The human carbonic anhydrase isoenzymes I and II inhibitory effects of some hydroperoxides,alcohols, and acetates

The carbonic anhydrases (CAs, EC 4.2.1.1) represent a superfamily of widespread enzymes, which catalyze a crucial biochemical reaction, the reversible hydration of carbon dioxide to bicarbonate and protons. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. In this study, a series of hydroperoxides, alcohols, and acetates were tested for the inhibition of the cytosolic hCA I and II isoenzymes. These compounds inhibited both hCA isozymes in the low nanomolar ranges. These compounds were good hCA I inhibitors (K_is in the range of 24.93–97.99?nM) and hCA II inhibitors (K_is in the range of 26.04–68.56?nM) compared to acetazolamide as CA inhibitor (K_i: 34.50?nM for hCA I and K_i: 28.93?nM for hCA II).     

Synthesis and biological evaluation of aminomethyl and alkoxymethyl derivatives as carbonic anhydrase,acetylcholinesterase and butyrylcholinesterase inhibitors

Compounds containing nitrogen and sulfur atoms can be widely used in various fields such as industry, medicine, biotechnology and chemical technology. Therefore, the reactions of aminomethylation and alkoxymethylation of mercaptobenzothiazole, mercaptobenzoxazole and 2-aminothiazole were developed. Additionally, the alkoxymethyl derivatives of mercaptobenzoxazole and 2-aminothiazole were synthesized by a reaction with hemiformals, which are prepared by the reaction of alcohols and formaldehyde. In this study, the inhibitory effects of these molecules were investigated against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) enzymes and carbonic anhydrase I, and II isoenzymes (hCA I and II). Both hCA isoenzymes were significantly inhibited by the recently synthesized molecules, with K_i values in the range of 58–157?nM for hCA I, and 81–215?nM for hCA II. Additionally, the K_i parameters of these molecules for BChE and AChE were calculated in the ranges 23–88 and 18–78?nM, respectively.     

Kinetics of the inhibition of acetylcholinesterase from pigeon brain by procaine hydrochloride

The kinetic parameters of the inhibition of pigeon brain acetylchlolinesterase (AChE) by procaine hydrochloride were investigated. Procaine (0·083–1·67 mM) reversibly inhibited AChE activity (15–83 percent) in a concentration dependent manner, the IC₅₀ being about 0·38 mM. The Michaelis-Menten constant (K_m) for the hydrolysis of acetylthiocholine iodide was found to be 1·53 × 10^?4 M and the V_max was 1·06 μmol min^?1 mg^?1 protein. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition is of the linear mixed type which is considered to be a mixture of partial competitive and pure non-competitive. The values of K_i(slope) and K_i (intercepts) were estimated as 0·14 mM and 0·22 mM respectively by the primary Dixon and by the secondary replots of the Lineweaver-Burk plot. The K_i′/K_i ratio shows that procaine has a greater affinity of binding for the peripheral than for the active site.     

The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles

New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A₁ and/or A_2A receptor subtypes. On the whole, the novel derivatives 1–24 shared scarce or no affinities for the off-target hA_2B and hA₃ ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (K_i =?150?nM) and the best selectivity for the hA_2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA_2A (K_i =?123?nM) and A₁ AR affinity (K_i =?25?nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (K_i =?11?nM) and good selectivity for the hA₁ AR. The 5-(N⁴-substituted-piperazin-1-yl) derivatives 15–24 bind the hA₁ AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.     

Synthesis,carbonic anhydrase I and II inhibition studies of the 1,3,5-trisubstituted-pyrazolines

4-(3-(4-Substituted-phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) benzenesulfonamides (9–16) were successfully synthesized and their chemical structures were confirmed by ¹H NMR, ¹³C NMR, and HRMS spectra. Carbonic anhydrase I and II inhibitory effects of the compounds were investigated. K_i values of the compounds were in the range of 316.7?±?9.6–533.1?±?187.8?nM towards hCA I and 412.5?±?115.4–624.6?±?168.2?nM towards hCA II isoenzymes. While K_i values of the reference compound Acetazolamide were 278.8?±?44.3?nM and 293.4?±?46.4?nM towards hCA I and hCA II izoenzymes, respectively. Compound 14 with bromine and compound 13 with fluorine substituents can be considered as the leader compounds of the series because of the lowest K_i values in series to make further detailed carbonic anhydrase inhibiton studies.     

