Cationic Inhibitors Research Articles

Characterization of the inhibitory mechanism of 1-methyl-4-phenylpyridinium and 4-phenylpyridine analogs in inner membrane preparations.

We have investigated the mechanism of the inhibition of membrane-bound NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) and a series of its 4'-alkyl-substituted analogs of increasing hydrophobicity, as well as their neutral, desmethyl congeners. Comparison of hydrophobicity, as measured by partition coefficients, with the IC50 for the inhibition of NADH oxidase activity in mitochondrial inner membrane preparations shows a negative correlation, but the cationic inhibitors are more effective than the neutral analogs with similar hydrophobicity. The presence of 10 microM tetraphenylboron (TPB-) potentiates the inhibitory power of positively charged analogs up to 4'-pentyl-MPP+, while the neutral inhibitors are unaffected by TPB-. Without TPB-, the more hydrophilic analogs give incomplete inhibition, but the inclusion of TPB- permits the attainment of complete inhibition, accompanied by the appearance of sigmoidal titration curves. These data support the hypothesis that MPP+ analogs, like rotenone, are bound at two sites on the enzyme and occupancy of both is required for complete inhibition. TPB-, by forming ion pairs with the cationic analogs, facilitates their equilibration to both sites in membrane preparations. When present in molar excess over the MPP+ analog, TPB- partially reverses the inhibition by decreasing its concentration in the more hydrophilic binding site. The effect of temperature and of pH on the IC50 values for inhibition support the concept of dual binding sites, and the pH dependence of the inhibition reveals the participation of two ionized protein groups in the binding, one of which may be a thiol group.

Acetylcholinesterase as Polyelectrolyte: Inhibition by Alkylammonium Ions

It is shown that the influence of alkylammonium salts on the acetylcholinesterase-catalyzed hydrolysis of cationic substrates is caused by two factors: (A) nonspecific binding (condensation) of inhibitory cations on the polyanionic enzyme molecule due to electrostatic interactions, and (B) binding of the inhibitor in the active site of the enzyme due to hydrophobic interaction, accompanied by the release of a fraction of condensed counterions during the binding process. The description of the electrostatic effect upon ligand binding as a polyionic field effect is in complete agreement with the recent structural data which have indicated the absence of the functionally important "anionic point" in the choline-binding pocket of acetylcholinesterase.

Acetylcholinesterase as polyelectrolyte: interaction with multivalent cationic inhibitors

Influence of inorganic salts on the interaction of cobra venom acetylcholinesterase (EC 3.1.1.7) with hexamethonium and gallamine has been studied. The observed negative electrostatic salt effect in the dissociation constant of the enzyme-ligand complex, K D, has been described by equation p K D = p K D° − Z L Ψ +Z log[Me +Z] following from Manning's polyelectrolyte theory, where Ψ +Z is the fraction of condensed counterions Me +Z per one negative charge of the polyanionic enzyme. The Z L Ψ +Z values for the complex formation between native acetylcholinesterase and hexamethonium ( Z L = + 2) or gallamine ( Z L = + 3) were in quantitative agreement with those predicted by the theory making use of Ψ +1 = 0.50 found earlier from the influence of salts upon the hydrolysis of acetylcholine by the enzyme. Increase in the number of negative charges in acetylcholinesterase by its modification with pyromellitic dianhydride resulted in an increase of Ψ +1 to 0.6. The data show that the influence of salts on the electrostatic contribution to the energy of binding of cationic substrates and inhibitors by acetylcholinesterase can be quantitatively described proceeding from the counterion condensation model of Manning by using only one empirical parameter Ψ +1 for a given subtype or modified form of the enzyme.

