Search

Toxoplasma gondii , the causative agent of toxoplasmosis, is a protozoan parasite capable of infecting a wide range of hosts, posing significant health risks, particularly to immunocompromised individuals and congenital transmission. Current therapeutic options primarily target the active tachyzoite stage but are limited by issues such as toxicity and incomplete efficacy. As a result, there is an urgent need for alternative therapies that can selectively target parasite-specific mechanisms critical for metabolic processes and host-parasite interactions. In this context, α-carbonic anhydrase (Tg_CA), an enzyme essential for T. gondii survival has emerged as a promising drug target. Tg_CA was successfully expressed and purified to evaluate its susceptibility to sulfonamide-based inhibitors, represented by compounds 1 - 24 and the AAZ - HCT series. These inhibitors demonstrated a broad spectrum of activity, with KI values ranging from 17.8 to 8450 nM. Several compounds exhibited moderate to high potency against Tg_CA; however, concerns regarding selectivity arose because of the inhibition of human isoforms, particularly CA I and CA II. Thus, although some inhibitors showed strong activity against Tg_CA, optimizing selectivity remains crucial for minimizing off-target effects and improving therapeutic efficacy. Further structural modifications may enhance selectivity and advance the development of effective treatments for toxoplasmosis.

Chronic wound resulting from diabetes mellitus is a significant cause of amputation worldwide. Secondary infections, lowering of nitric oxide synthase level, reduction of glucose-6-phosphate dehydrogenase levels, improper extracellular matrix remodelling, neuropathy, abnormality of endothelial cell function, and vasculopathy impedes the normal wound healing cycle during diabetes. Multiple studies have concluded that Ser9 phosphorylation causes inhibition of the glycogen synthase kinase-3β (GSK3-β) protein, which is essential for faster diabetic wound healing. Hence this protein could be a potential target for molecular interactions with prospective wound-healing molecules. Verbascoside, martynoside, echinacoside, crenatoside, and salidroside are a few phenylethanoid glycosides that have potential wound-healing ability by increasing extracellular matrix synthesis, angiogenesis, keratinocyte migration, and the functioning of macrophages and neutrophils. Thus, the five glycosides were subjected to molecular docking with GSK3-β protein (PDB ID: 1I09). This study revealed strong binding interactions with GSK3-β (between −10.2 and −7.3 kcal/mol) and inhibition constants (between 0.032 and 4.397 µM) which suggested potent inhibition of the target protein even at lower concentrations of these compounds. Further, the docked complexes were visualized to find the interaction of the ligands with the amino acid residues. However, further in vivo and in vitro studies are required to validate the activity of these phenylpropanoid glycosides in diabetic wound healing.

