Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. KI, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k3, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. KI values of 1.3 x 10(-4) M(-1), 5.6 x 10(-6) M(-1) and 7.2 x 10(-6)M(-1) were obtained for malathion, malaoxon and isomalathion, respectively. The IC50 values for free/immobilized AChE, (3.7 +/- 0.2) x 10(-4) M/(1.6 +/-0.1) x 10(-4), (2.4 +/- 0.3) x 10(-6)/(3.4 +/- 0.1) x 10(-6)M and (3.2 +/- 0.3) x 10(-6) M/(2.7 +/- 0.2) x 10(-6) M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 x 10(-7) M/2 x 10(-7) M, 2 x 10(-7) M/3 x 10(-7)M and 2 x 10(-7) M/4.5 x 109-7) M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.     

Inhibition of AChE by single and simultaneous exposure to malathion and its degradation products

In vitro inhibition of bovine erythrocytes acetylcholinesterase (AchE) by separate and simultaneous exposure to organophosphorous insecticide malathion and the transformation products, which are generally formed during the storage or natural as well as photochemical degradation pathways of malathion, was investigated. The increasing concentration of malathion, its oxidation product - malaoxon and isomerisation product - isomalathion inhibited AChE activity in a concentration-dependent manner. The half-maximum inhibitory concentrations (IC(50) values): (3.2 +/- 0.1) x 10(-5) mol/l, (4.7 +/- 0.8) x 10(-7) mol/l and (6.0 +/- 0.5) x 10(-7)mol/l were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl phosphorodithioic ester (OOS(S)) and O,O-dimethyl thiophosphate did not noticeably affect the enzyme activity at all investigated concentrations, while diethyl maleate inhibited the AChE activity at concentrations >10 mmol/l. By simultaneous exposure of the enzyme to malaoxon and isomalathion in various concentration combinations the additive effect was achieved by low concentration of inhibitors, while the antagonistic effect was obtained at high concentration (>or= 3 x 10(-7) mol/l) of inhibitors. Inhibitory power of irradiated samples of 1 +/- 10(-5) mol/l malathion can be attributed to the formation of malaoxon and isomalathion, organophosphates about 100 times more toxic than their parent compound, while the presence of non-inhibiting degradation product OOS(S) did not affect the inhibitor efficiency of inhibiting malathion by-products, malaoxon and isomalathion.     

Acetylcholinesterase from the horn fly (Diptera: Muscidae) II: Biochemical and molecular properties

Purified acetylcholinesterase (AChE) of the horn fly was characterized to elucidate the enzymological, inhibitory, and molecular properties of the enzyme. Maximum activity of the AChE against the substrate acetylthiocholine (ATCh) occurred when reactions were conducted at 37°C and pH 7.5. Km and V_max values were (9.2 ± 0.35) × 10^?6 M and 239.8 ± 10.8 units/mg, respectively, for ATCh and (1.5 ± 0.07) × 10^?5 M and 138.5 ± 5.5 units/mg, respectively, for butyrylthiocholine (BTCh). The activity of AChE decreased when concentrations of ATCh or BTCh were higher than 1 mM. Studies of the interaction of AChE with different inhibitors revealed pl₅₀ values of 8.88 for eserine, 6.90 for BW284C51, and 4.97 for ethopropazine. Bimolecular reaction constants (k_is) for the organophosphorus (OP) anticholinesterases were (2.74 ± 0.14) × 10⁶ M^?1 min^?1 for coroxon, (7.20 ± 0.28) × 10⁵ M^?1 min^?1 for paraoxon, and (2.33 ± 0.12) × 10⁵ M^?1 min^?1 for stirofos. Two major forms of native AChE molecules were found on non-denaturing polyacrylamide gel electrophoresis (PAGE) with Triton X-100, corresponding to bands AChE-2 and AChE-4 found on PAGE without Triton X-100. AChE-2 had an estimated molecular weight of 603,000 and was amphiphilic. AChE-4 had a molecular weight of 147,000 and was hydrophilic. Results of PAGE analyses indicated that the purified enzyme had two bands, one of about 123 kDa and the other greater than 320 kDa, prior to disulfide reduction and only one band at about 54 kDa after reduction on SDS-PAGE. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Acetylcholine potentiation of field stimulated rat vas deferens: A pre-synaptic muscarinic mechanism?