Binding of 125I-fasciculin to rat brain acetylcholinesterase. The complex still binds diisopropyl fluorophosphate

Iodination of fasciculin 3 (FAS3) from Dendroaspis viridis venom provided us with a fully active specific probe of fasciculin binding sites on rat brain acetylcholinesterase (AChE). Binding and inhibition are concomitant, as association and inhibition rate constants k1 and ki are identical. The 125I-FAS3.AChE complex dissociates very slowly (t 1/2 = 48 h) and is characterized by a dissociation constant, Kd, of 0.4 pM. All the specific binding of 125I-FAS3 to AChE is prevented by FAS3 as from D. angusticeps venom (Kd = 0.4, 14, and 25 pM, respectively). It is also prevented by propidium iodide, BW284C51, and d-tubocurarine, which bind to peripheral anionic sites of AChE, by Ca2+ and Mg2+, known to enhance AChE activity through an allosteric phenomenon and by acetylthiocholine concentrations which lead to excess substrate inhibition of the enzyme. Diisopropyl fluorphosphate and paroxon, which inhibit AChE by phosphorylating the catalytic serine, have no effect on either the binding rate or the number of binding sites of 125I-FAS3. O-Ethyl-S2-diisopropylaminoethyl methylphosphonothionate, however, which binds irreversibly to the AChE catalytic site but reversibly to a peripheral site, induces a 130% increase in the binding rate of 125I-FAS3, without changing the total number of 125I-FAS3 binding sites. Our results demonstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors and the substrate in excess bind. Since phosphorylation of the catalytic serine (esteratic subsite) by [1,3-3H]diisopropyl fluorophosphate can still occur on the FAS3.AChE complex, the structural modification induced by fasciculins may affect the anionic subsite of AChE catalytic site.

The nature of proton‐translocating ATPases in maize roots

Abstract The mechanisms of the coupling between ATP hydrolysis and proton transport catalyzed by the ATPases of the tonoplast and plasma membrane of maize (Lea mays L.) roots were investigated. Proton transport by the tonoplast ATPase was found to be much more sensitive to nitrate than ATP hydrolysis, being inhibited by 80% with almost no effect on hydrolysis at 5 mM NO3. Mercury was also found to be a potent inhibitor of this enzyme, inhibiting transport and hydrolysis by 50% at 60 and 100 μM, respectively. The same type of pattern was seen with other divalent cations. Millimolar concentrations of Cd2+, Co2+, Cu2+, and Zn2+ in the presence of Mg2+ inhibited proton transport significantly more than hydrolysis whereas Ba2+ and Ca2+ had little effect. Both free and ATP‐complexed species of these inhibitory cations appeared to be effective. When the influence of temperature was investigated, both the tonoplast and plasma membrane enzymes showed a similar pattern. ATP hydrolysis by both enzymes generally obey...

Inhibition of mitochondrial respiration by neutral, monocationic, and dicationic bis-pyridines related to the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium cation (MPP +)

The cytotoxic effect of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP +) is believed to be associated with a compromise in cellular energy arising as a consequence of its persistent inhibition of mitochondrial respiration. MPP + is a rather weak inhibitor of electron transport, but it undergoes passive accumulation inside actively respiring mitochondria in response to the transmembrane electrochemical potential gradient. In order to test the prediction that dicationic analogs of MPP + might be concentrated to a much greater extent and thereby exert especially potent inhibition of respiration on the intact organelle, we synthesized four differently spaced bis-pyridines, each in neutral, monocationic, and dicationic forms, and evaluated their inhibitory activities in intact mitochondria and in electron transport particles (ETP). Compared to the neutrals, the monocations and especially the dications exhibit reduced inhibition in ETP, but the inhibition in mitochondria is enhanced selectively for the cationic inhibitors presumably on account of their accumulation in the mitochondrial matrix. This enhancement is limited by the relatively poor ability of the cationic bis-pyridines to enter mitochondria, as judged from experiments which evaluated the rate of onset of inhibition (without preincubation), in the absence and presence of tetraphenylborate (TPB −). The dications appear to be transported less well than the monocations, and only the most lipophilic dication exhibited a substantially greater accumulation-dependent enhancement of inhibitory activity on mitochondria than did the corresponding monocation. The compounds studied here constitute a novel class of respiratory chain probes which may be useful for a variety of studies on mitochondria.

