Cationic Inhibitors Research Articles

K+ and Cl- conductances in the apical membrane from secreting oxyntic cells are concurrently inhibited by divalent cations.

This study concerns the properties of rapid K+ and Cl transport pathways that are present in the (H+ + K+)-ATPase membrane from stimulated, and secreting, gastric oxyntic cells. Ion permeabilities in the isolated stimulation-associated vesicles were monitored via the rates of H+ efflux under conditions of exclusive H+/K+ counterflux or H+ - Cl co-efflux, as well as by comparison of equilibration rates for 86Rb and 36Cl under conditions of equilibrium exchange and unidirectional salt flux. These latter studies suggest that Rb+ and Cl pathways are conductive and independent. In spite of the functional independence of the ion pathways, several divalent cations inhibit Rb+ and Cl isotopic exchange as well as the H+ efflux that is dependent on either K+ or anion (Cl, SCN, NO2) fluxes. Zn2+ is the more potent inhibitor, reducing by 50% the sensitive component of K+, Cl, and NO2 fluxes at about 20 microM; Mn2+ has a similar effect at 200 microM. Ni2+ and Co2+ were roughly equipotent to Mn2+ while Mg2+ and Ca2+ had no inhibitory effect. These results suggest that the stimulation-induced permeabilities, while functioning independently, may be physically linked, i.e., residing within a single entity. In similar studies carried out in (H+ + K+)-ATPase vesicles obtained from nonstimulated cells, no vestiges of sensitivity to the inhibitory divalent cations could be detected. The implications of these findings for the physiology of the oxyntic cell in the context of a model for membrane fusion are discussed.

The interaction between calcium and the activation of Na+, K+-ATPase by noradrenaline.

The interaction of noradrenaline, various cation chelators and calcium on Na+, K+-ATPase from rat cerebral cortex plasma membranes was studied. It was shown that chelation of inhibitory cations by EGTA, EDTA and dipyridyl activated Na+, K+-ATPase to the same extent as noradrenaline but at higher concentrations; increasing concentrations of EGTA depressed the activation by noradrenaline; calcium in the form of a calcium-EGTA buffer depressed Na+, K+-ATPase at physiological concentrations; the inhibition of Na+, K+-ATPase by calcium is dependent on the magnesium concentration in the assay and the inhibition by calcium was partially reversed by noradrenaline.

Membrane-specific inhibitors of the bovine heart mitochondrial ATPase

New cationic inhibitors of the bovine heart mitochondrial ATPase have been synthesized by quaternizing 1-dansylamido-3-dimethypropylamine with decyl and hexadecyl iodides. These ligands are unique in their mode of action because they inhibit the submitochondrial membrane-associated forms of the enzyme more potently than the soluble form of the enzyme (F 1). Derivatives prepared with propyl or hexyl iodides are weak inhibitors and exhibit little affinity for submitochondrial membranes particle. The inhibitory effectiveness of these derivatives measured either in the direction of ATP synthesis or ATP hydrolysis results from efficient insertion into the membrane. Other inhibitory organic cations such as the 3:1 4,7-diphenyl-1,10-phenanthroline-ferrous chelate and alkyl guanidines inhibit both the membrane-associated and soluble ATPase comparably.

Selectivity of the Ca binding site in synaptosome Ca channels. Inhibition of Ca influx by multivalent metal cations.

