Changes In Enzyme Concentration Research Articles

Changes in enzyme concentrations in mixed saliva during clinical manifestations of oral galvanosis

This article reviews the literature on the problem of the relationship between enzymes and changes in their concentrations in mixed saliva during clinical manifestations of galvanosis, as well as ways to determine their activity.

Effect of Pre-Sowing Magnetic Field Treatment on Enzymes and Phytohormones in Pea (Pisum sativum L.) Seeds and Seedlings

The aim of the presented studies was to evaluate the magnetic field (MF) effect on changes in some enzymes and phytohormones that takes place in the process of seed germination and growth of seedlings. Studies were led in the climatic chambers HERAEUS, on Petri dishes during six consecutive days. Pea seeds were divided into three groups from which one was the control (without stimulation) and two were treated with different doses of magnetic field (30 and 85 mT, respectively). Contents of amylolytic enzymes (AE) and phytohormones were determined at seven terms (0; 24; 48; 72; 96; 120 and 144 h) after placing them on the dishes. A favorable effect of seed stimulation with MF was found on the biochemical processes in the germinating seeds and pea seedlings. The size of changes in enzyme concentration was dependent mainly on the advancement of germination process (i.e., on length of time in which the seeds were on the dishes) and the MF treatment. The use of MF also had an effect on the increase in hormone content in the seeds and organs of seedlings, but values from objects treated with different doses did not differ significantly.

Complexity of a peroxidase-oxidase reaction model.

The peroxidase-oxidase oscillating reaction was the first (bio)chemical reaction to show chaotic behaviour. The reaction is rich in bifurcation scenarios, from period-doubling to peak-adding mixed mode oscillations. Here, we study a state-of-the-art model of the peroxidase-oxidase reaction. Using the model, we report systematic numerical experiments exploring the impact of changing the enzyme concentration on the dynamics of the reaction. Specifically, we report high-resolution phase diagrams predicting and describing how the reaction unfolds over a quite extended range of enzyme concentrations. Surprisingly, such diagrams reveal that the enzyme concentration has a huge impact on the reaction evolution. The highly intricate dynamical behaviours predicted here are difficult to establish theoretically due to the total absence of an adequate framework to solve nonlinearly coupled differential equations. But such behaviours may be validated experimentally.

Genetic Diversity in the Environmental Conditioning of <i>Gossypium hirsutum</i> and <i>Gossypium barbadense</i> Cultivars

Enzyme adaptations to temperature occur constantly as temperature patterns modulate diurnally and seasonally. These adaptations entail qualitative and/or quantitative metabolic changes that often provide a competitive advantage, impact adjustment to new environments, and effect the survival of the species. Changes in isozymes or allozymes, changes in enzyme concentration, modification by substrate and effectors, and metabolic regulation of enzyme function without changing enzyme composition are all possible strategies for adaptation to changes in temperature. The degree of adaptation among cotton cultivars to a specific thermal regime may be difficult to determine from phenotypic responses of the plants. The present study evaluated the thermal sensitivity of Gossypium hirsutum L. and Gossypium barbadense L. cultivars following growth under distinct thermal environments. The metabolic fitness of Gossypium hirsutum L. and Gossypium barbadense L. cultivars showed that the Gossypium hirsutum L. cultivars grown in a 28°C/20°C day/night cycle tended to be better equipped to cope with a 16 h - 38°C treatment than the same cultivars grown in a 38°C/32°C day/night cycle. The Gossypium barbadense L. cultivars, on the other hand, grown in a 38°C/32°C day/night cycle tended to be equipped to cope with a 16 h - 38°C treatment than the same cultivars grown in a 28°C/20°C day/night cycle. The Gossypium hirsutum L. line TX 303 is an exception to these general trends as its responses were similar to the Gossypium barbadense L. St. Vincent and Pima S-7 cottons.