Demonstration of a voltage dependent calcium current in follicles of the cockroachNauphoeta cinerea

Summary The electrical properties of vitellogenic cockroach follicles have been studied using a two-electrode voltage-clamp technique. The resting potential of freshly isolated follicles was usually between −30 mV and −45 mV. A voltage-dependent inward current could be activated when the follicles were placed in a medium containing high concentrations of either Ca2+ or Ba2+. Maximal current amplitudes were 0.8μA in a medium containing 30 mM Ca2+ and 2 μA in 30 mM Ba2+. Maximal amplitudes could be elicited with depolarizing pulses to about +15 mV. The steady-state inactivation curve indicated half maximal current at a holding potential of about −20 mV. Co2+, Ni2+ and Mn2+ inhibited the divalent cation currents in a concentration dependent manner. Half maximal inhibition was observed at about 3 mM concentration for each of the inhibitory cations.

Stability and reaction characteristics of reverse‐phase evaporation vesicles (REVs) as enzyme containers

The ion-dependent enzymes, activities of which are affected by ions, were entrapped in the inside aqueous phase of reverse-phase evaporation vesicles (REVs) to protect the enzyme activities from inhibitory cations. Their activities were still preserved in the presence of the inhibitory cations because the permeabilities of the inhibitory cations through the lipid membranes of REVs were much lower than those of substrates and products. The REVs were relatively stable in hypertonic condition, while they were unstable in hypotonic condition. Changes in osmotic pressure difference largely affected solute permeabilities of REVs. The shrinking and swelling of REVs resulting from osmotic pressure differences led to these changes in stabilities and solute permeabilities. Although the entrapment of the enzyme into REVs showed good protective effects against divalent cations, it was not effective against univalent cations. The measurements of cation permeabilities revealed that the enzyme trapped in the bilayer regions of REVs acts as a selective channel for the univalent cations. The REVs containing the enzyme could be used without any activity losses in a hollow fiber module.

Ionic Inhibitors of Human Leukocyte Elastase: Pyridinium and Phenyl Carboxylate Derivatives of 3-Alkyl-N-Hydroxysuccinimide

A series of pyridinium and phenyl carboxylate derivatives of 3-alkyl-N-hydroxysuccinimide has been synthesized; the compounds have been shown to be highly effective, time-dependent inactivators of human leukocyte elastase. The cationic inhibitor having an isobutyl side chain as the P1 residue (3) was found to be the most effective. Human leukocyte cathepsin G and chymotrypsin are also inactivated by these compounds.

Low molecular weight serine proteinase inhibitors of human articular cartilage. Isolation, Characterization, and Biosynthesis

The major low molecular weight serine proteinase inhibitor of human articular cartilage was purified to homogeneity as determined by single-peak elution with 4 high resolution techniques. The purified protein was found to be a potent inhibitor of human leukocyte elastase and cathepsin G, as well as the native serine proteinases derived from human articular cartilage and intervertebral disc. The inhibitor and lysozymes were synthesized by human articular cartilage in vitro. These properties and the ability of this cationic inhibitor to bind to cartilage matrix components suggest a possible role in the modulation of matrix catabolism in normal and pathologic states.

The Influence of Inorganic Salts on the Inhibition of Acetylcholinesterase by O,O-Diethylthiophosphates

