Different Types Of Inhibition Research Articles

Synthesis of novel glycoconjugates and evaluation as inhibitors against β-glucosidase from almond

Hydrolytic activity of glycosidases can be influenced by inhibitors of glycosidic nature. We have synthetized a serie of glycoconjugates consisting of galactose, glucosaminide or lactose 1- O-linked with allyl alcohol, pentenol or tetraethyleneglycol moieties. These compounds were found to be inhibitors of the hydrolase activity of the β-glucosidase from almond, with K i in the range of 7.7–107 mM and presenting different types of inhibition. Based on the structure of the functionalized glycosides and the inhibition studies performed, we suggest that the nature of the carbohydrate is determinant in the inhibitory strength of these glycoconjugates.

A Kinetic Comparison on the Inhibition of Adenosine Deaminase by Purine Drugs

The effects of allopurinol, acyclovir and theophylline on the activity of adenosine deaminase (ADA) were studied in 50 mM sodium phosphate buffer pH 7.5 at 27 °C, using a UV– Vis spectrophotometer. Adenosine deaminase is inhibited by these ligands, via different types of inhibition. Allopurinol, as a transition state analog of xanthine oxidase, and acyclovir competitively inhibit the catalytic activity of ADA. Inhibition constant values are 285 and 231 µM for allopurinol and acyclovir, respectively. Theophylline acts as a non-competitive inhibitor for ADA, which shows different affinity binding sites at various drug concentrations. There were two different types of inhibition constant, one of them due to a low concentration of the drug (K i = 56 μM) and the other appearing at higher concentrations of theophylline (K i = 201 μM). Thermodynamic parameters also show that ADA has two binding sites for theophylline. The comparison of inhibition constant for inosine (K i = 143 μM) and acyclovir (K i = 231 μM) elucidates the critical role of the ribose ring within the inosine structure, relative to the open ring of acyclovir. Comparison of the inhibition constant of theobromine (K i = 311 μM) with inosine (K i = 143 μM) shows the critical binding role of N 7 position within the purine ring. Interestingly, the N 7 position in allopurinol is replaced by a CH 2 group, which demonstrates the lower inhibiting potency of allopurinol (K i = 285 μM) relative to inosine (K i = 143 μM). In a structural sense, a comparison made between the structure of theophylline and theobromine besides a comparison between the inhibition constant of theophylline (K i = 56 μM at low and 201 μM at higher concentrations) and caffeine (K i = 342 μM) indicate that substitution of a bulky group in N 1 and N 7 positions of purine has a critical role in the binding affinity of the above- mentioned inhibitors to the enzyme.

Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies

The median-effect equation derived from the mass-action law principle at equilibrium-steady state via mathematical induction and deduction for different reaction sequences and mechanisms and different types of inhibition has been shown to be the unified theory for the Michaelis-Menten equation, Hill equation, Henderson-Hasselbalch equation, and Scatchard equation. It is shown that dose and effect are interchangeable via defined parameters. This general equation for the single drug effect has been extended to the multiple drug effect equation for n drugs. These equations provide the theoretical basis for the combination index (CI)-isobologram equation that allows quantitative determination of drug interactions, where CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively. Based on these algorithms, computer software has been developed to allow automated simulation of synergism and antagonism at all dose or effect levels. It displays the dose-effect curve, median-effect plot, combination index plot, isobologram, dose-reduction index plot, and polygonogram for in vitro or in vivo studies. This theoretical development, experimental design, and computerized data analysis have facilitated dose-effect analysis for single drug evaluation or carcinogen and radiation risk assessment, as well as for drug or other entity combinations in a vast field of disciplines of biomedical sciences. In this review, selected examples of applications are given, and step-by-step examples of experimental designs and real data analysis are also illustrated. The merging of the mass-action law principle with mathematical induction-deduction has been proven to be a unique and effective scientific method for general theory development. The median-effect principle and its mass-action law based computer software are gaining increased applications in biomedical sciences, from how to effectively evaluate a single compound or entity to how to beneficially use multiple drugs or modalities in combination therapies.