BACKGROUND: Aldo‐keto isomerizations of D‐xylose and D‐glucose catalyzed by xylose isomerase (XI) is commercially carried out to produce bio‐ethanol and high fructose corn syrup. XI is a metalloenzyme. In this paper, kinetics and thermodynamics of the activity and stability of hyperthermophilic recombinant XI from Thermotoga naphthophila RKU‐10T (TnapXI) were studied in the presence and absence of metal ions. RESULTS: Kinetics parameters of recombinant XI from TnapXI were calculated as Km = 0.99 mmol L–1, Vmax = 500 μmol.mg−1.min−1, and kcat = 68.3 min−1 for the isomerization of D‐xylose(aldo)⇌D‐xylulose(keto), whereas for D‐glucose(aldo)⇌D‐fructose(keto) conversion Km = 7.72 mmol L–1, Vmax = 90 μmol.mg−1.min−1, and kcat = 12.4 min−1. Ionization constants were calculated as pKa1 = 6.0 and pKa2 = 7.6, whereas activation constants (Ka) of TnapXI were 0.3, 0.5, and 4.0 mmol L–1 for Co2+, Mn2+, and Mg2+, respectively. Inhibition constant (Ki) was 0.119 mmol L–1 for Ca2+. Q10 was 2.8 and Ea = 82.25 kJ.mol−1. Thermodynamics for isomerization were calculated as ∆H* = 79.2 kJ.mol−1, ∆G* = 83 kJ.mol−1, and ∆S* = −10.5 J.mol−1.K−1. The z‐value was 12.6 °C for apo‐TnapXI and 32.6 °C for holo‐TnapXI. Deactivation parameters were ∆G*(d) = 100 kJ.mol−1, ∆H*(d) = 206 kJ.mol−1, ∆S*(d) = 0.28 kJ.mol−1.K−1 for apo‐TnapXI and t1/2 = 18 min at 95 °C, while ∆G*(d) = 104 kJ.mol−1, ∆H*(d) = 767 kJ.mol−1, ∆S*(d) = 1.8 kJ.mol−1.K−1 for holo‐TnapXI and t1/2 = 65 min at 95 °C. CONCLUSION: These noteworthy features make this enzyme an attractive candidate for the industrial production of fuel ethanol and high fructose corn syrup at high temperatures to maximize the product yield. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Aim. Study of the inhibitory effect of polyelectrolytes with antiviral effect against influenza neuraminidase. Determination of the type and inhibition constant. The study of the effect of polyelectrolytes (PE) on the process of oxidative phosphorylation in the mitochondria of cells host organism. Materials and methods. Purified influenza virus strains were used: A/VPCH/Weybridge (H7N7), A/Mallard Pennsylvania/10218/84 (H5N2), A/NIBRG-14 (H5N1) with an initial infectious titer of 4,5 lgTCD50/ml. PE solutions of polystyrenesulfonate with a degree of polymerization of 8 (PSS-8) in concentrations of 0,5—4,0 mM and polyallylamine (6 kDa) PAA (6 kDa) in concentrations of 0,5—4,0 μM. To determine the activity of influenza neuraminidase, influenza virus strains were used after the removal of low molecular weight inhibitors of neuraminidase by dialysis against water. The neuraminidase substrate was fetuin at final concentrations of 0,052 to 1,2 μM for PAA (6 kDa) and from 0,052 to 1,2 mM for PSS-8. As quantitative characteristics of respiration and phosphorylation of mitochondria, respiratory coefficients according to Lardi-Velman and Chans-Williams, as well as the ratio of ADP/O were used. Mitochondria were isolated from skeletal muscle. The determination of respiratory coefficients and the ratio of ADP/O) was determined by the polarographic method. Results. A noncompetitive type of inhibition of these PEs was detected in relation to the neuraminidase activity of influenza viruses with inhibition constants KI = 1,6 ± 0.08 μM for PAA (6 kDa) and KI = 1,7 ± 0.085 mM for PSS-8. Respiratory coefficients and the ratio of ADP / 0 were determined in the absence and after addition of PSS-8 and PAA (6 kDa) to mitochondria at concentrations of 20 mM and 10 μM, respectively. A decrease in respiratory coefficients and an ADP/O ratio was observed, indicating an inhibition of the enzymes of the electron-transport chain of mitochondria. At concentrations of less than 20 mM and 10 μM for PSS-8 and PAA (6 kDa), all indicators did not significantly change. Conclusion. The non-competitive mechanism of inhibition of the neuraminidase activity of the influenza PE viruses is explained by the conformational changes in the molecules of the enzyme and/or enzyme-substrate complex and, accordingly, the structural and functional changes in its secondary structure. When going beyond the range of non-toxic concentrations of20 mM for PSS-8 and 10 μM for PAA (6 kDa), inhibition of the mitochondrial respiratory chain enzymes was observed while maintaining the antiviral effect.