Vinyl functionalized 5,6-dimethylbenzimidazolium salts: Synthesis and biological activities

A series of vinyl functionalized 5,6-dimethylbenzimidazolium salts are synthesized. All compounds were fully characterized by elemental analyses, MS, ¹H-NMR, ¹³C-NMR, and IR spectroscopy techniques. Enzyme inhibition is a very active area of research in drug design and development. In this study, the synthesized novel benzimidazolium salts were evaluated toward the human erythrocyte carbonic anhydrase I (hCA I), and II (hCA II) isoenzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. They demonstrated highly potent inhibition ability against hCA I with K_i values of 484.8 ± 62.6–1389.7 ± 243.2 nM, hCA II with K_i values of 298.9 ± 55.7–926.1 ± 330.0 nM, α-glycosidase with K_i values of 170.3 ± 27–760.1 ± 269 μM, AChE with K_i values of 27.1 ± 3–77.6 ± 1.7 nM, and BChE with K_i values of 21.0 ± 5–61.3 ± 15 nM. As a result, novel vinyl functionalized 5,6-dimethylbenzimidazolium salts (1a–g) exhibited effective inhibition profiles toward studied metabolic enzymes. Therefore, we believe that these results may contribute to the development of new drugs particularly to treat some global disorders including glaucoma, Alzheimer's disease, and diabetes.     

Properties of brain L-glutamate decarboxylase: inhibition studies

—l -Glutamate decarboxylase purified from mouse brain was found to be highly sensitive to the sulfhydryl reagents, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) and p-chloromerburibenzoate (PCMB), which were competitive inhibitors (K_i for DTNB is 1·1 · 10^?8m ). Iodoacetamide and iodoacetic acid were less effective inhibitors than DTNB and PCMB. The mercapto acids, 3-mercaptopropionic, 2-mercaptopropionic, and 2-mercaptoacetic acids were potent competitive inhibitors with K_i values of 1·8, 53 and 300 μm , respectively. 2-Mercaptoethanol was less effective. Aminooxyacetic acid was the most potent carbonyl-trapping reagent tested inhibiting the enzyme activity completely at 1·6 μm , followed by hydroxylamine, hydrazine, semicarbazide, and d -penicillamine. Carboxylic acids with a net negative charge were strong competitive inhibitors e.g. d -glutamate (K_i 0·9 mm ), α-ketoglutarate (K_i, l·2mm ), fumarate (K_i,1·8 mm ), dl -β-hydroxyglutamate (K_i, 2·8 mm ), l -aspartate (k_i, 3·1 mm ) and glutarate (K_i, 3·5 mm ). 2-Aminophosphonobutyric and 2-aminophosphonopropionic acids, phosphonic analogs of glutamate and aspartate, respectively, had no effect at l0mm . γ-Aminobutyric acid, l -glutamine, l -γ-methylene-glutamine, and α,γ-diaminoglutaric acid, amino acids with no net negative charge at neutral pH, had no effect at 5 mm . Glutaric and α-ketoglutaric acids were the most potent inhibitors among the various dicarboxylic and α-keto-dicarboxylic acids tested (K_i, 3·5 and 1·2 mm , respectively). Compounds with one carbon less, succinic and oxalacetic acids, or with one carbon more, adipic and α-ketoadipic acids, were less inhibitory. The monovalent cations, Li⁺, Na⁺, NH₄⁺, and Cs⁺ had no effect on l -glutamate decarboxylase activity in concentrations up to 10mm . Divalent cations, on the other hand, were very potent inhibitors. Among eleven divalent cations tested, Zn²⁺ was the most potent inhibitor, inhibiting to the extent of 50 per cent at 10μm . The decreasing order of inhibitory potency was: Zn²⁺ > Cd²⁺, Hg²⁺, Cu²⁺ > Ni²⁺ > Mn²⁺ Co²⁺ > Ba²⁺ > Ca²⁺ > Mg²⁺ > Sr⁺², The anions, I^?, Br^?, Cl^? and F^? were only weak inhibitors. The K_i value for Cl^? was 17mm . The above findings suggest minimally the presence of aldehyde, sulfhydryl and positively charged groups at or near the active site of the holoenzyme. Intermediates of glycolysis had little effect on l -glutamate decarboxylase activity, but intermediates of the tricarboxylic acid cycle, e.g. α-ketoglutarate (K_i= 1·2 mm ) and fumarate (K_i= 1·8 mm ) were relatively potent inhibitors. The nucleotides, ATP, ADP, AMP, cyclic AMP, GTP, GDP, GMP, and cyclic GMP were weak inhibitors. l -Norepinephrine (K_i= 1·3 mm ) and serotonin were potent inhibitors, while acetylcholine, dopamine and histamine were less effective. Ethanol and dioxane inhibited the enzyme activity to the extent of 20-50 per cent at 10 per cent (v/v), while slight activation was observed at low concentrations (0·1-1 per cent) of both solvents. The possible role of Zn²⁺ and some metabolites in the regulation of steady-state levels of γ-aminobutyric acid also was discussed.     