Discovery of Different Types of Inhibition between the Human and Thermotoga maritima α-Fucosidases by Fuconojirimycin-Based Derivatives

An efficient method for examining the selectivity of inhibitors on two alpha-fucosidases, one from Thermotoga maritima and the other from human, was established. The X-ray crystal structure of the former enzyme makes possible the homology modeling of the human alpha-fucosidase, indicating the major difference between both enzymes in the periphery of the catalytic site. To investigate the difference at the molecular level, a variety of fuconojirimycin (FNJ) derivatives with substitution at C1, C2, C6, or N were rapidly prepared in microplates and screened without purification for the inhibition activities of the two alpha-fucosidases. Among the molecules that were tested, only the substitution at C1 can significantly enhance the inhibitory potency, in contrast to the control (no substitution) and compounds with substitution at other positions. The majority of C1-substituted FNJs were found to be slow tight-binding inhibitors of the Thermotoga enzyme, while acting as the reversible inhibitors of the human fucosidase. The best inhibitor exhibited 13,700-fold difference in affinity between the two enzymes, which was attributed to the dissimilar aglycon binding site. Further investigations were carried out, including site-directed mutagenesis, the comparison of K(i) values among the wild type and mutants, and the intrinsic fluorescence change upon inhibitor titration, all supporting the idea that Tyr64 and Tyr267 of the Thermotoga alpha-fucosidase are critically involved in closely interacting with the aglycon of inhibitors. The increased level of contact thus induced conformational change, leading to the observed slow tight-binding inhibition.

Formation of nisin, plant-derived biomolecules and antimicrobial activity in starter culture fermentations of sauerkraut

The study investigated the formation of glucosinolate breakdown products and other biomolecules in sauerkraut using different starter cultures of lactic acid bacteria ( Lactobacillus plantarum, Lactobacillus sakei, Leuconostoc mesenteroides, Pediococcus pentosaceus, Lactococcus lactis N8 and L. lactis LAC67). Moreover, we examined the antimicrobial effects of cabbage juice containing nisin and glucosinolate breakdown products. The major glucosinolates were sinigrin, glucoiberin, and glucobrassicin. Several breakdown products were formed during fermentation. When L. sakei was used as the starter culture, the amount of total breakdown products in sauerkraut was 2–3 times that of other strains. Especially the concentrations of 3-methylsulphinylpropyl-isothiocyanate (ion ratio 195 100 g −1 FW), allyl-isothiocyanate (980 μg 100 g −1 FW), sulphoraphane (ion ratio 60 100 g −1 FW) were significantly higher in the fermented end product of L. sakei than in the other end products. Goitrin was found in relatively high concentrations (480–1090 μg 100 g −1 FW) in all end products. We detected different types of inhibition on indicator strains in the fermented end products: juice fermented with L. sakei strain effectively inhibited the growth of Candida lambica, whereas Listeria monocytogenes was not affected. Escherichia coli was strongly inhibited in all cabbage juices. L. lactis N8 produced up to 1400 IU ml −1 of nisin in cabbage brine within 24 h, and nisin was detectable even after 13 days (250 IU ml −1).

Pyruvate shuttle in muscle cells: high-affinity pyruvate transport sites insensitive to trans-lactate efflux.