Abstract The influence of various inorganic salts (NaCl, KCl, CsCl, KNO3, Na2SO4, K2SO4, MgCl2, CaCl2) on the kinetics of the reactions of acetylcholinesterase with neutral and cationic 0,O-diethylthiophosphates, (C2H50)2 P(0)SX, where × = (CH2)n and (CH2)n S+ (CH3) C2H5 (n=1–6), has been studied at 25° C and pH 7.5. The salt effect in the second-order rate constant can be quantitatively described by the equation log(k2/Ks) = log(k2 /KS)°-ψlog[M] + bc where bc is the term for the kinetic salting effect, ψlog[M] is the electrostatic term, c and [MI are the molar concentrations of salt and salt cation. The salting effect is observed in the binding step (Ks) as well as in the enzyme phosphorylation step (k2). Parallel increase of b with increasing n has been observed for the series of both ionic and neutral inhibitors. Electrostatic effect is observed only in the binding step for cationic inhibitors and does not depend on the distance between the onium atom and the reaction center in the inhibitor molecule. The observation of uniform Y is in accordance with the Manning's polyelectrolyte theory, suggesting that acetylcholinesterase can be considered as negatively charged polyelectrolyte. According to the theory the release of condensed counterions from the polyelectrolyte provides electrostatic contribution to the binding of cationic ligands, and the presence of localized anionic site on the acetylcholinesterase molecule is not required to account for the observed electrostatic effect. The obtained mean value of ψ = 0.49 ± 0.08 can be used as a quantitative parameter to characterize the effective charge density of acetylcholinesterase.

Effect of ATP on the activity of bovine heart mitochondrial b-c1 complex

The effect of ATP on the reductase activity of purified bovine heart b-c 1 complex was studied. ATP stimulates the steady-state activity and the antimycin-insensitive pre-steady-state reduction of b and c 1 cytochromes, also causing changes of kinetic properties of the enzyme. There is no absolute specificity for ATP since other polyvalent anions such as EDTA and EGTA produce similar effects in the micromolar range. It is proposed that ATP stimulates the activity of the b-c 1 complex, chelating inhibitory cation(s), exerting a modulatory action on the enzyme.

Dihydropyridine receptor regulation of acetylcholinesterase biosynthesis.

The dihydropyridine calcium channel antagonist nifedipine causes marked reductions in the amounts of acetylcholinesterase (AchE) molecular forms in primary tissue cultures of avian pectoral muscle. These reductions are time-dependent, requiring passage of 3 h prior to any observable response, dose-dependent, with principal actions occurring in the 1-100 nM range, are greater on the 7 S and 19 S forms than on the 11.4 S form, and, based on susceptibility of AchE to irreversible inhibition by a cationic inhibitor, occur almost exclusively with intracellular AchE coincident with a 2-fold reduction in the rate of secretion. The effects are markedly more pronounced in skeletal muscle than in neurons and differ from those observed for verapamil, diltiazem, and the calcium ionophore A23187. These reductions are incompatible with accelerated protein degradation, alterations in posttranslational processing and assembly in the Golgi complex, or enhanced loss of enzyme to the medium, but instead indicate that nifedipine causes a reduction in AchE biosynthesis. Since AchE forms are thought to arise from a single gene, these findings imply a linkage in skeletal muscle between transcription and posttranscriptional processing of mRNA and ligand occupation of the dihydropyridine receptor.

The effect of several pH control chemicals on the dry batch digestion of the organic fraction of municipal solid waste

Methane production in dry batch digestion of the organic fraction of municipal solid waste (MSW) was studied as a more complete method for management of MSW than landfilling. For start-up of the dry digestion of the organic fraction of MSW the addition of a methanogenic inoculum is essential. The first start-up of a dry anaerobic digestion batch reactor appeared to be unbalanced when a seed/substrate solids ratio of 0.08 was applied. Higher seed/substrate solids ratios were not employed because the total solids concentration is too low to speak of dry digestion under these conditions. The imbalance resulted in pH values below 6, and organic acid concentrations of 60 g COD (chemical oxygen demand) per liter. At these conditions methane production was negligible. To accelerate the start-up of the dry digestion process, several pH control chemicals were investigated. NaHCO 3 gave the best results when the buffer/ substrate solids ratio amounted to 0.06. The potential methane yield of 80 1 STP of methane per kg of organic fraction was obtained within 6 months. Ca (OH) 2 had a minor effect on the pH. CaCO 3 did not control the pH and inhibited the biogas production. The amount of pH control chemicals which were found to provide optimum pH control resulted in toxic levels of the cations for the methane production. The period required for adaptation of the methane-producing bacteria to these inhibitory cation concentrations limits the benefits of buffer additions during dry digestion.