A graphical method for determining inhibition constants

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.     

Characterization of the G protein coupling of SRIF and β-adrenergic receptors to the maxi KCa channel in insulin-secreting cells

Modulation of the Ca- and voltage-dependent K channel—K_Ca—by receptors coupled to the G proteins G _i /G _o and G _s has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the K_Ca channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous subunits of either G _i or G _o -type G proteins also inhibited the K_Ca channel (IC₅₀ 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses K_Ca channel activity via a membrane-delimited pathway that involves the subunits of PTX-sensitive G proteins G _i and/or G _o . In outside-out patches, activation of G _s either by -agonists or with cholera toxin (CTX) increased K_Ca channel activity, consistent with a membrane-delimited stimulatory pathway linking the -adrenergic receptor to the K_Ca channel via G _s . In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of -agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous _i or _o . Taken together these data suggest that K_Ca channel activity is enhanced by activation of G _s and blocked by activated G _i and/or G _o . Further, K_Ca channel stimulation by activated G _s may be direct, while inhibition by G _i /G _o may involve deactivation of G _s . In inside-out patches K_Ca channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the K_Ca channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous _i or _o . These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G _i /G _o -induced deactivation of G _s .The authors would like to thank Dr. S. Ciani for many helpful discussions, Dr. A.E. Boyd III for supplying the HIT cells, Drs. J. Codina and L. Birnbaumer for supplying the alpha subunits of the G proteins G _i and G _o , and Mrs. Satoko Hagiwara for preparing and maintaining the cell cultures.This work was supported by grant DCB-8919368 from the National Science Foundation and a research grant (W-P 880513) from the American Diabetes Association to B.R., and by grant RO1-DK39652 from the National Institutes of Health to G.T.E.     

Kinetics of phosphate absorption by mycorrhizal and non-mycorrhizal Scots pine seedlings

Kinetics of net phosphate (P_i) uptake was measured on intact ectomycorrhizal and non‐mycorrhizal Pinus sylvestris seedlings using a semihydroponic cultivation method. The depletion of P_i in a nutrient solution was assessed over a 160–0.2 μM P_i gradient. Growth of the pine seedlings was P limited and measurements were performed 7 and 9 weeks after inoculation. Three ectomycorrhizal fungi were studied: Paxillus involutus, Suillus bovinus and Thelephoraterrestris. P_i uptake was extremely fast in plants colonised by P. involutus. The P_i concentration dropped below 0.2 μM within 4–5 h. In plants colonised with S. bovinus this occurred in 5–6 h and in plants associated with T. terrestris 8 h were needed to run through the whole concentration range. Non‐mycorrhizal plants of similar size and nutrient status decreased P_i to a concentration between 1 and 2 μM in 18 h. Data were curve fitted to a two‐phase Michaelis‐Menten equation. The apparent kinetic constants, K_m and V_max, for the high affinity P_i uptake system of the pine roots could be estimated accurately. V_max of this system was up to 7 times higher in pines associated with P. involutus than in non‐mycorrhizal seedlings. The intact extraradical mycelium greatly increased the absorption surface area of the roots (V_max). Non‐mycorrhizal plants had a K_m between 7.8 and 16.4 μM P_i. Plants mycorrhizal with P. involutus had K_m values between 2.4 and 7.2, plants colonised with S. bovinus had a K_m between 5.1 and 12.3, and seedlings associated with T. terrestris had a K_m from 4.6 to 10.1 μM P_i. All 3 ectomycorrhizal fungi had a strong impact on the P_i absorption capacity of the pine seedlings. The results also demonstrated that there is substantial heterogeneity in kinetic parameters among the different mycorrhizal root systems.     