K-stimulated (voltage-dependent) influx of 45Ca was measured in synaptosomes (isolated presynaptic nerve terminals) from rat brain. Influx was terminated at 1 s with a rapid-filtration technique, so that most of the Ca uptake was mediated by inactivating ("fast") Ca channels (Nachshen, D. A., and Blaustein, M. P., 1980, J. Gen. Physiol., 76:709-728). This influx was blocked by multivalent cations with half-inhibition constants (K1) that clustered in three distinct groups: (a) K1 greater than 1 mM (Mg2+, Sr2+, and Ba2+); (b) K1 = 30-100 microM (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Hg2+); (c) K1 less than 1 micro M (Cd2+, Y3+, La3+ and the trivalent lanthanides, and Pb2+). Most of these ions had very little effect on synaptosome steady state membrane potential, which was monitored with a voltage-sensitive fluorescent dye, or on the voltage dependence of Ca influx, which was assessed by measuring voltage-dependent Ca uptake at two levels of depolarization. The blockers inhibited Ca influx by competing with Ca for the channel site that is involved in the transport of divalent cations. Onset of fast channel inhibition by Mg, Co, Ni, Cu, Zn, Cd, La, Hg, and Pb was rapid, occurring within 1 s; inhibition was similar after 1 s or 30 min of exposure to these ions. The inhibition produced by Co, Cu, Zn, Cd, La, and Pb could be substantially reversed within 1 s by removing the inhibitory cation. The relative efficacies of the lanthanides as fast channel blockers were compared; there was a decrease in inhibitory potency with decreasing ionic radius. A model of the Ca channel binding site is considered, in which inhibitory polyvalent cation selectivity is determined primarily by coulombic interactions between the binding site and the different cations. The site is envisaged as consisting of two anions (radius 1 A) with a separation of 2 A between them. Small cations are unable to bind effectively to both anions. The selectivity sequences predicted for the alkaline earth cations, lanthanides, and transition metals are in substantial agreement with the selectivity sequences observed for inhibition of the fast Ca channel.

Effects of valinomycin on hexose transport and cellular ATP pools in mouse fibroblasts.

The K+ ionophore valinomycin at concentrations of 1 X 10(-8) M and over, stimulated 2-deoxy-D-glucose (2DG) and 3-O-methylglucose (3OMG) uptake in Swiss 3T3 fibroblasts. The rate-limiting step of 2DG uptake was transport rather than phosphorylation, in the control or valinomycin-treated cells. Kinetic analysis showed that valinomycin increased the Vmax for 2DG uptake without change of the Km. The valinomycin-stimulated 2DG uptake was insensitive to 10 micrograms/ml cycloheximide, and extracellular K+ concentrations between 0.1 and 50 mM. On the other hand, valinomycin at the concentration of 1 X 10(-8) M and over, induced a rapid decrease in cellular ATP content, followed by stimulation of 2DG uptake and recovery of the ATP content. A similar relationship between the reduction of cellular ATP content and the subsequent stimulation of 2DG uptake was observed when the cells were treated not only with 2,4-dinitrophenol and iodoacetic acid, but also with other monovalent cation ionophores or inhibitors of oxidative phosphorylation. These results suggest that valinomycin may posttranslationally stimulate hexose transport by increasing the number of functional carriers of hexose or changing their mobility, and the rapid decrease in cellular ATP pools by valinomycin may be a trigger of the stimulation of the hexose transport in Swiss 3T3 fibroblasts.

Effect of the surface potential upon ion selectivity found in competitive inhibition of divalent cation uptake. A theoretical approach

The sequence of inhibition of cation uptake caused by a series of cationic inhibitors may shift dramatically on varying the experimental conditions. This sequence depends upon the concentration of the inhibitory cation, the concentration of the substrate cation, the charge density of the cell membrane, the ionic strength of the medium and the affinity of the substrate cation for the negative groups located on the cell membrane. A shift in sequence between inhibitory cations can only occur if one of the inhibitory cations shows a greater affinity for the translocation site than the other, while the affinity for the negative groups on the cell membrane is lower.

1-Bromopinacolone, an active site-directed covalent inhibitor for acetylcholinesterase.

1-Bromopinacolone, BrPin, acts initially as a reversible competitive inhibitor for acetylcholinesterase, KI = 0.18 mM in hydrolysis of acetylcholine. Unlike bromoacetone, with time it acts as an irreversible covalent inhibitor. BrPin has a hydrolytic half-life of 30 h at the pH of incubation, 7.8. The enzyme-BrPin complex is 50% inactivated in 2 h. First order kinetics are observed; the rate constant is proportional to the concentration of complex. Retardation by cationic inhibitors of the inactivation is consistent with inactivation occurring as a result of binding of BrPin to the active site. Efficiency of irreversible inhibition by BrPin is essentially the same for hydrolysis of cationic and uncharged substrates, acetylcholine, 3,3-dimethylbutyl acetate, phenyl acetate, n-butyl acetate, and indophenyl acetate. In contrast, a cationic alkylating agent, N,N-dimethyl-2-phenylaziridinium ion, DPA, acts noncompetitively; it inactivates completely toward cationic, and partially toward uncharged substrates, and does so slightly more rapidly than BrPin, but less than would be commensurate with its greater intrinsic reactivity. Enzyme first treated with DPA is inactivated by BrPin toward hydrolysis of 3,3-dimethylbutyl acetate. It is proposed that BrPin, and not DPA, binds and reacts in, and may be a useful labeling agent for, the active site.