Kinetics and specificity of serine proteases in peptide synthesis catalyzed in organic solvents.

Initial rates of peptide-bond synthesis catalyzed by poly(ethylene glycol)-modified chymotrypsin in benzene were determined using high-performance liquid chromatography. Enzymatic synthesis of N-benzoyl-L-tyrosyl-L-phenylalanine amide from N-benzoyl-L-tyrosine ethyl ester and L-phenylalanine amide was found to obey Michaelis-Menten kinetics an to be consistent with a ping-pong mechanism modified by a hydrolytic branch. The catalytic activity of modified chymotrypsin was dependent on both water concentration and type of organic solvent, the highest synthesis rate being obtained in toluene. Since the chymotrypsin specificity in the organic phase was actually altered, the enzyme's apparent kinetic parameters were determined for different substrates and compared to those obtained with other serine proteases in benzene. Both N-benzoyl-L-tyrosine ethyl ester and N-alpha-benzoyl-L-lysine methyl ester were comparable acyl donors in benzene and the (kcat/Km)app value of modified chymotrypsin was only 10-fold smaller than that obtained with poly(ethylene glycol)-modified trypsin in the synthesis of N-alpha-benzoyl-L-lysyl-L-phenylalanine amide. The change in chymotrypsin specificity was also confirmed through the binding of trypsin inhibitors in benzene. The overall results suggest that hydrophobic bonding between the enzyme and its substrate should not be taken into account during catalysis in the organic phase. In general, if hydrophobic interactions are involved in the binding of substrates to the active site in aqueous media, the replacement of water by hydrophobic solvents will induce some change in enzyme specificity. Moreover, secondary residues of enzyme-binding sites may also exert a significant influence on specificity since, as observed in this study, chymotrypsin exhibited high affinity for cationic substrates and cationic inhibitors as well in apolar solvents.

Metal ion binding to D-xylose isomerase from Streptomyces violaceoruber.

The binding of two activating cations, Co2+ and Mg2+, and of one inhibitory cation, Ca2+, to D-xylose isomerase from Streptomyces violaceoruber was investigated. Equilibrium-dialysis and spectrometric studies revealed that the enzyme binds 2 mol of Co2+/mol of monomer. Difference absorption spectrometry in the u.v. and visible regions indicated that the environment of the first Co2+ ion is markedly different from that of the second Co2+ ion. The first Co2+ appears to have a six-co-ordinate. The conformational change induced by binding of Co2+ to the first site is maximum after the addition of 1 equivalent of Co2+ and yields a binding constant greater than or equal to 3.3 x 10(6) M-1. Binding of Co2+ to the second, weaker-binding, site caused a visible difference spectrum. The association constant estimated from Co2+ titrations at 585 nm agrees satisfactorily with the value of 4 x 10(4) M-1 obtained from equilibrium dialysis. Similarly, the enzyme undergoes a conformational change on binding of Mg2+ or Ca2+, the binding constants being estimated as 1 x 10(5) M-1 and 5 x 10(5) M-1 respectively. Competition between the activating Mg2+ and Co2+ and the inhibitory Ca2+ ion for both sites was further evidenced by equilibrium dialysis and by spectral displacement studies.

Interactions in vitro and in vivo between Human and Porcine Cationic Pancreatic Elastase and Plasma Protease Inhibitors

Trace amounts of porcine pancreatic elastase mixed with porcine serum, or injected intravenously into the pig, were found to be bound mainly to alpha 1- and alpha 2-macroglobulin (90%). Alpha 1-macroglobulin approached saturation with elastase before significant binding to alpha 2-macroglobulin was demonstrable. Human pancreatic cationic elastase showed in human serum preferential binding to alpha 2-macroglobulin, but the elastase was also bound by alpha 1-protease inhibitor and by alpha 1-antichymotrypsin. The porcine elastase-alpha 1 alpha 2-macroglobulin complexes injected intravenously or formed in vivo in the pig were rapidly eliminated from the blood stream following a first order reaction with t 1/2 = 8 min. Porcine alpha 1-protease-inhibitor-bound elastase disappeared considerably more slowly.