Anticancer drugs are widely used cancer treatment. Human carbonic anhydrase I and II were shown widely distribution with erythrocytes so it is important to understand how effect is anticancer drugs these isozymes. In this aim, paclitaxel, irinotecan, oxaliplatin, docetaxel and epirubicin studied in vitro effect on human carbonic anhydrases I and II obtained from human erythrocytes. Carbonic anhydrase isozymes were purified with affinity chromatography by Sepharose 4B-ethylene diamine-4-thioureido benzenesulfanamide gel and the purity of the isozymes has been checked a single band by sodium dodecyl sulphate polyacrylamide gel electrophoresis. IC50 values, inhibition constants (Ki) and inhibition types were determined with anticancer drugs on human carbonic anhydrase I and II. Competitive type inhibition was obtained with irinotecan on human carbonic anhydrase I but uncompetitive inhibition was seen on human carbonic anhydrase II using p-nitrophenyl acetate as a substrate. Non-competitive inhibition type was determined with docataxel, paclitaxel and oxaliplatin on human carbonic anhydrase I and II. Epirubicin was shown uncompetitive inhibition type on studied isozymes. Docataxel showed the greatest inhibition with the IC50 value 8.59 μmol/L on human carbonic anhydrase I. But there is less inhibition was seen with docataxel for human carbonic anhydrase II. The results of study show that anticancer drugs also have an important role in inhibiting human carbonic anhydrases I and II. [ABSTRACT FROM AUTHOR]

Hydroxycitrate from the fruit of Garcinia cambogia [ i.e. (2 S,3 S)-2-hydroxycitrate] is the best-known inhibitor of ATP-citrate lyase. Well diffracting crystals showing how the inhibitor binds to human ATP-citrate lyase were grown by modifying the protein. The protein was modified by introducing cleavage sites for Tobacco etch virus protease on either side of a disordered linker. The protein crystallized consisted of residues 2-425-ENLYFQ and S-488-810 of human ATP-citrate lyase. (2 S,3 S)-2-Hydroxycitrate binds in the same orientation as citrate, but the citrate-binding domain (residues 248-421) adopts a different orientation with respect to the rest of the protein (residues 4-247, 490-746 and 748-809) from that previously seen. For the first time, electron density was evident for the loop that contains His760, which is phosphorylated as part of the catalytic mechanism. The pro- S carboxylate of (2 S,3 S)-2-hydroxycitrate is available to accept a phosphoryl group from His760. However, when co-crystals were grown with ATP and magnesium ions as well as either the inhibitor or citrate, Mg2+-ADP was bound and His760 was phosphorylated. The phosphoryl group was not transferred to the organic acid. This led to the interpretation that the active site is trapped in an open conformation. The strategy of designing cleavage sites to remove disordered residues could be useful in determining the crystal structures of other proteins. [ABSTRACT FROM AUTHOR]

Elemental copper (Cu 0 ) and zinc oxide (ZnO) nanoparticle (NP) toxicity to methanogens has been attributed to the release of soluble metal ions. Iron sulfide (FeS) partially controls the soluble concentration of heavy metals and their toxicity in aquatic environments. Heavy metals displace the Fe from FeS forming poorly soluble metal sulfides in the FeS matrix. Therefore, FeS may be expected to attenuate the NP toxicity. This work assessed FeS as an attenuator of the methanogenic toxicity of Cu 0 and ZnO NPs and their soluble salt analogs. The toxicity attenuation capacity of fine (25–75 μm) and coarse (500 to 1200 μm) preparations of FeS (FeS-f and FeS-c respectively) was tested in the presence of highly inhibitory concentrations of CuCl 2 , ZnCl 2 Cu 0 and ZnO NPs. FeS-f attenuated methanogenic toxicity better than FeS-c. The results revealed that 2.5 × less FeS-f than FeS-c was required to recover the methanogenic activity to 50% (activity normalized to uninhibited controls). The results also indicated that a molar FeS-f/Cu 0 NP, FeS-f/ZnO NP, FeS-f/ZnCl 2 , and FeS-f/CuCl 2 ratio of 2.14, 2.14, 4.28, and 8.56 respectively, was necessary to recover the methanogenic activity to > 75%. Displacement experiments demonstrated that CuCl 2 and ZnCl 2 partially displaced Fe from FeS. As a whole, the results indicate that not all the sulfide in FeS was readily available to react with the soluble Cu and Zn ions which may explain the need for a large stoichiometric excess of FeS to highly attenuate Cu and Zn toxicity. Overall, this study provides evidence that FeS attenuates the toxicity caused by Cu 0 and ZnO NPs and their soluble ion analogs to methanogens. [ABSTRACT FROM AUTHOR]