On the relative roles of Ca2+ and Mg2+ in regulating the endogenous K+/H+ exchanger of rat liver mitochondria.

1. A23187 and ionomycin cause the release of Ca2+, Mg2+, and K+ from the mitochondrial matrix. The electroneutral K+ efflux does not reflect direct transport by these ionophores but, rather, results from release of the endogenous K+/H+ exchanger from inhibition by divalent cations. 2. A23187 and ionomycin differ in their affinities for CA2+ and Mg2+, having relative affinity ratios (Ca2+/Mg2+) in respiring mitochondria of 3 and 35, respectively. 3. This difference in Ca2+/Mg2+ affinities was exploited in dose-response studies designed to determine which cation exerts primary control over K+/H+ exchange activity. The results of such studies demonstrate that removal of Ca2+ has no effect on K+ efflux, and that K+ efflux follows Mg2+ efflux whether induced by ionomycin or A23187. 4. K+ steady states, in which K+ uniport equals K+/H+ antiport, were induced by adding low levels of valinomycin to respiring mitochondria. The addition of Ca2+ perturbed the steady state in the direction of increased K+/H+ exchange. 5. Addition of Ca2+ to respiring mitochondria resulted in electroneutral K+ efflux which was strongly affected by the anion composition of the medium. We propose that Ca2+ turns on the K+/H+ exchanger indirectly, through a decrease in matrix [Mg2+] secondary to swelling and uptake of chelating anions. This phenomenon may be of physiological importance as a temporary protection against excessive swelling due to Ca2+ uptake. 6. These studies support the contention that Mg2+, not Ca2+, is the inhibitory cation responsible for the physiological regulation of mitochondrial K+/H+ exchange.

Cystic fibrosis. II. The urinary mucociliary inhibitor.

In the current study, the cystic fibrosis cationic mucociliary inhibitor has been purified from urine by ion exchange chromatography, gel filtration, lectin affinity chromatography, isoelectric focusing, and high performance liquid chromatography. The molecular size of the cationic mucociliary inhibitor was estimated to be in the range of 4,000 to 13,500 MW, by its elution on Sephadex G-50, and between 7,500 and 2,750 MW, by urea-sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition to the cationic mucociliary inhibitor, an anionic mucociliary inhibitor was also detected in the urinary fraction isoelectrically focused between pH 4.5 and 4.9. The identity of the mucociliary inhibitor as a glycoprotein was established in the current study by affinity chromatography on Phaseolus lunatus lectin, by radiolabeling the carbohydrate with galactose oxidase and tritiated sodium borohydride, and by determining the presence of a large concentration of glucosamine and small amounts of galactosamine by amino acid analysis. The amino acid analysis of the purified major component of the cationic mucociliary inhibitor reveals that the glucosamine concentrations represents a high percentage of the composition of the glycoprotein.

Amiloride-resistant Madin-Darby canine kidney (MDCK) cells exhibit decreased cation transport.

Cation transport systems were investigated in mutant Madin-Darby Canine Kidney (MDCK) cells resistant to the diuretic drug amiloride. The mutants were isolated previously as clones resistant to the cytotoxic effects of 3 X 10(-4) M amiloride. Decreased amiloride transport by the Na+ channel was implicated as the basis of the resistance (Taub, '78). Consistent with this hypothesis, Na+ accumulation was lower in amiloride-resistant cells than in normal sensitive MDCK cells. Kinetic studies indicated that Na+ uptake in MDCK cells occurs by a single ATP independent transport system--the Na+ channel. In several amiloride-resistant clones, including clone Amr2, the decreased Na+ uptake was associated with a decrease in both the Km and Vmax for Na+ uptake by the Na+ channel. In Amr2 cells no significant alteration in the inhibitory effect of amiloride on Na+ uptake was observed. As the Na+ channel may actually be a general uptake system for monovalent cations (a number of cations inhibit Na+ uptake), the uptake of these inhibitory cations was examined in Amr2 cells. Both Ca++ and ouabain-insensitive Rb+ uptake occurred at decreased rates in Amr2 cells as compared with normal MDCK cells. However, further uptake studies suggested that Na+, Ca++ and ouabain-insensitive Rb+ uptake all occur by different systems. Thus several transport systems may be defective in Amr2 cells. Amr2 cells were also resistant to the inhibitory effects of amiloride on CO2 evolution from pyruvate. These observations indicate that alterations at a number of molecular sites may be associated with defective Na+ transport via the Na+ channel in amiloride-resistant cells. Thus the amiloride-resistant cells are potentially valuable in examining the interrelationships between Na+ transport and other cellular functions.

