount and activity are closely linked with NO3 −–N. To a certain extent, the NRA is positively correlated to the NO3 −–N accumulation in plants. NH4 +–N as the sole N source inhibits NRA. Nitrate increases or maintains, whereas NH4 +–N decreases the leaf water potential and turgor pressure. Different N forms may produce different effects on the reactive (free) oxygen species (ROS) levels, antioxidant enzyme, and protective enzyme activities. When supplied with NH4 +–N and NO3 −–N together, the activities of the superoxide dismutase (SOD) and catalase (CAT) are the highest in leaves. Nitrate addition can promote the survival and restoration of plants damaged by waterlogging; and can produce NO, a suitable amount of which increases SOD and CAT activities under a salt stress, decreases the forming-velocity and cumulative amounts of H2O2 and O2 − as well as malondialdehyde, and increases antioxidant substances. The two N forms may affect the formation and activity of hormones and thereby may inhibit or promote cell elongation and plant growth. Compared to NH4 +, application of NO3 − significantly and rapidly promotes the synthesis and transport of root-originated hormones, such as cytokinin (CTK) and auxin, especially indole-3-acetic acid (IAA), and increases their activities. The IAA content of tobacco seedlings grown in 100% NO3 −–N solution was higher than those grown in 50% NH4 +–N+50% NO3 −–N solution, and the auxin concentration in the aboveground part of plants cultured by NO3 − was higher than that for NH4 +. [Copyright &y& Elsevier]

Abstract: Unlimited ultrasensitivity in a kinase/phosphatase “futile cycle” has been a paradigmatic example of collective behaviour in multi-enzyme systems. However, its analysis has relied on the Michaelis–Menten reaction mechanism, which remains widely used despite a century of new knowledge. Modifying and demodifying enzymes accomplish different biochemical tasks; the donor that contributes the modifying group is often ignored without the impact of this time-scale separation being taken into account; and new forms of reversible modification are now known. We exploit new algebraic methods of steady-state analysis to reconcile the analysis of multi-enzyme systems with single-enzyme biochemistry using zero-order ultrasensitivity as an example. We identify the property of “strong irreversibility”, in which product re-binding is disallowed. We show that unlimited ultrasensitivity is preserved for a class of complex, strongly irreversible reaction mechanisms and determine the corresponding saturation conditions. We show further that unlimited ultrasensitivity arises from a singularity in a novel “invariant” that summarises the algebraic relationship between modified and unmodified substrate. We find that this singularity also underlies knife-edge behaviour in allocation of substrate between modification states, which has implications for the coherence of futile cycles within an integrated tissue. When the enzymes are irreversible, but not strongly so, the singularity disappears in the form found here and unlimited ultrasensitivity may no longer be preserved. The methods introduced here are widely applicable to other reversible modification systems. [Copyright &y& Elsevier]

Abstract: The selective oxidation of d-glucose to d-gluconic acid was performed at atmospheric pressure, controlled pH value, and constant oxygen concentration, in the temperature range from 273.2 to 303.2 K, using Hyderase (a commercial glucose oxidase and catalase preparation). Measurements of initial rate as a function of initial glucose concentration were interpreted by a simplified version of the already proposed reaction mechanism, having the form of a simple equation of the Michaelis–Menten type, with two kinetic parameters, i.e., referring to the reaction between glucose and oxidized enzyme, and referring to a combination of the other steps. The activation energy for was found to be ; an apparent activation energy of was obtained for . [Copyright &y& Elsevier]

A straightforward and empirical regression method based on a logarithmic approximation has been developed to accurately estimate initial rates from nonlinear progress curves of enzyme reactions. The principle of this parametric approach is to use a relatively large number of observations, while averaging out random errors, to predict the curvature at time zero, which has the highest rate of change. The usual linear regression of a few initial time points lacks prediction power at time zero and therefore underestimates the true initial rate. Application of this nonlinear regression approach to enzyme reactions demonstrated satisfactory results. This approach is less subjective in choosing initial time points to be used for rate determination, and much more robust to random errors. Moreover, it is relatively easy to realize with commonly available software. [Copyright &y& Elsevier]

Snake venom prothrombin activators such as Ecarin are readily assayed by continuous spectrophotometric monitoring of p -nitroaniline production in a one step assay containing prothrombin and a p -nitroanilide peptide substrate for thrombin. The coupled reactions result in accelerating p -nitroaniline (pNA) production over the course of the assay giving non-linear progress curves, from which initial velocities are not readily obtained. Most studies therefore resort to approximate estimates of activity, based on the absorbance reached at an arbitrary time. A simple kinetic analysis of the coupled reactions shows that the early points of such curves should be fitted by second order polynomials, representing the accelerating reaction rate in μmol pNA/min/min. The first derivative of the polynomial then gives the increasing velocity of pNA production in μmol pNA/min over the time course of the assay. We demonstrate here that, with the substrate S2238, these rates can be converted to absolute thrombin concentrations using the Michaelis-Menten equation, substituted with values for kcat and Km. These thrombin concentrations increase linearly over the time course of the assay allowing the activity to be expressed in units, defined as μmol product/min, most commonly used to report enzyme activity. Image 1 • Readily implemented Spectrophotometric Microtitre Plate assay for prothrombin activators. • Continuous monitoring of pNA production by thrombin produced in a coupled reaction assay containing prothrombin and S2238. • Utilizes non-linear Progress Curve Analysis based on a fitted equation derived from the coupled reaction model. • Expresses prothrombin activator activity in μmol of thrombin produced per minute. [ABSTRACT FROM AUTHOR]

Nutrient pollution and algal blooms in coastal waters have long been a major concern, and understanding the response of algae to nutrient dynamics is thus essential. The minimum concentration under which Ulva prolifera will not grow, estimated from a nutrient-alga coupling model developed in this study, was 6.5 μmol L−1 for nitrate and 0.27 μmol L−1 for phosphate. The model was parameterized with cultural experiments to examine the effects of nitrate and phosphate on the growth of Ulva prolifera and the development of green tide. The cultural experiments showed that flux of nitrate absorbed by Ulva prolifera increased significantly with the concentration of nitrate or phosphate, which followed the Michaelis-Menten equation, while the flux of phosphate linearly increased with its concentrations. Nitrate concentrations at >25 μmol L−1 had little influence on the absorption of phosphate, but compensating absorption of phosphate occurred when the nitrate concentration was below 15 μmol L−1. Phosphate concentrations had a remarkable enhancement to the absorption of nitrate when phosphate concentration was <1.4 μmol L−1. The absorption rates of nitrate and phosphate by Ulva prolifera followed the same variation trend as nutrient absorption fluxes. The increase in nitrate absorption rates has a limited impact on the relative growth rate of Ulva prolifera , while the increase in phosphate absorption rates has a marked impact on the relative growth rate. Our results provide new insights into the growth mechanism of Ulva prolifera and the development and evolution of green tides in coastal marine environments. Applications of our model should help governmental agencies in environmental remediation and policy making. [ABSTRACT FROM AUTHOR]