Acetylcholine (ACh) was found to markedly enhance the nerve stimulation induced twitch response of isolated, field-stimulated rat vas deferens (RVD). The ED₂₀₀ (concentration which enhances the twitch response to 200% of control) for this potentiation was 6 × 10^?6M with the maximum twitch response being increased by more than 3 fold (325 ± 30%). Carbachol (ED₂₀₀ = 8.5 × 10^?7M) showed identical results. With each drug the potentiation was competitively antagonized by atropine (10^?7?10^?5M). Physostigmine 10^?8?10^?6M) both enhanced the basal twitch response (215 ± 8% of control at 10^?5M) and the sensitivity of the RVD to ACh (ED₂₀₀ = 3.3 × 10^?7M) but not to carbachol. Atropine, on the other hand reduced the basal twitch response by 18 ± 3% at 10^?5M. Hemicholinium (10^?4M) also reduced the basal twitch responses by 23 ± 5%. ACh (10^?7M?10^?5M) did not modify the responses of unstimulated RVD to norepinephrine or KCl suggesting a pre-synaptic site of action. Taken together these results are compatible with the presence of a pre-junctional, excitatory muscarinic mechanism in the field stimulated RVD. That this cholinergic system may be of physiological significance is supported by the observations that atropine and hemicholinium depress while physostigmine enhances the twitch response in the absence of exogenous ACh.     

In vitro studies on the genotoxicity of the organophosphorus insecticide malathion and its two analogues

Malathion [S-(1,2-dicarboethoxyethyl)O,O-dimethyl phosphorodithioate] is a commonly used organophosphorus insecticide reported to be genotoxic both in vivo and in vitro, but the reports are conflicting. In order to elucidate the genotoxic potency of the main compounds present in commercial preparations of malathion, the DNA-damaging effect of this insecticide, its major metabolite malaoxon [S-(1,2-dicarboethoxyethyl)O,O-dimethyl phosphorothiolate] and its isomer isomalathion [S-(1,2-dicarboethoxyethyl)O,S-dimethyl phosphorodithioate], all at purity of at least 99.8%, was investigated by use of the alkaline single cell gel electrophoresis (comet assay). Freshly isolated human peripheral blood lymphocytes were incubated with 25, 75 and 200 microM of the chemicals for 1 h at 37 degrees C. The concentrations used are comparable to those found in blood following various non-lethal human exposures to pesticides. Malathion did not cause any significant changes in the comet length of the lymphocytes, throughout the range of concentrations tested. Malaoxon and isomalathion introduced damage to DNA in a dose-dependent manner. The effect induced by malaoxon was more pronounced than that caused by isomalathion. Treated cells were able to recover within a 60-min incubation in insecticide-free medium at 37 degrees C except the lymphocytes exposed to malaoxon at 200 microM, which did not show measurable DNA repair. The latter result suggests a considerable cytotoxic effect (cell death) of malaoxon at the highest concentration used. The reported genotoxicity of malathion might, therefore, be a consequence of its metabolic biotransformation to malaoxon or the presence of malaoxon and/or isomalathion as well as other unspecified impurities in commercial formulations of malathion. In this regard, the results of our study clearly indicate that malathion used as commercial product, i.e., containing malaoxon and isomalathion, can be considered as a genotoxic substance in vitro. This means that it may also produce DNA disturbances in vivo, such as DNA breakage at sites of oncogenes or tumor suppressor genes, thus playing a role in the induction of malignancies in individuals exposed to this agent. Therefore, malathion can be regarded as a potential mutagen/carcinogen and requires further investigation.     

Heterologous expression,purification, and biochemical characterization of a greenbug (Schizaphis graminum) acetylcholinesterase encoded by a paralogous gene (ace‐1)

Acetylcholinesterase is a critical enzyme in the regulation of cholinergic neurotransmission in insects. To produce Schizaphis graminum acetylcholinesterase‐1 for structure–function analysis, we constructed a recombinant baculovirus to infect Sf9 cells, which secreted the soluble protein at a final concentration of 4.0 mg/L. The purified enzyme had an apparent M_r of 70 and 130 kDa in the reducing and nonreducing SDS‐polyacrylamide gels, respectively, indicating that it formed a dimer via an intermolecular disulfide bond. The fresh enzyme had a specific activity of 245 U/mg, which stabilized at a lower level (115 U/mg) in storage. The Michaelis constant and maximum velocity were 88.3 ± 9.6 μM and 133.2 ± 1.6 U/mg for acetylthiocholine iodide, 113.9 ± 12.5 μM and 106.4 ± 3.0 U/mg for acetyl(β‐methyl)thiocholine iodide, 68.9 ± 7.8 μM and 76.7 ± 1.0 U/mg for propionylthiocholine iodide, and 201.1 ± 21.0 μM and 4.4 ± 0.1 U/mg for S‐butyrylthiocholine iodide, respectively. The IC₅₀ values (5 min, room temperature) of ethopropazine, BW284C51, carbaryl, eserine, malaoxon, and paraoxon were 102, 1.66, 0.94, 0.20, 0.061, 0.016 μM, respectively. The bimolecular reaction constants (k_i) were (6.50 ± 0.40) × 10⁴ for carbaryl, (1.00 ± 0.16) × 10⁵ for eserine, (4.70 ± 0.13) × 10⁵ for malaoxon, and (9.06 ± 0.23) × 10⁵ M^?1 min^?1 for paraoxon. The enzyme was also inhibited by one of its products, choline, at concentrations higher than 20 mM, suggesting that choline bound to an anionic site and regulated the enzymatic activity. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:51–59, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20311     