A study of some karst waters in Norway Spatial variation in solute concentrations and equilibrium parameters in limestone dissolution

Lauritzen, S.-E. 1981. A study of some karst waters in Norway. Spatial variation in solute concentrations and equilibrium parameters in limestone dissolution. Norsk geogr. Tidsskr. Vol. 35, 1–19. Oslo. ISSN 0029-1951. 104 July and August samples of karst waters from Norway were analysed for their solute contents and for their aggresiveness towards calcium carbonate. The most important cations were Ca and Mg and sometimes Na, while bicarbonate was the most abundant counter-ion. Few waters were in equilibrium with calcium carbonate; they were either under- or supersaturated. There was a clear difference between karst systems above and below the local tree-line, and the investigated areas could be classified according to total hardness, aggressiveness, and altitude. Above the tree-line no supersaturated waters were found and the undersaturated ones saturated towards 80 ppm total hardness, due to aerial PCO2 control only. Below the tree-line, there was a hardness of up to 120 ppm with both under- and supersaturated spring waters, most probably caused by biogenic CO2 and proton supply. Because of low concentrations of some cationic inhibitors (Pb, Cu, Fe, Zn) in the samples, the disequilibrium of the waters probably reflects flow conditions in the karst aquifer rather than inhibition.

Effect of cations on the human creatine kinase isoenzymes.

We report our findings on the effect of Ca2+, K+, Na+, Fe2+, Mn2+, Zn2+, and Cu2+ on the activity of creatine kinase isoenzymes derived from human tissues. The only inhibitory cation was Ca2+, and Dixon plots of the data indicate the inhibition to be competitive for the Mg2+ in the reaction assay mixture. This effect of Ca2+ on all three isoenzymes is completely reversed by including Mg2+ (10 mmol/L) and ethylenediaminetetraacetate (2 mmol/L) in the assay mixture. Our findings suggest that it is unnecessary to add chelators to the patient's sample.

Tests of an electrostatic screening hypothesis of the inhibition of neurotransmitter release by cations at the frog neuromuscular junction

We have investigated an electrostatic screening hypothesis of cationic inhibition of quantal release at the neuromuscular junction of the frog (Rana pipiens). According to this hypothesis, increasing the extracellular concentration of an inhibitory cation reduces the quantal content (m) of the end-plate potential by reducing the ability of negative surface charge to attract Ca2+ to the external surface of the presynaptic membrane. The inhibitory power of various cations should depend only on their net ionic charge and should increase strongly with increasing charge. We have demonstrated, in Ringer's solutions containing modified concentrations of Na+, Ca+, and Mg2+, that at fixed concentrations of Ca2+ and Na+ (a) the dependence of m on [Mg2+]0 is satisfactorily accounted for by electrostatic theory and (b) the dependence of m on the univalent cation concentration of the modified Ringer's solution is satisfactorily predicted from the Mg2+ inhibition of m. (Glucosamine or arginine was used to replace a fraction of the Na+ content of Ringer's solution in the latter experiments.) These results are consistent with electrostatic screening actions of Mg2+ and univalent cations in the inhibition of m. We have also re-examined the inhibition of m caused by the addition to Ringer's solution of two trace concentration divalent cations, Mn2+ and Sr2+. Our data suggest that the inhibition of m by Sr2+ at high quantal contents may also be due to surface charge screening, while the potent inhibitory actions of Mn2+ may be due to its ability to bind negative surface charge.