Absolute Quantitative Profiling of the Key Metabolic Pathways in Slow and Fast Skeletal Muscle

Slow and fast skeletal muscles are composed of, respectively, mainly oxidative and glycolytic muscle fibers, which are the basic cellular motor units of the motility apparatus. They largely differ in excitability, contraction mechanism, and metabolism. Because of their pivotal role in body motion and homeostasis, the skeletal muscles have been extensively studied using biochemical and molecular biology approaches. Here we describe a simple analytical and computational approach to estimate titers of enzymes of basic metabolic pathways and proteins of the contractile machinery in the skeletal muscles. Proteomic analysis of mouse slow and fast muscles allowed estimation of the titers of enzymes involved in the carbohydrate, lipid, and energy metabolism. Notably, we observed that differences observed between the two muscle types occur simultaneously for all proteins involved in a specific process such as glycolysis, free fatty acid catabolism, Krebs cycle, or oxidative phosphorylation. These differences are in a good agreement with the well-established biochemical picture of the muscle types. We show a correlation between maximal activity and the enzyme titer, suggesting that change in enzyme concentration is a good proxy for its catalytic potential in vivo. As a consequence, proteomic profiling of enzyme titers can be used to monitor metabolic changes in cells. Additionally, quantitative data of structural proteins allowed studying muscle type specific cell architecture and its remodeling. The presented proteomic approach can be applied to study metabolism in any other tissue or cell line.

Genetic Dominance & Cellular Processes

In learning genetics, many students misunderstand and misinterpret what “dominance” means. Understanding is easier if students realize that dominance is not a mechanism, but rather a consequence of underlying cellular processes. For example, metabolic pathways are often little affected by changes in enzyme concentration. This means that enzyme-producing alleles usually show complete dominance. For genes producing nonenzymatic proteins such as collagen or hemoglobin, the amount of product matters, and dominance relationships are more complicated. Furthermore, with hemoglobin, dominance can change depending on what aspect of the phenotype is being studied and on the environmental conditions. X-linked genes are a special case, whether enzymatic or not. Because of X-chromosome inactivation, only one X-linked allele can be active in a cell, which means that the concept of dominance cannot be applied at the cellular level. Instead, a type of dominance is demonstrated at the individual level; but even so, dominant traits may fail to be expressed, and recessive traits can be expressed. Teaching not only what is happening but why it's happening will give students a deeper understanding, not only of dominance relationships, but of the underlying cellular processes as well.

Engineering and adaptive evolution of Escherichia coli for d-lactate fermentation reveals GatC as a xylose transporter

Despite the abundance of xylose in nature, the production of chemicals from C5 sugars remains challenging in metabolic engineering. By deleting xylFGH genes and using adaptive evolution, an efficient E. coli strain capable of producing d-lactate from xylose was engineered. Quantitative proteomics and genome sequencing were used to understand the new phenotype and the metabolic limitations of xylose conversion to d-lactate. Proteomics identified major changes in enzyme concentration in the glycolytic and tricarboxylic acid pathways. Whole genome sequencing of the evolved strain identified a point mutation in the gatC gene, which resulted in a change from serine to leucine at position 184 of the GatC protein. The knockout of gatC in a number of strains and the insertion of the mutation in the non-evolved strain confirmed its activity as a xylose transporter and demonstrated that the mutation is responsible for the high xylose consumption phenotype in the evolved strain. The newly found xylose transporter is a candidate for future strain engineering for converting C5–C6 syrups into valuable chemicals.

Exploring the effect of variable enzyme concentrations in a kinetic model of yeast glycolysis.

Metabolism is one of the best studied fields of biochemistry, but its regulation involves processes on many different levels, some of which are still not understood well enough to allow for quantitative modeling and prediction. Glycolysis in yeast is a good example: although high-quality quantitative data are available, well-established mathematical models typically only cover direct regulation of the involved enzymes by metabolite binding. The effect of various metabolites on the enzyme kinetics is summarized in carefully developed mathematical formulae. However, this approach implicitly assumes that the enzyme concentrations themselves are constant, thus neglecting other regulatory levels--e.g. transcriptional and translational regulation--involved in the regulation of enzyme activities. It is believed, however, that different experimental conditions result in different enzyme activities regulated by the above mechanisms. Detailed modeling of all regulatory levels is still out of reach since some of the necessary data--e.g. quantitative large scale enzyme concentration data sets--are lacking or rare. Nevertheless, a viable approach is to include the regulation of enzyme concentrations into an established model and to investigate whether this improves the predictive capabilities. Proteome data are usually hard to obtain, but levels of mRNA transcripts may be used instead as clues for changes in enzyme concentrations. Here we investigate whether including mRNA data into an established model of yeast glycolysis allows to predict the steady state metabolic concentrations for different experimental conditions. To this end, we modified an established ODE model for the glycolytic pathway of yeast to include changes of enzyme concentrations. Presumable changes were inferred from mRNA transcript level measurement data. We investigate how this approach can be used to predict metabolite concentrations for steady-state yeast cultures at five different oxygen levels ranging from anaerobic to fully aerobic conditions. We were partly able to reproduce the experimental data and present a number of changes that were necessary to improve the modeling result.