Malathion detoxification by human hepatic carboxylesterases and its inhibition by isomalathion and other pesticides

The organophosphorothioate (OPT) pesticide malathion (MAL) in mammals is readily hydrolyzed by mammalian carboxylesterases (CE). The reaction competes with the CYP‐catalyzed formation of malaoxon (MOX), the toxic metabolite. Alterations or individual variations in CE activity may result in increased MOX formation, enhancing MAL toxicity. We have characterized the human hepatic CE activity in a panel of 18 human liver microsomes as well as the inhibitory effect of IsoMAL, a major impurity of MAL commercial formulations, parathion (PAR), chlorpyrifos (CPF), and chlorpyrifos‐oxon (CPFO). CE activity showed a low level of variation among individuals (4‐fold). The reaction consists of two different phases, differing in their affinity for MAL (K_m1 = 0.25–0.69 μM; K_m2 = 10.3–26.8 μM). The relatively low K_m1 values confirmed that human CE efficiently detoxify MAL. IsoMAL was shown to be a potent noncompetitive inhibitor of MAL detoxification (K_i = 0.6 μM), with a higher inhibitory potency than CPF and PAR (K_i = 7.5 μM and 50 μM, respectively). These two latter compounds very likely act as mixed inhibitors. CPFO showed the highest inhibitory potency toward CE‐mediated detoxification, being characterized by a K_i = 22 nM. The present results provide useful information for a better understanding of possible interactions between different OPTs and for assessing the potential cumulative risk for exposure to OPT mixtures. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:406–414, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20106     

Kinetic study of the quenching reaction of singlet oxygen by α-, β-, γ-, δ-tocotrienols,and palm oil and soybean extracts in solution

Measurements of the singlet oxygen (¹O₂) quenching rates (k_Q (S)) and the relative singlet oxygen absorption capacity (SOAC) values were performed for 11 antioxidants (AOs) (eight vitamin E homologues (α-, β-, γ-, and δ-tocopherols and -tocotrienols (-Tocs and -Toc-3s)), two vitamin E metabolites (α- and γ-carboxyethyl-6-hydroxychroman), and trolox) in ethanol/chloroform/D₂O (50:50:1, v/v/v) and ethanol solutions at 35?°C. Similar measurements were performed for five palm oil extracts 1–5 and one soybean extract 6, which included different concentrations of Tocs, Toc-3s, and carotenoids. Furthermore, the concentrations (wt%) of Tocs, Toc-3s, and carotenoids included in extracts 1–6 were determined. From the results, it has been clarified that the ¹O₂-quenching rates (k_Q (S)) (that is, the relative SOAC value) obtained for extracts 1–6 may be explained as the sum of the product {Σ k_Q^AO-i (S) [AO-i]/100} of the rate constant (k_Q^AO-i (S)) and the concentration ([AO-i]/100) of AO-i (Tocs, Toc-3s, and carotenoid) included.     

Ethanol production using nuclear petite yeast mutants

Two respiratory-deficient nuclear petites, FY23Δpet191 and FY23Δcox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23ρ⁰. Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant. K _i, of 2.3% (w/v) and a specific rate of ethanol production, q _p, of 0.90 g ethanol g dry cells⁻¹ h⁻¹. FY23ρ⁰ was the most sensitive to ethanol, exhibiting a K _i of 1.71% (w/v) and q _p of 0.87 g ethanol g dry cells⁻¹ h⁻¹. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23Δpet191, having a K _i of 2.14% (w/v) and the 85% respiratory-deficient FY23Δcox5a, having a K _i of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23ρ⁰ is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation. Received: 22 September 1997 / Accepted: 7 December 1997     