The specificity of the transport mechanisms for pyruvate and lactate and their sensitivity to inhibitors were studied in L6 skeletal muscle cells. Trans- and cis-lactate effects on pyruvate transport kinetic parameters were examined. Pyruvate and lactate were transported by a multisite carrier system, i.e., by two families of sites, one with low affinity and high capacity (type I sites) and the other with high affinity and low capacity (type II). The multisite character of transport kinetics was not modified by either hydroxycinnamic acid (CIN) or p-chloromercuribenzylsulfonic acid (PCMBS), which exert different types of inhibition. The transport efficiency (TE) ratios of maximal velocity to the trans-activation dissociation constant (Kt) showed that lactate and pyruvate were preferentially transported by types I and II sites, respectively. The cis-lactate effect was observed with high Ki values for both sites. The trans-lactate effect on pyruvate transport occurred only on type I sites and exhibited an asymmetric interaction pattern (Kt of inward lactate > Kt of outward lactate). The inability of lactate to trans-stimulate type II sites suggests that intracellular lactate cannot recruit these sites. The high-affinity type II sites act as a specific pyruvate shuttle and constitute an essential relay for the intracellular lactate shuttle.

Pteridines as inhibitors of xanthine oxidase: structural requirements

Different pteridine derivatives were investigated for their inhibitory action on xanthine oxidase. From 27 investigated compounds, 13 showed concentration-dependent inhibition of the enzyme. Concentrations necessary for 50% inhibition ranged from <0.1 up to >100 μM. Different types of inhibition were found concerning xanthine and pterin as substrates: competitive, noncompetitive and mixed type. Out of 18 aromatic compounds tested, 12 were inhibitors. Only one out of nine reduced derivatives served as inhibitor. A simple regression model was used to specify the structural requirements for a pteridine to be an inhibitor. The most characteristic features of an inhibitor are aromaticity and no substitution at position 7 of the pteridine ring.

A dynamic and generic model of gas exchange of respiring produce: the effects of oxygen, carbon dioxide and temperature

Abstract A generic model is developed that describes rates of respiration and fermentation, in response to temperature and environmental gas conditions (oxygen and carbon dioxide). The mathematics of the model are based on Michaelis–Menten kinetics. Respiration is simplified as the effect of one enzymic reaction, inhibited by its own product, carbon dioxide. Both competitive, and uncompetitive inhibition are incorporated. Fermentation is modelled including the competitive inhibition of fermentation by both oxygen and carbon dioxide. Temperature dependence is introduced using Arrhenius equations for the maximum rates of oxygen consumption and carbon dioxide production. The model is generic in nature: it is applicable to different types of products (apple, chicory and tomato) at different conditions (in terms of O2, CO2 and temperature) and for different types of inhibition. This fitness is an indirect validation of the assumptions on which the model is based.

Interaction of the Clathrin-coated Vesicle V-ATPase with ADP and Sodium Azide

The kinetics of adenosine triphosphate (ATP)-dependent proton transport into clathrin-coated vesicles from bovine brain have been studied. We observe that the vacuolar proton-translocating ATPase (V-ATPase) from clathrin-coated vesicles is subject to two different types of inhibition by ADP. The first is competitive inhibition with respect to ATP, with a Ki for ADP of 11 microM. The second type of inhibition occurs after preincubation of the V-ATPase in the presence of ADP and Mg2+, which results in inhibition of the initial rate of proton transport followed by reactivation over the course of several minutes. The second effect is observed at ADP concentrations as low as 0.1-0.2 microM, indicating that a high affinity inhibitory complex is formed between ADP and the V-ATPase and is only slowly dissociated after the addition of ATP. We have further investigated the effect of sodium azide, an inhibitor of the F-ATPases that has been shown to stabilize an inactive complex between ADP and the F1-F0-ATP synthase (F-ATPase). We observed that azide inhibited ATP-dependent proton transport by the purified, reconstituted V-ATPase with a K0.5 of 0.2-0.4 mM but had no effect on ATP hydrolysis. Azide was shown not to increase the passive proton permeability of reconstituted vesicles and did not stimulate ATP hydrolysis by the reconstituted enzyme, in contrast with CCCP, which both abolished the proton gradient and stimulated hydrolysis. Thus, azide does not appear to act as a simple uncoupler of proton transport and ATP hydrolysis. Rather, azide may have some more direct effect on V-ATPase activity. Possible mechanisms by which azide could exert this effect on the V-ATPase and the contrasting effects of azide on the F- and V-ATPases are discussed.