Sex Differences in the Rapid Control of Aromatase Activity in the Quail Preoptic Area

Adult male quail show high levels of aromatase activity in the preoptic area-hypothalamus (POA-HYP), which parallels the high number of aromatase-immunoreactive cells and elevated mRNA concentrations detected in this brain region by in situ hybridisation. Interestingly, females display considerably lower aromatase activity than males but have almost equal numbers of aromatase-immunoreactive cells and express similar levels of aromatase mRNA. Aromatase activity in the male POA-HYP can be rapidly regulated by calcium-dependent phosphorylations, in the absence of changes in enzyme concentration. In the present study, we investigated whether aromatase activity is differentially regulated by phosphorylations in males and females. A linear increase in accumulation of aromatisation products was observed in both sexes as a function of time but the rate of conversion was slower in females. Saturation analysis confirmed the lower maximum velocities (V(max) ) in females but indicated a similar affinity (K(m) ) in both sexes. Aromatase activity in females reacted differentially to manipulations of intracellular calcium. In particular, chelating calcium with ethylene glycol tetraacetic acid (EGTA) resulted in a larger increase of enzymatic activity in males than in females, especially in the presence of ATP. A differential reaction to kinase inhibitors was also observed between males and females (i.e. a larger increase in aromatase activity in females than in males after exposure to specific inhibitors). These findings suggest that the nature of aromatase is conserved between the sexes, although the control of its activity by calcium appears to be different. Additional characterizations of intracellular calcium in both sexes would therefore be appropriate to better understand aromatase regulation.

Optimization of Enzymatic Hydrolysis with Cryotin F on Antioxidative Activities for Shrimp Hydrolysate Using Response Surface Methodology

Cryotin F could be used for hydrolyzing shrimp byproducts into bioactive ingredients, which could be used as value-added products. The objective of this study was to investigate the optimum condition for antioxidative activities of the enzymatic hydrolysate produced with Cryotin F using response surface methodology with central composite rotatable design. Shrimp byproducts (shells and heads) were hydrolyzed with Cryotin F. The experimental ranges of the independent variables for 20 experimental runs were 28.2～61.8℃ reaction temperature, pH 6～10 and 0.5～5.5% enzyme concentration. The degree of hydrolysis for the reaction products was measured. Their antioxidative activities were measured using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging activity and Fe-chelating activity. The experimental method with central composite rotatable design was well designed to investigate the optimum condition for biofunctional ingredients with antioxidative activities using Cryotin F because of their high R² values of 0.97 and 0.95 for DPPH-scavenging activity and Fe-chelating activity, respectively. Change in enzyme concentration did not significantly affect their antioxidative activities (p＜0.05). Both DPPH scavenging activity and chelating activity against Fe for the enzyme hydrolysates were more affected by the pH of enzyme hydrolysis than by their action temperature. DPPH-scavenging activity was higher at acidic pH than alkali pH, while chelating activity against Few was inversely affected. Hydrolysate of shrimp byproducts showed high antioxidative activities depending on the treatment condition, so the optimum treatment of enzymatic hydrolysate with Cryotin F and other proteases can be applied to shrimp byproducts (shells) and other protein sources for biofunctional ingredients.