Elemental copper nanoparticles (Cu 0 NPs) are potentially inhibitory to the different key microbial trophic groups involved in biological wastewater treatment processes. Cu-based NPs are known to be toxic to methanogens at low concentrations. However, very little is known about the toxic effect of Cu 0 NPs on other microbial groups involved in either upper trophic levels of anaerobic digestion or anoxic nitrogen removal processes. This study evaluated the toxicity of Cu 0 NPs to glucose fermentation, syntrophic propionate oxidation and denitrification in shaken batch bioassays with soluble substrates. Batch experiments were also supplemented with CuCl 2 to evaluate the inhibitory impact of soluble Cu(II) ions. Syntrophic propionate oxidation and glucose fermentation were the least and most inhibited processes with inhibition constant ( K i ) values of 0.202 and 0.047 mM of added Cu 0 NPs, respectively. Further analyses revealed that the K i values calculated as a function of the free soluble Cu concentration were < 0.003 mM for every biological process tested and most of these K i values were similar in order of magnitude regardless of whether the Cu source was CuCl 2 or Cu 0 NPs. The results taken as a whole indicate that Cu 0 NPs are toxic to all the microbial processes studied. Therefore, Cu 0 NPs can potentially be an important inhibitor of anaerobic wastewater treatment processes that rely on these trophic groups. The evidence suggests that the inhibitory impact of Cu 0 NPs was mainly due to the release of toxic Cu(II) ions originating from the corrosion and dissolution of Cu 0 NPs. [ABSTRACT FROM AUTHOR]

To identify the common inhibition types, the putative decision system is unsatisfactory. In a new decision system, Michaelis-Menten constants and maximal reaction rates were plotted versus inhibitor concentrations for deriving Kik and Kiv as the inhibition constants, respectively; their difference was quantified as the ratio of the larger one to the smaller one. Such ratios below 2.0 suggested uncompetitive inhibitors, over 5.0 suggested noncompetitive or competitive inhibitors, and from 2.0 to 5.0 suggested mixed inhibitors. By the new decision system, (i) the simulation recovery of uncompetitive inhibitors under CVs of 2% or 5% was improved by four times, but that of competitive or noncompetitive inhibitors was improved slightly; (ii) the recovery of L-phenylalanine as an uncompetitive inhibitor of intestinal alkaline phosphatase reached 38%, while the putative decision system lost all; the recovery of xanthine as a competitive inhibitor of uricase was improved slightly. Therefore, the new decision system was better. [ABSTRACT FROM AUTHOR]

Abstract: The transport activity of a membrane protein, bilitranslocase (T.C. # 2.A.65.1.1), which acts as a transporter of bilirubin from blood to liver cells, was experimentally determined for a large set of various endogenous compounds, drugs, purine and pyrimidine derivatives. On these grounds, the structure–activity models were developed following the OECD principles of QSAR models and their predictive ability for new chemicals was evaluated. The applicability domain of the models was estimated by Euclidean distances criteria according to the applied modeling method. The selection of the most influential structural variables was an important stage in the adopted modeling methodology. The interpretation of selected variables was performed in order to get an insight into the mechanism of transport through the cell membrane via bilitranslocase. Validation of the optimized models was performed by a previously determined validation set. The classification model was build to separate active from inactive compounds. The resulting accuracy, sensitivity, and specificity were 0.73, 0.89, and 0.64, respectively. Only active compounds were used to develop a predictive model for bilitranslocase inhibition constants. The model showed good predictive ability; Root Mean Squared error of the validation set, RMSV =0.29log units. [Copyright &y& Elsevier]