Mechanism of inhibition of bovine pineal gland hydroxyindole-O-methyltransferase (EC 2.1.1.4) by divalent cations.

The effect of inhibitory cations on hydroxyindole-O-methyltransferase (HIOMT) was investigated in order to elucidate the mechanisms involved. Copper inhibition of HIOMT can be classified as partial, classic noncompetitive inhibition with respect to S-adenosyl methionine and as partial, mixed noncompetitive inhibition with respect to N-acetylserotonin. Binding of copper to the enzyme impaired but did not prevent catalysis, and the difference in inhibitory pattern observed with both substrates may be due to the obligatory order of substrate binding to HIOMT. Calcium and magnesium were uncompetitive inhibitors of HIOMT with respect to both substrates and binding of these cations to the enzyme prevents catalysis. The cations presumably bind to HIOMT in the vicinity of the catalytic site and in some way prevent or impair the catalytic process, possibly by interfering with substrate binding to the enzyme, interfering with methyl transfer or product release after catalysis, or a combination of these effects. The different inhibitory patterns observed may result from differences in molecular size or outer electron shell valence of the cations.

Potassium Transport Across the Membranes ofChara

Smith, J. R. and Kerr, R. J. 1987. Potassium transport across the membranes of Chara. IV. Interactions with other cations.—J. exp. Bot. 38: 788–799. The 42K influx (⁠ϕKin⁠) and the membrane electrical conductance (Gm were measured simultaneously for intemodal cells of Chara australis bathed in solutions containing different concentrations of various cationic species. It was found that the potassium permeability (Pk,) of the membrane was reduced significantly when the bathing [CaSO4 exceeded 01 mol m−1. Concentrations of tetra-ethylammonium ions (TEA) exceeding 0·3 mol m−3 were found to reduce significantly both ϕKin and ϕKoff⁠, but even high concentrations (10 mol m−3) usually did not reduce the fluxes by more than a factor of 3. Na+ ions were found to be capable of reducing PK by a factor of 5·6 to a value of ∼ 4 nm s−1. This appeared to be a minimum value for PK which was not reduced even if several inhibitory cations were present simultaneously. This suggests that possibly only one of two different modes of K+ transport can be inhibited by cations. The possible geometry of the inhibitable K+ channel is briefly discussed and the implications of the presence of Na+ and Ca2+ ions in many common bathing solutions are considered.

Leaving group effects in binding and reaction steps of acetylcholinesterase inhibition by O,O-diethylthiophosphates

Affinity and phosphorylation constants for the inhibition of acetylcholinesterase by a series of O,O-diethylthiophosphates, (C 2H 5O) 2P(O)SX, where X = C n H 2 n+1 ( n = 4–8), (CH 2) n SC 2H 5 ( n = 2–6), and (CH 2) n S +(CH 3)C 2H 5 ( n = 3–6), have been measured at 25°C and pH 7.0 in order to quantify the specificity-determining factors in the binding and reaction steps of the process. In contrast to the influence of the leaving group of the acetic ester substrates, where the hydrophobicity of the substituent affects the binding and acetylation steps equally, it has been found that the hydrophobicity of the leaving group of the nonionic organophosphorus inhibitor affects only the binding in the enzyme's active site whereas the rate of the phosphorylation reaction is governed solely by the inductive effect of substituent X. A common phosphorylation rate constant logarithm-σ x ∗ relationship holds for the cationic inhibitors and their nonionic analogs; however, an extra effect of the cationic group in X appears in the dissociation constant for the enzyme-inhibitor complex and points to the putative influence of the enzyme's anionic site in the binding step. The data obtained suggest that the hydrophobic cleft for the binding of the inhibitors' leaving group is not “shut” in the transition state of the phosphorylation of the enzyme's active site, in contrast to the acetylation reaction in which the closed hydrophobic slit in the transition state is shown to stabilize the complex.