The Influence of Protein Concentration on Oligomer Structure and Catalytic Function of Two Pyruvate Decarboxylases

As a general rule protein concentration typical for structural studies differs considerably from that chosen for kinetic investigations. Consequently, structure-function relationships are often postulated without appropriate knowledge, whether the functional behaviour of the enzyme is the same in both protein concentration ranges. To deal with this question, substrate activation kinetics of two well-characterised yeast pyruvate decarboxylases, from Saccharomyces cerevisiae and from Kluyveromyces lactis, were analysed over the broad protein concentration range 2-2,000 microg/mL. Analytical ultracentrifugation and small-angle X-ray scattering were used to analyse the enzymes' oligomer structure in aqueous solution. For the upper part of the concentration range the determined parameters, like catalytic activity, observed substrate activation rates, sedimentation coefficients and scattering parameters are independent on enzyme concentration changes. No indication of protein aggregation is detectable. However, significant changes occur at low enzyme concentration. The catalytically active tetramer dissociates progressively into dimers with comparable catalytic activity, but with significantly accelerated substrate activation.

Evolution of dominance in metabolic pathways.

Dominance is a form of phenotypic robustness to mutations. Understanding how such robustness can evolve provides a window into how the relation between genotype and phenotype can evolve. As such, the issue of dominance evolution is a question about the evolution of inheritance systems. Attempts at explaining the evolution of dominance have run into two problems. One is that selection for dominance is sensitive to the frequency of heterozygotes. Accordingly, dominance cannot evolve unless special conditions lead to the presence of a high frequency of mutant alleles in the population. Second, on the basis of theoretical results in metabolic control analysis, it has been proposed that metabolic systems possess inherent constraints. These hypothetical constraints imply the default manifestation of dominance of the wild type with respect to the effects of mutations at most loci. Hence, some biologists have maintained that an evolutionary explanation is not relevant to dominance. In this article, we put into question the hypothetical assumption of default metabolic constraints. We show that this assumption is based on an exclusion of important nonlinear interactions that can occur between enzymes in a pathway. With an a priori exclusion of such interactions, the possibility of epistasis and hence dominance modification is eliminated. We present a theoretical model that integrates enzyme kinetics and population genetics to address dominance evolution in metabolic pathways. In the case of mutations that decrease enzyme concentrations, and given the mechanistic constraints of Michaelis-Menten-type catalysis, it is shown that dominance of the wild type can be extensively modified in a two-enzyme pathway. Moreover, we discuss analytical results indicating that the conclusions from the two-enzyme case can be generalized to any number of enzymes. Dominance modification is achieved chiefly through changes in enzyme concentrations or kinetic parameters such as k(cat), both of which can alter saturation levels. Low saturation translates into higher levels of dominance with respect to mutations that decrease enzyme concentrations. Furthermore, it is shown that in the two-enzyme example, dominance evolves as a by-product of selection in a manner that is insensitive to the frequency of heterozygotes. Using variation in k(cat) as an example of modifier mutations, it is shown that the latter can have direct fitness effects in addition to dominance modification effects. Dominance evolution can occur in a frequency-insensitive manner as a result of selection for such dual-effects alleles. This type of selection may prove to be a common pattern for the evolution of phenotypic robustness to mutations.

Estimation of temperature profiles in microwaved particulates using enzyme and vision system

A new method, which used enzyme (peroxidase) as a time–temperature indicator (TTI) and color vision system to detect enzyme concentration was developed in this study. Peroxidase was immobilized in particulates containing potato starch and alginate. The particulates were heated in a microwave generation and transmission system from 15 to 85 s at 5 s intervals. The enzyme concentration was assayed by a color reaction, and detected by a color vision system. The temperature profiles of particulate foods heated in the microwave cavity were obtained through the calculation based on the results of changes in enzyme concentration. A z-value of 34.5°C and D ref of 1.62 s at reference temperature of 121.1°C were obtained. Matching the results of temperature measurements with a fiber optic probe validated the temperatures estimated by this new method. Furthermore, the color vision system adequately provided the temperature distribution within the particulates including heating time. Therefore, the temperature of particulates could be calculated at any location and heating time.