Eight chiral esters of quinuclidin-3-ol and butyric, acetic, pivalic and benzoic acid were synthesized as well as their racemic and chiral, quaternary N-benzyl derivatives. All racemic and chiral quaternary compounds were studied as substrates and/or inhibitors of horse serum butyrylcholinesterase (BChE). The best substrate for the enzyme was (R)-N-benzyl butyrate. The rates of hydrolysis decreased in order (R)-butyrate >> (R)-acetate (7-fold slower) > (R)-pivalate (8-fold slower) > (R)-benzoate (9-fold slower reaction), while (S)-N-benzyl esters were much poorer substrates (320 (butyrate) - 4360-fold slower (pivalate) than the appropriate (R)-enantiomer). For all (S)-N-benzyl esters excluding (S)-N-benzyl acetate inhibition constants were determined (Ka = 3.3-60 µmol dm-3). The hydrolysis of racemic mixtures of N-benzyl esters proceeded 1.4 (for acetate) - 5.1 (for benzoate) times slower than that of pure (R)-enantiomers of the corresponding concentrations due to the inhibition with (S)-enantiomers. Change of the acyl moiety of the substrate effected both activity and stereoselectivity of the BChE. [ABSTRACT FROM AUTHOR]

A comparative kinetic study of the inhibition of urea hydrolysis by 9 substituted 1,5,6,7-tetrahydro-4 H-benzimidazol-4-ones (BI I–IX) has been carried out. The inhibition had reversible competitive mode; the inhibition constants K i, varied from 29 to 754 μM in dependence of structure of BI I–IX. Three BI I–IX, characterized by the K i values ranged from 29 to 82 μM, may be used as potential therapeutic agents in gastroenterology for treatment of gastroduodenal ulcers. [ABSTRACT FROM AUTHOR]

Abstract: Duggleby [Duggleby, R.G., 1979. Experimental designs for estimating kinetic parameters for enzyme-catalyzed reactions. J. Theor. Biol. 81, 672–684] discussed the “design of several replicate measurements of the velocity at as many experimental conditions as there are parameters to be estimated.” He discussed the application of this method to A→products, without and with competitive inhibition, and commented briefly on A+B→products. The availability of computer applications that can solve large sets of simultaneous equations makes it possible to use this method to calculate kinetic parameters for more complicated enzyme mechanisms. This article is concerned with rapid-equilibrium rate equations, but this method can also be used with steady-state rate equations. Computer programs are provided for the calculation of the three kinetic parameters for ordered A+B→products from three velocity measurements and for the calculation of the four kinetic parameters for random A+B→products from four velocity measurements. Computer programs are also provided for competitive inhibition, uncompetitive inhibition, and mixed inhibition of ordered A+B→products. [Copyright &y& Elsevier]

Peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) was inhibited byo-aminophenol (AP), 2-amino-4-tert-butylphenol (ATBP), 2-amino-4,6-di-tert-butylphenol (ADTBP), and 4-tert-butylpyrocatechol (TBP). Inhibitors were characterized by inhibition constantKi and stoichiometric coefficientf, the number of radicals terminated by one inhibitor molecule. The most efficient inhibitor is ADTBP characterized byKi = 36 µM in 0.015 M phosphate citrate buffer, pH 6.0, at 20°C. According to their antiradical efficiency, the studied inhibitors can be arranged as follows: ADTBP>ATBP>AP>TBP. The role of the NH2 group in the inhibitory capacity of aminophenols is discussed. Using gas-liquid chromatography, kinetics of consumption of the initial components and accumulation of the reaction products on peroxidase-catalyzed oxidation of the TMB-TBP pair was studied; the data clarify the stages of a complex process of co-oxidation of amines and phenols. [ABSTRACT FROM AUTHOR]