Sensitivity of Patterns of Molecular Evolution to Alterations in Methodology: A Critique of Hughes and Yeager

Employing a set of 43 othologous mouse and rat genes, Hughes and Yeager (J. Mol. Evol. 45:125–130, 1997) reported (1) no correlation between synonymous and nonsynonymous rates of nucleotide substitution, (2) a positive correlation between intronic GC contents (GC _i) and intronic substitution rates (K _i), (3) that the average K _i value was very similar to the average K _s value, and (4) that the compositional correlation between the rat and the mouse genes is stronger at the third codon position (GC₃) than at the first and second codon positions (GC₁₂). We have examined the robustness of these results to alterations in substitution rate estimation protocol, alignment protocol, and statistical procedure. We find that a significant correlation between K _a and K _s is observed either if a rank correlation statistic is used instead of regression analysis, if one outlier is excluded from the analysis, or if a regression weighted by gene size is employed. The correlation between K _i and GC _i we find to be sensitive to changes in alignment protocol and disappears on the use of weighted means. The finding that K _s and K _i are approximately the same is dependent on the method for estimating K _s values. Finally, the variance around the regression line of rat GC₃ versus mouse GC₃ we find to be significantly higher than that in GC₁₂. The source of the discrepancy between this and Hughes and Yeager's result is unclear. The variance around the line for GC₄ is higher still, as might be expected. Using a methodology that may be considered preferable to that of Hughes and Yeager, we find that all four of their results are contradicted. More importantly this analysis reinforces the need for caution in assembling and analyzing data sets, as the degree of sensitivity to what many might consider minor methodological alterations is unexpected. Received: 2 February 1998 / Accepted: 23 March 1998     

Purification and kinetic properties of ATP: citrate oxaloacetate lyase from rat brain.

Abstract— A method for a partial purification of ATP:citrate oxaloacetate lyase from rat brain is described. The Lineweaver–Burk plots of velocity vs citrate concentration are biphasic in the presence of fixed concentrations of MgCl₂. Therefore two values of K_m, corresponding to low and high concentrations of citrate, can be determined. When MgCl₂ is added in equimolar concentrations with citrate, a monophasic plot with one K_m of 0.13 mm is obtained. The K_m value for MgATP^2- was independent of citrate concentration, being equal to 0.40–0.43 mm. The K_m for CoA was 0.0007 mm. ADP and P_i are competitive inhibitors with respect to ATP. K_i for MgADP⁻ is equal to 0.13 mm. dl -isocitrate and cis-aconitate are partially competitive inhibitors with respect to citrate with K_i values of 5.8 and 4.8 mm, respectively. α-Ketoglutarate and pyruvate are noncompetitive inhibitors with respect to ATP and citrate, with K_i values equal to 9 and 45 mm, respectively. The physiological significance of these effectors for the regulation of citrate lyase activity in brain is discussed.     

Syntheses of 1-[2-(Phosphonomethoxy)Alkyl]Thymine Monophosphates and an Evaluation of their Inhibitory Activity Toward Human Thymidine Phosphorylase

A series of new monophosphates of 1-[2-(phosphonomethoxy)alkyl]thymines, such as PMPTp_, 3-MeO-PMPTp, HPMPTp, and FPMPTp, were synthesized and tested for their ability to inhibit human thymidine phosphorylase. Kinetic measurements of enzyme activity were performed using thymidine and inorganic phosphate as the substrates. The data show that some monophosphates provide a considerable increase of the multisubstrate inhibitory effect. The highest inhibitory potency was found with (R)-FPMPTp 4c (K _i ^dT = 4.09 ± 0.47 μM, K _i(P_i) = 2.13 ± 0.29 μM) and (R) 3-MeO-PMPTp 4d (K _i ^dT = 5.78 ± 0.71 μM, K _i(P_i) = 2.71 ± 0.37 μM).     