Rapid and Reversible Inhibition of Brain Aromatase Activity

Many actions of androgens require their conversion via the enzyme aromatase into oestrogens. Changes in brain aromatase activity are thought to take place via changes in enzyme concentration mediated by effects of sex steroids on aromatase transcription. These changes are relatively slow which fits in well with the fact that oestrogens are generally viewed as slow-acting messengers that act via changes in gene transcription. More recently, fast actions of oestrogens, presumably at the level of the cell membrane, have been described both in the female brain and in the male brain after the conversion of testosterone to oestradiol. It is difficult to reconcile the slow regulation of oestrogen synthesis (that occurs via changes in aromatase concentration) with a rapid action at the membrane level. Even if fast transduction mechanisms are available, this will not result in rapid changes in brain function if the availability of the ligand does not also change rapidly. Here, we report that aromatase activity in neural tissue of male Japanese quail (Coturnix japonica) is rapidly downregulated in the presence of Mg(2+), Ca(2+) and ATP in hypothalamic homogenates and in brain explants exposed to high Ca(2+) levels following a K(+)-induced depolarization or the stimulation of glutamate receptors. The K(+)-induced inhibition of aromatase activity is observed within minutes and reversible. Given that aromatase is present in presynaptic boutons, it is possible that rapidly changing levels of locally produced oestrogen are available for nongenomic regulation of neuronal physiology in a manner more akin to the action of a neuropeptide than previously hypothesized.

Rapid and Reversible Inhibition of Brain Aromatase Activity

AbstractMany actions of androgens require their conversion via the enzyme aromatase into oestrogens. Changes in brain aromatase activity are thought to take place via changes in enzyme concentration mediated by effects of sex steroids on aromatase transcription. These changes are relatively slow which fits in well with the fact that oestrogens are generally viewed as slow‐acting messengers that act via changes in gene transcription. More recently, fast actions of oestrogens, presumably at the level of the cell membrane, have been described both in the female brain and in the male brain after the conversion of testosterone to oestradiol. It is difficult to reconcile the slow regulation of oestrogen synthesis (that occurs via changes in aromatase concentration) with a rapid action at the membrane level. Even if fast transduction mechanisms are available, this will not result in rapid changes in brain function if the availability of the ligand does not also change rapidly. Here, we report that aromatase activity in neural tissue of male Japanese quail (Coturnix japonica) is rapidly downregulated in the presence of Mg2+, Ca2+ and ATP in hypothalamic homogenates and in brain explants exposed to high Ca2+ levels following a K+‐induced depolarization or the stimulation of glutamate receptors. The K+‐induced inhibition of aromatase activity is observed within minutes and reversible. Given that aromatase is present in presynaptic boutons, it is possible that rapidly changing levels of locally produced oestrogen are available for nongenomic regulation of neuronal physiology in a manner more akin to the action of a neuropeptide than previously hypothesized.

Design of Large Metabolic Responses. Constraints and Sensitivity Analysis

Metabolic control analysis (Kacser & Burns (1973). Symp. Soc. Exp. Biol.27, 65–104; Heinrich & Rapoport (1974). Eur. J. Biochem.42, 89–95) has been extensively used to describe the response of metabolic concentrations and fluxes to small (infinitesimal) changes in enzyme concentrations and effectors. Similarly, metabolic control design (Acerenza (1993). J. theor. Biol.165, 63–85) has been proposed to design small metabolic responses. These approaches have the limitation that they were not devised to deal with large (non-infinitesimal) responses. Here we develop a strategy to design large changes in the metabolic variables. The only assumption made is that, for all the parameter values under consideration, the system has a unique stable steady state. The procedure renders the kinetic parameters of the rate equations that when embedded in the metabolic network produce the pattern of large changes in the steady-state variables that we aim to design. Structural and kinetic constraints impose restrictions on the type of responses that could be designed. We show that these conditions can be transformed into the language of mean-sensitivity coefficients and, as a consequence, a sensitivity analysis of large metabolic responses can be performed after the system has been designed. The mean-sensitivity coefficients fulfil conservation and summation relationships that in the limit reduce to the well-known theorems for infinitesimal changes. Finally, it is shown that the same procedure that was used to design metabolic responses and analyse their sensitivity properties can also be used to determine the values of kinetic parameters of the rate laws operating “in situ”.