Mass transport with enzyme reactions

A mathematical approach is presented for modelling the reactions catalyzed by enzymes attached to the inner surface of a tube through which the substrate solution passes. In a classical paper of Kobayashi and Laidler [4] a theoretical treatment of this problem was considered but the resulting nonlinear equation appeared to be intractable, except for the simple cases. In the present paper, the central integro-differential equation is reduced to an integral Volterra equation. The analysis is extended to different transport situations and to reactions with classical Michaelis-Menten kinetics and with different types of inhibition. Explicit power series representations of the solutions are established for several reaction velocity models. The consistency of the mathematical model used is established by proving the existence and the uniqueness of the continuous solution of the basic integral equation.

The distribution of the GABA Aβ2, β3 subunit receptor in the cat superior colliculus using antibody immunocytochemistry

GABA-containing synaptic terminals in the cat superior colliculus include two varieties of presynaptic dendrite and at least one type of axon terminal with flattened vesicles. These anatomically distinct synaptic profiles probably also mediate different types of inhibition. Whether they are associated with different types of GABA receptor is unknown and one objective of the present paper. We used the antibody mAb 62-3G1 directed against the β2, β3 subunits of the GABA A receptor complex to determine whether the distribution of this receptor subunit is specific to one or more types of GABA-containing synapse. At the light microscope level, β2, β3 immunoreactivity was densely distributed within the neuropil of the zonal and superficial gray layers, and more lightly within the optic, intermediate, and deep gray layers. No cell bodies were labelled by the antibody in the zonal and superficial gray layers, but numerous cells contained internalized cytoplasmic immunoreactivity in the optic, intermediate gray, and deeper layers. At the ultrastructural level, synaptic sites opposite axon terminals that contained flattened synaptic vesicles (F profiles) were often β2, β3 immunoreactive, while postsynaptic sites opposite presynaptic dendrites (PSD profiles) were never immunoreactive. The label at F profiles usually filled the synaptic cleft and coated the postsynaptic plasma membrane. Some membrane-associated label was also found at non-synaptic sites. We conclude that this receptor subunit is selectively associated with flattened vesicle axon terminals and not with presynaptic dendrites, a result which supports evidence that those terminal types mediate different types of inhibition.

Evaluation of four types of inhibition for modelling the influence of carbon dioxide on oxygen consumption of fruits and vegetables

Abstract High carbon dioxide (CO 2 ) concentrations can reduce the oxygen (O 2 ) consumption rate of a number of fruits and vegetables. This reduction can be modelled by four types of inhibition in an enzyme kinetics model: (1) the competitive type; (2) the uncompetitive type; (3) a combination of both previous types; and (4) the non-competitive type. These different types of inhibition were tested for describing the CO 2 influence on O 2 consumption using experimental data supplemented with data from literature. The gas exchange rates of apples (cv. Golden Delicious and Elstar), asparagus, broccoli, mungbean sprouts and cut chicory were measured under a wide range of O 2 and CO 2 concentrations. With the range of CO 2 concentrations used, no influence was found on gas exchange rates of apples. There was a clear influence of high CO 2 on the gas exchange rates of the other produce. A good estimation of C 2 consumption could be obtained with the inhibition models. This supports the use of Michaelis-Menten kinetics for modelling O 2 consumption. Depending on the product the statistical analysis gave good results for the competitive and the uncompetitive type of inhibition. Based on gas exchange data only, no distinction between the competitive and uncompetitive type of inhibition could be made. It suggests the simultaneous existence of both types of inhibition. The idea is that the combined inhibition equation is most closely related to what is actually occurring in plant tissues. However, for reasons of simplicity the non-competitive type of inhibition is preferred, showing similar results to the combined inhibition, and giving good results for all the products tested.