Effects of papaverine on basal and on isoproterenol-stimulated renin secretion from rat kidney slices

Papaverine inhibited the basal renin secretion of rat kidney slices incubated in a physiological salt solution at 37°C. Inhibition was concentration-dependent; secretion was 99 ± 0.2 % inhibited by 5 × 10^?4 M papaverine, and 8 × 10^?5 M was the estimated ED₅₀. In contrast, 2 × 10^?4 M IBMx (3-isobutyl-1-methyl-xanthine) increased the rate of secretion from 215 ± 17 to 366 ± 30 ng hr^?1mg^?1/20 min (p < 0.001). Isoprotenol (4 × 10^?7, 8 × 10^?7, and 5 × 10^?6 M) stimulated renin secretion in a concentration-dependent manner; the stimulatory effects were antagonized by papaverine but unaffected by IBMx. Thus, two known inhibitors of phosphodiesterase--IBMx and papaverine--produce sharply contrasting effects on basal and on isoproterenol-stimulated renin secretion from rat kidney slices.     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures

Sodium selenite (Na₂Se0₃) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetyl aminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 × 10^-6 M Na₂SeO₃ resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na₂SeO₃ concentrations (7.90 × 10^?6 and 1.19 × 10^?5 M) resulted in a three-fold increase in the SCE frequency above background level (6–7 SCEs/cell). Exposure of lymphocytes to 1 × 10^?4 M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 ± 0.75 while a similar exposure to 2.7 × 10^?5 M N-OH-AAF resulted in 13.61 ± 0.43 SCEs/cell. Simultaneous addition of the high Na₂Se0₃ concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25–30% and 11–17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

Abnormality of 2-deoxyglucose uptake kinetics in fibroblasts at low concentrations

2-deoxyglucose uptake rates at low sugar concentrations (less than 500 μM) appeared to be lower than those predicted by the Michaelis-Menten model which correctly described higher concentrations. This phenomenon which we will call concentration-dependent transport lag, was also observed for L-glucose uptake which suggest that this phenomenon is carrier-independent. A model involving the perimembrane space is developed which, for L-glucose, gives k₁ = 0.931 ± 0.072 × 10^?6 l. mg protein^?1. minute^?1, k₂ = 2.97 ± 0.19 × 10^?7 l. mg protein^?1. minute^?1 and So = 88,8 ± 4,3 μM; where k₁ is the diffusion constant in the cell membrane, k₂ is the diffusion constant in the perimembrane space and S_o the sugar concentration required in the external medium in order to provide an équivalent sugar concentration in the transport carrier area.     

Mutagenic and alkylating activities of organophosphate impurities of commercial malathion

The purpose of this study was to determine if 4 major organophosphate impurities of malathion were active as alkylators of nitrobenzylpyridine (NBP) or as mutagens in the Salmonella typhimurium bioassay. Malathion, isomalathion, O,O,O-trimethyl phosphorothioate, O,O,S-trimethyl phosphorothioate, and O,S,S-trimethyl phosphorodithioate produced alkylated NBP at varying rates. In order of increasing NBP reactivity, the compounds ranked: O,O,O-trimethyl phosphorothioate = O,O,S-trimethyl phosphorothioate less than O,S,S-trimethyl phosphorodithioate less than isomalathion = malathion. At 37 degrees C, the most reactive compounds produced an NBP alkylation rate equal to approximately 25% of the rate produced by methyl methanesulfonate, a potent Salmonella mutagen. However, none of the organophosphates were mutagenic in S. typhimurium TA97, TA98 and TA100 when tested by the standard plate-incorporation method or by the preincubation modification of the plate-incorporation method. The possible relationships between NBP reactivity and the biological activities of these organophosphates are discussed.     