A Two-Step Computer-Assisted Method for Deriving Steady-State Rate Equations

A number of computer-assisted methods have been described for the derivation of enzyme-catalyzed steady-state rate equations [K. R. Runyan and R. B. Gunn (1989) Methods Enzymol. 171, 164–190; R. Varon, F. Garcia-Seville, M. Garvia-Moreno, F. Garcia-Canovas, R. Peyro, and R. G. Duggleby (1997) Comput. Appl. Biosci. 13, 159–167]; however, the required programs are either not readily available or require special software. We present here a two-step computer-assisted procedure for deriving steady-state rate equations using the widely available program Mathematica. In the first step, the differential equations for a particular kinetic mechanism that describe changes in enzyme concentration as a function of time are set equal to zero and entered into Mathematica in matrix form. In the second step, a single command allows for the computation of the distribution equations for the free enzyme and each enzyme–ligand complex.

Mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo.

Little is known about mechanisms of regulation of cysteine dioxygenase (CDO), gamma-glutamylcysteine synthetase (GCS), and cysteine-sulfinate decarboxylase (CSDC) in response to diet. Enzyme activity and Western and Northern or dot blot analyses were conducted on liver samples from rats fed a basal low-protein diet or diets with graded levels of protein or methionine for 2 wk. Higher levels of CDO activity and CDO protein but not of CDO mRNA were observed in liver of rats fed methionine or protein-supplemented diets, indicating that CDO activity is regulated by changes in enzyme concentration. Lower concentrations of the heavy or catalytic subunit of GCS (GCS-HS) mRNA and protein, as well as a lower activity state of GCS-HS in rats fed methionine- or protein-supplemented diets, indicated that dietary regulation of GCS occurs by both pretranslational and posttranslational mechanisms. Lower CSDC activity, CSDC protein concentration, and CSDC mRNA concentration were found in rats fed the highest level of protein, and regulation appeared to involve changes in mRNA concentration. Regulation of key enzymes of cysteine metabolism in response to diet determines the use of cysteine for synthesis of its essential metabolites.

Survival of Lactic Acid Bacteria in a Dynamic Model of the Stomach and Small Intestine: Validation and the Effects of Bile

This study was conducted to validate a dynamic model of the stomach and small intestine to quantify the survival of lactic acid bacteria and to assess the influence of gastrointestinal secretions. The survival of a single strain of each of the following species, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus bulgaricus, and Streptococcus thermophilus, was measured under physiological conditions (e.g., peristalsis, changes in pH, and changes in concentrations of enzymes and bile) and were compared with data obtained from humans. No significant differences were found between the in vitro and in vivo data, indicating that the model has a predictive value for the survival of these bacteria in humans.The survival of these strains of lactic acid bacteria in the gastrointestinal model was investigated under two different conditions in the small intestine: simulation of physiological secretion of bile and low bile secretion. Reductions in viability were significantly different between the bacterial species. The dose-response effect of bile on the survival of the tested bacteria was significant, demonstrating the bactericidal effect of bile salts. This study demonstrates the differences among bacterial species in their sensitivity to gastric and intestinal secretions.

Control Analysis in Terms of Generalized Variables Characterizing Metabolic Systems

Metabolic Control Analysis had originally been devised to quantify the effects of changes in enzyme concentrations on steady-state fluxesand metabolite concentrations. In many situations, fluxes and concentrations are not the only relevant variables. A formalism is presented by which the control of generalized variables characterizing biochemical systems can be described. The concepts of “state variables” and “response variables” are introduced. Formulae linking generalized control coefficients to generalized elasticities are established. From these, unified summation and connectivity theorems are derived. These formulae result in some of the well-known equations of Metabolic Control Analysis as special cases when the variables are specified to concentrations or fluxes. It is shown that if the response variables only depend on the state variables or the reaction rates or both, control coefficients do not depend on the special choice of the perturbation parameters. This is no longer the case if the response variables do depend on the parameters directly. The formalism provides a framework for dealing with proton-motive force, energy charge and many other variables as special cases. We illustrate the analysis by specifying it to the control analysis of concentration ratios, free-energy differences, transition times, and growth rate.