The effects of temperature and pH during extraction of toxin(s) from the cyanobacterial genus Anabaena were studied. Different extracts were compared for their anticholinesterase potency against purified eel acetylcholinesterase. A lyophilized powder from Anabaeba spiroides cells was dissolved in ethanol, sonicated, and filtered. This solution was then evaporated at three different temperatures (40, 60, or 80°C), resuspended in chloroform, and washed in deionized water at three different pH values (3.3, 5.0, or 8.5). At 40°C, the estimated 50% inhibition concentration (IC50) was 2.06, 2.19, and 8.64 mg lyophilized A. spiroides cells ml-1 for the extraction pH of 3.3, 5.0, and 8.5, respectively. At 60°C, the values were 4.82, 3.06, and 12.0 mg/ml at increasing pH values. Finally, at 80°C, the IC50 values were 3.52, 4.35, and 30.1 mg/ml at the same pH values. These results indicate that pH 3.3 and a temperature of 40°C should be employed to preserve toxin content. The optimized protocol was employed to evaluate the anticholinesterase potency of environmental samples of water containing different numbers of filaments of cyanobacterial species. Registered inhibition ranged from 5.11 to 11.1%, supporting the feasibility of the method. [ABSTRACT FROM AUTHOR]

Dissociation constants and stoichiometry of binding for interaction of trimeric calf spleen purine nucleoside phosphorylase with potent multisubstrate analogue inhibitors were studied by kinetic and spectrofluorimetric methods. [ABSTRACT FROM AUTHOR]

A comparative study of the kinetics of peroxidase-catalyzed oxidation of 3,3",5,5"-tetramethylbenzidine (TMB) in the presence of 2,4-dinitrosoresorcinol (DNR), its polydisulfide derivative [poly(DNRDS)], and resorcinol polydisulfide [poly(RDS)], substances that competitively inhibit the formation of TMB conversion product, was carried out. The inhibition constants Ki for DNR, poly(DNRDS), and poly(RSD) were determined at 20°C and pH 6.4 to be 110, 13.5, and 0.78 μM, respectively. The stoichiometric coefficients of inhibition were calculated to be 0.38 and 76 for poly(DNRDS) and poly(RDS), respectively. In the pH range 6.4–7.0, the initial rates of the peroxidative oxidation of TMB, and its mixtures with DNR and poly(DNRDS) and the Ki value for poly(RDS) substantially decreased with increasing pH. The kinetic parameters of poly(RDS) ( Ki 0.22–0.78 μM and f 76) suggest that it is the most efficient inhibitor of peroxidase oxidation of TMB: in micromolar concentrations, it completely stops this process and can be used in EIA. [ABSTRACT FROM AUTHOR]

Rates of free radical initiation were determined at 20°C in 10 mM phosphate buffer (pH 7.4) in the systems metmyoglobin (methemoglobin)–H2O2 using 2,2"-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) as the diammonium salt (ABTS). The catalytic activity of MetMb was 2-3-fold higher than that of MetHb. The process can be described by the Michaelis–Menten equation, from which effective values of Km and Vmax were calculated. Comparative kinetic studies on the inhibition of ABTS oxidation were carried out using Trolox, propylgallate (PG), polydisulfide of gallic acid (poly(DSG)), polydisulfide of (2-amino-4-nitrophenol) (poly(ADSNP)), and its conjugate with human serum albumin (HSA–poly(ADSNP)). The inhibitors were characterized by inhibition constants Ki and stoichiometric inhibition coefficients f (the number of radicals terminated by a single molecule of inhibitor). The minimum Ki and the maximum f values were obtained for poly(DSG), and in the system of MetHb–H2O2–ABTS they were 0.08 μM and 27.5, respectively. According to their antiradical activities, the inhibitors can be arranged as follows: poly(DSG) > poly(ADSNP) > PG > Trolox. PG, poly(DSG), poly(ADSNP), and its conjugate with HSA are suggested as “calibrators”, i.e., inhibition standards for evaluation of antioxidant status of biological fluids in possible test systems based on the free radical-generating pair MetMb–H2O2 with ABTS as the acceptor of the active radicals. [ABSTRACT FROM AUTHOR]