Effects of prostacyclin on the canine isolated basilar artery

Prostacyclin (PGI₂) produced a biphasic response in canine isolated basilar arteries. In low doses (1 × 10^?8M?1 × 10^?7M) PGI₂ caused a slight but consistent relaxation of resting muscle tone. In low concentrations (1 × 10^?8M?1 × 10^?6M) PGI₂ antagonized muscle contractions caused by serotonin or prostaglandin (PG) F_2α. This relaxant effect with low doses of PGI₂ on the isolated cerebral artery contrasts with findings obtained with other PGs and supports the hypothesis that PGI₂ is a mediator of vasodilatation. However, in 1 × 10^?5M concentrations PGI₂ contracted the arterial muscle and did not antagonize contractions induced by serotonin or PGF_2α.     

Expression and kinetic analysis of carboxylesterase LmCesA1 from Locusta migratoria

Objective

To investigate the biochemical characterization of the carboxylesterase LmCesA1 from Locusta migratoria.

Results

We expressed recombinant LmCesA1 in Sf9 cells by using the Bac-to-bac baculovirus expression system. Enzyme kinetic assays showed that the K_m values of LmCesA1 for α-naphthyl acetate (α-NA) and β-naphthyl acetate (β-NA) were 0.08?±?0.01 mM and 0.22?±?0.03 mM, respectively, suggesting that LmCesA1 has a higher affinity for α-NA. LmCesA1 retained its enzymatic activity during incubations at pH 7–10 and at 10–30 °C. In an inhibition experiment, two organophosphate pesticides (malaoxon and malathion) and one pyrethroid pesticide (deltamethrin) showed different inhibition profiles against purified LmCesA1. Recombinant LmCesA1 activity was significantly inhibited by malaoxon in vitro. UPLC analysis showed that no metabolites were detected.

Conclusions

These results suggest that overexpression of LmCesA1 enhances malathion sequestration to confer malathion tolerance in L. migratoria.

     

Revealing binding interaction between seven drugs and immobilized β2‐adrenoceptor by high‐performance affinity chromatography using frontal analysis

The development of new approaches to study the affinity between ligands and G‐protein‐coupled receptors proves to be of growing interest for pharmacologists, chemists, and biologists. The aim of this work was to determine the binding of seven drugs to β₂‐adrenoceptors by frontal analysis using immobilized receptor stationary phase. The dissociation constants (K_d) were determined to be (3.16 ± 0.09) × 10^?4 M for salbutamol, (4.29 ± 0.12) × 10^?4 M for terbutaline, (6.19 ± 0.16) × 10^?4 M for methoxyphenamine, (2.11 ± 0.07) × 10^?4 M for tulobuterol, (1.82 ± 0.11) × 10^?4 M for fenoterol, (9.75 ± 0.24) × 10^?6 M formoterol, and (9.84 ± 0.26) × 10^?5 M for clenbuterol. These results showed a good correlation with the data determined by radioligand binding assay. Further investigations revealed that the dissociation constant mainly attributed to the number of hydrogen bonds in the structures of ligands. This study indicates that affinity chromatography using immobilized receptor stationary phase can be used for the direct determination of drug‐receptor binding interactions and has the potential to become a reliable alternative for quantitative studies of ligand–receptor interactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)‐expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293‐EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (K_D, 8.49 ± 0.11 × 10^?7 M) > erlotinib (K_D, 1.07 ± 0.02 × 10^?6 M) > canertinib (K_D, 1.41 ± 0.07 × 10^?6 M) > afatinib (K_D, 1.80 ± 0.12 × 10^?6 M) > vandetanib (K_D, 1.99 ± 0.03 × 10^?6 M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.     

Inhibition of the neutrophil oxidative response to a chemotactic peptide by inhibitors of arachidonic acid oxygenation

N-formylmethionylphenylalanine stimulates a short burst of antimycin A-insensitive O₂ uptake, O₂^? production and hexosemonophosphate shunt oxidation of glucose by guinea pig peritoneal neutrophils. The stimulated oxidative metabolism, as well as release of lysosomal enzymes ± cytochalasin B, are inhibited by 5,8,11,14-eicosatetraynoic acid (ID₅₀ 1.5 × 10^?5 M). High concentrations of indomethacin inhibit the peptide-stimulated oxidations (ID₅₀ 1.6 × 10^?4 M) while acetylsalicylic acid (2.5 × 10^?3 M) does not. Digitonin-stimulated oxidative metabolism and enzyme release are not inhibited by 5,8,11,14-eicosatetraynoic acid or indomethacin at concentrations that depress effects of the N-formylated peptide.     

IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

Cytochalasin B binding by human platelets

Intact human platelets bind cytochalasin B (CB) with a capacity of 100– 120 p mols CB/mg protein or approximately 7 × 10⁴ molecules/cell and dissociation constants (K_D) ranging from 2 × 10^?8 to 10^?6 M. Up to 85% of this saturable binding is displaced by 10^?5 M cytochalasin E (CE). This CE-sensitive binding also appears heterogeneous with K_D similar to those of the overall binding. The CE-insensitive binding, however, appears as a single component with K_D ≌ 4 × 10^?7 M. The sedimentable constituents from frozen, thawed, and washed cells also bind CB with K_D ranging from 2.4 × 10^?8 to 1.5 × 10^?6 M and a total capacity of approximately 39 p mols/mg protein which accounts for only 4% of the ligand binding to the intact cell. The major portion (60–80%) of this CB binding is displaceable by 500 mM D-glucose and has a K_D of 1.5 × 10^?6M, while only 10–15% is CE-sensitive with a K_D of 2.4 ± 10^?8 M. It is concluded that 95% of the saturable CB binding in platelets is associated with the cytosol of which 80–85% is sensitive to CE and that only 3% of the cellular binding is glucose sensitive, membrane-associated binding. If the CE-sensitive binding associated with the cytosol is entirely to actin, the stoichiometry of this binding is approximately one CB to 30 actin monomers, which is greater by an order of magnitude than that for CB binding to muscle actin.     

Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).     

The optical biosensor study of the redox partner interaction with the cytochrome P450 2B4-containing monooxygenase system under hydroxylation conditions

The interactions between cytochrome P450 2B4 (d-2B4), NADPH:cytochrome P450 reductase and cytochrome b5 have been investigated in the monomeric reconstituted P450 2B4-containing monooxygenase system in the presence of a substrate (7-pentoxyresorufin) and an electron donor, NADPH. Each partner was immobilized via its amino groups on the carboxymethyldextran biochip surface of the optical biosensor IAsys+. Such mode immobilization was not accompanied by any loss of activities of the immobilized proteins. The formation of binary d-Fp/d-2B4 complexes was registered. The association/dissociation rate constants (k_on/k_off) were (0.013 ± 0.005) × 10⁶ M^?1 s^?1/0.05 ± 0.02 s^?1, and dissociation constant (K_D) was (0.26 ± 0.13) × 10^?6 M. Comparison of k_on, k_off and K_D values for d-Fp/d-2B4 complexes formed under hydroxylation (O-dealkylation) with corresponding constants obtained for the oxidized proteins of (0.10 ± 0.03) × 10⁶ M^?1 s^?1/(0.14 ± 0.06) s^?1, and (0.71 ± 0.37) × 10^?6 M, respectively shows that the decrease in k_on and an insignificant decrease in K_D are associated with the increase of complex lifetime during transition from the oxidized to hydroxylation conditions. Complex formation between d-Fp and d-b5 was not registered in both hydroxylation conditions and in the case of oxidized forms of these proteins. In both cases formation of the ternary d-Fp/d-2B4/d-b5 complexes occurred.     

Noncompetitive Amine Uptake Inhibition by the New Antidepressant Pridefine

Abstract: Pridefine (AHR-1118) is a pyrrolidine derivative with clinically established antidepressant efficacy. Previous work from this laboratory indicates that pridefine is a reuptake blocker of catecholamines and serotonin with weak releasing activity. This study characterized the mode of amine uptake inhibition by pridefine as noncompetitive. The uptake experiments were performed utilizing ouabain instead of zero-degree controls to differentiate between the passive and active components of uptake. Furthermore, the passive component was resolved into diffusion and binding of substrate. Correction was made for the effects of ouabain on binding. Kinetic constants determined from Lineweaver-Burk plots were: K_m= 3 × 10^?7 M for NE, K_m= 9 × 10^?8 M for DA, and K_m= 3 × 10^?8 M for 5-HT. Dixon analyses of uptake at various pridefine concentrations indicated noncompetitive inhibition with K_i= 2.5 × 10^?6 M for NE uptake, K_i= 2.0 × 10^?6 M for DA uptake, and K_i= 1 × 10^?5 M for 5-HT uptake. These constants compare well with IC₅₀ values for the same transmitters: NE, IC₅₀= 2.4 × 10^?6 M; DA, IC₅₀= 2.8 × 10^?6 M; 5-HT, IC₅₀= 1.0 × 10^?5 M. The in vitro results indicate that pridefine is relatively specific as a catecholamine uptake blocker. It differs from tricyclic antidepressants which are reportedly competitive inhibitors of monoamine uptake. The possible mechanisms by which pridefine acts as a noncompetitive inhibitor are discussed.