Synthesis and inhibitory properties of some carbamates on carbonic anhydrase and acetylcholine esterase

A series of carbamate derivatives were synthesized and their carbonic anhydrase I and II isoenzymes and acetylcholinesterase enzyme (AChE) inhibitory effects were investigated. All carbamates were synthesized from the corresponding carboxylic acids via the Curtius reactions of the acids with diphenyl phosphoryl azide followed by addition of benzyl alcohol. The carbamates were determined to be very good inhibitors against for AChE and hCA I, and II isoenzymes. AChE inhibition was determined in the range 0.209–0.291?nM. On the other hand, tacrine, which is used in the treatment of Alzheimer’s disease possessed lower inhibition effect (K_i: 0.398?nM). Also, hCA I and II isoenzymes were effectively inhibited by the carbamates, with inhibition constants (K_i) in the range of 4.49–5.61?nM for hCA I, and 4.94–7.66?nM for hCA II, respectively. Acetazolamide, which was clinically used carbonic anhydrase (CA) inhibitor demonstrated K_i values of 281.33?nM for hCA I and 9.07?nM for hCA II. The results clearly showed that AChE and both CA isoenzymes were effectively inhibited by carbamates at the low nanomolar levels.     

Comparative study of the effects of cromakalim (BRL 34915) and diazoxide on membrane potential, [Ca2+] i and ATP-sensitive potassium currents in insulin-secreting cells

Summary Patch-clamp and single cell [Ca²⁺] _i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of –62±2 mV (n=42) and an average basal [Ca²⁺] _i of 102±6nm (n=37), glucose (2.5–10mm): (i) depolarized the membrane, through a decrease in the outward K_ATP current, (ii) evoked Ca²⁺ spike potentials, and (iii) caused a sharp rise in [Ca²⁺] _i . In the continued presence of glucose both cromakalim (100–200 m) and diazoxide (100 m) repolarized the membrane, terminated Ca²⁺ spike potentials and attenuated the secretagogue-induced rise in [Ca²⁺] _i . In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K⁺ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K⁺ channels, reversed the effects of cromakalim on membrane potential and K_ATP currents.     

Branched respiratory chain in aerobically grown Staphylococcus aureus —oxidation of ethanol by cells and protoplasts

Addition of ethanol and some other primary alcohols, except methanol, to cells and protoplasts (but not membrane particles) considerably stimulated the rate of oxygen consumption. This additional respiration was strongly inhibited by 0.1 mM KCN. The cyanide inhibition curve of endogenous substrate oxidation was slightly biphasic while in the presence of ethanol it became clearly biphasic having K _i values of approx. 0.1 and 0.5 mM. Based on the steady-state cytochrome spectra in the presence of 0.1 mM KCN, we attributed the lower K _i to cytochrome a ₆₀₂. Proteolysis of protoplasts external membrane proteins did not change the rate of endogeneous substrate oxidation but prevented the inhibition of this respiration by low concentrations of KCN and stimulation of oxygen consumption by ethanol. The activity of NAD⁺-dependent ethanol dehydrogenase in the cytoplasm was found to be 520 nmol NADH-x min^–1 x mg^–1 protein. Proteolysis of external membrane proteins apparently inhibits the operation of the cytochrome a ₆₀₂-containing electron transport branch inducing the suppression of electron flow from NADH to oxygen.     

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro

Elevated glutathione transferase (GST) E2 activity is associated with DDT resistance in the mosquito Anopheles gambiae. The search for chemomodulators that inhibit the function of AgGSTE2 would enhance the insecticidal activity of DDT. Therefore, we examined the interaction of novel natural plant products with heterologously expressed An. gambiae GSTE 2 in vitro. Five of the ten compounds, epiphyllocoumarin (Tral-1), knipholone anthrone, isofuranonaphthoquinones (Mr 13/2, Mr13/4) and the polyprenylated benzophenone (GG1) were shown to be potent inhibitors of AgGSTE2 with IC₅₀ values of 1.5 μM, 3.5 μM, 4 μM, 4.3 μM and 4.8 μM respectively. Non-competitive inhibition was obtained for Tral 1 and GG1 with regards to GSH (K_i of 0.24 μM and 0.14 μM respectively). Competitive inhibition for Tral1 was obtained with CDNB (K_i = 0.4 μM) whilst GG1 produced mixed type of inhibition. The K_i and K_i' for GSH for Tral-1 and GG1 were 0.2 μM and 0.1 μM respectively. These results suggest that the novel natural plant products, particularly Tral-1, represent potent AgGSTE2 in vitro inhibitors.