A resonant mirror biosensor was used to study cyclic nucleotide-receptor interactions. In particular, a novel method was developed to determine inhibition constants ( Ki) from initial rates of ligate association to immobilized ligand. This approach was applied to the comparison of cyclic nucleotide-binding properties of the wild-type isolated B domain of the cAMP-dependent protein kinase type Iα regulatory subunit and its Ala-334-Thr (A334T) variant that has altered cyclic nucleotide specificity. A cUMP-saturated form of the B domain was used for all measurements. Under the conditions used, cUMP did not affect the kinetics of B domain association to immobilized cAMP. Triton X-100 was required to stabilize the protein at nanomolar concentrations. The association and dissociation rate constants for wild-type and A334T B domains yielded equilibrium dissociation constants of 11 and 16 nM. Heterogeneity of ligate and immobilized ligand, mass transport effects, and other factors were evaluated for their influence on biosensor-determined kinetic constants. Biosensor-determined relative inhibition constants ( K'i = KicAMP/ K'ianalog) for 16 cyclic nucleotide analogs correlated well with those determined by a [3H]cAMP binding assay. Previously published K'i values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The K'i values for the wild-type and A334T B domains were essentially unchanged except for dramatic enhancements in affinity of cGMP analogs for the A334T B domain. These observations validate the isolated B domain as a simple model system for studying cyclic nucleotide-receptor interactions. [ABSTRACT FROM AUTHOR]

The effect of imperatorin on hepatic microsomal mixed function oxidases (MFO) was investigated. On acute treatment, imperatorin (30 mg/kg, i.p.) caused a significant reduction in activities of hepatic aminopyrine N-demethylase, hexobarbital hydroxylase and aniline hydroxylase as well as cytochrome p-450 content in rats and mice. Kinetic studies on rat liver enzymes revealed that imperatorin appeared to be a competitive inhibitor of aminopyrine N-demethylase (Ki, 0.007 mM), whereas a non-competitive inhibitor of hexobarbital hydroxylase (Ki, 0.0148mM). Imperatorin also inhibited non-competitively aniline metabolism (Ki, 0.2mM). Imperatorin binds to phenobarbital-induced cytochrome p-450 to give a typical type 1 binding spectrum (max. 388nm, min 422nm). Multiple administrations of imperatorin (30 mg/kg, i.p. daily for 7 days) to mice shortened markedly the duration of hexobarbital narcosis and increased activities of hepatic aminopyrine N-demethylase and hexobarbital hydroxylase and the level of cytochrome p-450 whereas aniline hydroxylase activity was unaffected. [ABSTRACT FROM AUTHOR]

A new simple graphical method is described for the determination of inhibition type and inhibition constants of an enzyme reaction without any replot. The method consists of plotting experimental data as ( V– v)/ v versus the inhibitor concentration at two or more concentrations of substrate, where V and v represent the maximal velocity and the velocity in the absence and presence of inhibitor with given concentrations of the substrate, respectively. Competitive inhibition gives straight lines that converge on the abscissa at a point where [I] = − K i. Uncompetitive inhibition gives parallel lines with the slope of 1/ K’ i. For mixed type inhibition, the intersection in the plot is given by [I] = − K i and ( V–v)/ v = − K i /K’ i in the third quadrant, and in the special case where K i = K’ i (noncompetitive inhibition) the intersections occur at the point where [I] = − K i and ( V– v)/ v = −1. The present method, the “quotient velocity plot,” provides a simple way of determining the inhibition constants of all types of inhibitors. [ABSTRACT FROM AUTHOR]

This chapter deals with practical considerations on key issues such as choosing an enzyme source, determining linear conditions, and choosing appropriate substrate and organic solvent concentrations. Practical solutions for working with limited resources and carrying out inhibition experiments are also addressed. Thus, after reading this chapter, the novice reader should have a better idea of how to design, develop, and interpret basic experiments using drug metabolism enzymes., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)