Differences in the kinetic properties, effect of calcium and sensitivity to inhibitors of paraoxon hydrolase activity in rat plasma and microsomal fraction from rat liver

The properties of a rat hepatic microsomal enzyme that hydrolyses O, O-diethyl- p-nitro-phenylphosphate (paraoxon) were studied and compared to the paraoxon hydrolase activity found in rat plasma. The pH stability for both enzyme activities was optimum between pH 6.0 and 9.0. An overall analysis of the data showed that the microsomal fraction was less resistant to the effect of the pH than plasma. The kinetic constants for heat inactivation evaluated for paraoxonase in rat plasma and liver microsomal fraction indicate that paraoxonase tends to inactivate faster in rat liver microsomes than in rat plasma. The apparent activation energies of the heat inactivation process were 77.7 and 61.1 kcal/mol for rat plasma and microsomal fraction, respectively. Enzyme activity was lost after both dialysis and incubation with EDTA and partially restored by the addition of calcium. In rat plasma samples the requirement for calcium was absolute (essential activator) while in the microsomal fraction the reaction may occur, to a minimum extent, in the absence of the activator (non-essential activator). Calcium restored 85% activity when added immediately after EDTA; restored activity decreased when the time interval between addition of EDTA and calcium was increased. Other metals were not able to restore activity previously inhibited by EDTA or dialysis. The response to several inhibitors (EDTA, Mn, Co, Zn, Ba, Mg, Cu, La, Hg and p-hydroxy-mercuribenzoate) of rat plasma and microsomal fraction was studied, determining the type of inhibition and the inhibition constants. Plasma enzyme was always more resistant than liver sample to the effect of the inhibitors and showed different types of inhibition than the liver microsomal fraction. In general we found more differences than analogies between the rat plasma and liver enzymes which suggests the presence of two enzymes or two different forms of the same enzyme. Furthermore the existence of an EDTA-resistant fraction in rat liver microsomes suggests that more than one enzyme capable of hydrolysing paraoxon is present in the microsomal fraction of rat liver.

Inhibitory effects of cysteine and aromatic acids on tyrosinase activity

Cysteine and a wide range of aromatic carboxylic acids were inhibitors for the activity of a commercially purified mushroom tyrosinase. These compounds exhibited different types of inhibition including competitive, noncompetitive, mixed or uncompetitive. The variations in type and degree of inhibition were dependent upon the nature of the inhibitor and the method used for the determination of enzymatic activity, spectrophotometric or polarographic.

Quantitative analysis of interleukin-2-induced proliferation in the presence of inhibitors using a mathematical model

The proliferative response of CTLL-2 cells to human recombinant interleukin-2 (IL-2) can be modeled mathematically using enzyme kinetic equations. This approach has been used to analyze dose-response curves (IL-2 concentration vs. level of proliferation) measured by MTT and [ 3H]TdR assays. The values of functional dissociation constants, equivalent to IL-2 concentrations giving 50% of the maximal response, depended on the cell concentration and increased from 4 to 60 pM for the [ 3H]TdR assay and from 40 to 140 pM for the MTT assay when the cell concentration was increased from 2 × 10 3 to 4 × 10 4 cells/well. The types of inhibition and dissociation constants for various inhibitors of IL-2-dependent proliferation such as mAbs against IL-2 receptor (7D4 and AMT13) and normal mouse serum (NMS) were also analyzed. Both mAbs exhibited competitive mechanisms of inhibition whereas NMS inhibited IL-2-driven proliferation in a mixed manner. Two gel-filtration fractions of NMS with inhibitory activity manifested different types of inhibition: purely competitive type of inhibition in the case of a 10–15 kDa fraction and a mixed type of inhibition for a 100–150 kDa fraction. The proposed model can also be used for quantitative analysis of the influence of various factors (pH, temperature, cultivation condition) on the level of proliferation.

Contents of Postharvest Biology and Technology, vol. 1

A generic model is developed that describes rates of respiration and fermentation, in response to temperature and environmental gas conditions (oxygen and carbon dioxide). The mathematics of the model are based on Michaelis–Menten kinetics. Respiration is simplified as the effect of one enzymic reaction, inhibited by its own product, carbon dioxide. Both competitive, and uncompetitive inhibition are incorporated. Fermentation is modelled including the competitive inhibition of fermentation by both oxygen and carbon dioxide. Temperature dependence is introduced using Arrhenius equations for the maximum rates of oxygen consumption and carbon dioxide production. The model is generic in nature: it is applicable to different types of products (apple, chicory and tomato) at different conditions (in terms of O2, CO2 and temperature) and for different types of inhibition. This fitness is an indirect validation of the assumptions on which the model is based.

Theoretical treatment of tight-binding inhibition of an enzyme. Ribonuclease inhibitor as special case.

A general treatment of very tight-binding inhibition is described. It was applied to purified endogenous RNAase inhibitor from rat testis. This treatment discriminates among the different types of inhibition and allows for calculation of the inhibition parameters. When very tight-binding inhibitions are studied at similar molar concentrations of both enzyme and inhibitor, a further approach is required. This is also described and applied to the RNAase inhibitor. A Ki value of 3.2 x 10(-12) M was found for this inhibitor protein. On the basis of this result, it was considered inappropriate to classify this type of inhibitor in terms of competitive or non-competitive, as has been done for such inhibitors so far. Functional consequences of this analysis are discussed for the RNAase-RNAase inhibitor system.

Oxidation mechanism of mesoxalic acid on a gold electrode

Mesoxalic acid decarboxylates on a polycrystalline gold electrode giving CO 2 and H 3O + as the only products. A well-developed four-electron wave is obtained and a mechanism is given in agreement with the experimental results. Two different types of inhibition are detected. The first is caused by intermediate-poison adsorption and the second by oxide formation.

Die Auswirkung von Tensiden und das Verhalten von Bakterien gegen‐über grenzflächenaktiven Verbindungen

AbstractAn investigation was made into the effects of seven anion‐active tensides and one non‐ionic tenside on the growth and respiration of 19 strains of the bacteria genera Proteus, Pseudomonas, Aerobacter and Sarcina. A nutrient broth with phosphate buffer and partly glucose addition was used as the culture medium. Tenside was added either at the beginning of tests in the lag‐phase of cultures or during the logarithmic growth phase. In addition, the development of resistance or adaptation were tested by establishing subcultures. The inhibitory effect of tensides depends on the combination of tenside/species of bacteria, gram‐positive bacteria being more sensitive and the inhibitory effect being lower in their logarithmic growth phase. The time course of the inhibition of respiration after the addition of tenside to the medium shows different types of inhibition and adaptation, considerably extended lag‐phases occurring frequently.

Aspartate transcarbamylase de la coquille Saint-Jacques Pecten maximus 1. (Mollusque lamellibranche): Méthode de dosage et variations de l'activité dans le manteau et la gonade

The search of an index for the instantaneous estimate of the in situ growth rate of marine animals led us to attempt to measure the specific activity of aspartate transcarbamylase (ATC). The experiments to test the value of the index were carried out on the scallop Pecten maximus L. p]The first step was to find the optimum conditions for enzyme activity measurement. At 35 °C. the scallop ATC shows an optimum pH of 9 and a K m of 4.6 × 10 −3 M for aspartate and of 8.0 × 10 −4 M for carbamyl phosphate. The different types of inhibition by the substrates high concentrations and the products suggest an ordered sequential mechanism for the reaction. The decrease in enzyme activity due to metallic ions (Cu 2+ and Zn 2+) and to parahydroxymercuribenzoate is compatible with the presence of a sulphydryl group in the active site. p]The variations in ATC levels within the gonad and within the mantle of the scallop were measured and compared with the processes of the sexual maturation and somatic growth in a natural population. For the two tissues, a correlation between the ATC specific activity and the relative growth rate is demonstrated.