A study of the mechanism of radiation‐induced polymerization of carbon suboxide

AbstractThe γ radiolysis of solid C3O2 at 77 K produces paramagnetic centers of several types. The ESR spectrum constitutes a number of narrow lines with an anisotropic g factor. Some of the centers are photounstable and are lost after optical bleaching. Heating the sample results initially in the loss of photounstable centers and then, in the temperature region in which solid C3O2 undergoes a phase transition (140 K), loss of the centers responsible for the center section of the ESR spectrum. In the temperature region 140–270 K concentration of the centers remained at the level of 1015−1016 g−1. At higher temperatures an intense polymerization of liquid C3O2 followed by a sharp increase in concentration of the paramagnetic centers was observed. It is assumed that in radiolysis at 77 K and on subsequent heating the formation of oligomeric centers in solid C3O2 results in a decreasing probability of their loss. Using a calorimetric technique, we studied the kinetics of the postradiation polymerization of C3O2. Small additions of acetone (cationic inhibitor) brought about the loss of the oligomeric centers and suppressed completely all postpolymerization. The polymerization rate increased with the concentration of the resulting polymer. An autocatalytic character of the process is supposed to be connected with the formation of polymer‐monomer complexes.

Chemotactic activity of C5a des Arg: Evidence of a requirement for an anionic peptide ‘helper factor’ and inhibition by a cationic protein in serum from patients with systemic lupus erythematosus

Sera from some patients with systemic lupus erythematosus (SLE) ∗ contain a uniquely specific, reversible inhibitor of complement (C5)-derived chemotactic activity for polymorphonuclear leukocytes. SLE serum, proven capable of significantly inhibiting C5-derived chemotactic activity in zymosan-treated serum (ZTS), was found incapable of inhibiting the chemotactic activity of the highly purified human anaphylatoxin, C5a (10–20 ng/ml). Similarly, SLE serum was found incapable of inhibiting the chemotactic activity generated in ZTS containing the carboxypeptidase inhibitor, epsilon amino-caproic acid (EACA). EACA protects C5a from the action of the anaphylatoxin inactivator in serum and thereby prevents conversion of C5a to a peptide (C5a des Arg) which is completely devoid of anaphylatoxin activity. Highly purified human C5a des Arg (40–160 μg/ml) was not chemotactic unless assayed in the presence of small amounts of normal human serum. The ‘helper factor’ in normal human serum which permits C5a des Arg to exhibit chemotactic activity was isolated and found to be an anionic polypeptide. The chemotactic activity of C5a des Arg plus normal serum was inhibited significantly by SLE serum. The inhibitor in SLE serum was isolated and determined to be a 69,000 mol. wt cationic protein. These data suggest strongly that the cationic inhibitor in SLE serum acts not on C5a but only on the ‘complex’ of C5a des Arg plus a specific peptide ‘helper factor’. This ‘complex’ accounts for a substantial proportion of the chemotactic activity in ZTS.

The effect of metal ions on mitochondrial pyridine dinucleotide transhydrogenase

Bovine heart submitochondrial particle transhydrogenase is inhibited by cations in a concentration and pH-dependent manner, and non-energy-linked transhydrogenation is inhibited to a greater extent by metals than the energy-linked reaction. The inhibition of the enzyme by Mg2+ is competitive with the NADP substrate and non-competitive with the NAD substrate. Mg2+ stimulates inactivation of the enzyme by 5,5′-dithiobis(2-nitrobenzoic acid), and protects against thermal and proteolytic inactivation. This suggests that Mg2+ binding in the NADP site alters transhydrogenase to a more thermostable conformation, which is less susceptible to attack by trypsin and more reactive with 5,5′-dithiobis(2-nitrobenzoic acid). Other cation inhibitors mimic Mg2+ in these properties. The order of effectiveness of the inhibitors tested is La3+ > Mn2+ > Ca2+ ⋍ Mg2+ > Sr2+ > Na+ ⋍ K+. This order is described by the Irving-Williams order for the stability of metal-ligand complexes, suggesting that carboxylates or amines may comprise the inhibitory cation binding site.

Catalysis by β-glucosidase A3of Aspergillus wentii

(1)α-Deuterium kinetic isotope effects on Vmax. for hydrolysis of both β-D-glucopyranosylpyridinium ions and aryl β-D-glucopyranosides are in the range kH/kD 1.08–1.14, indicating that bond-breaking limits the rate of hydrolysis of both sets of substrates. The variation of kcat with aglycone acidity, expressed by log kcat=A+βlgpKa, is governed by a βlg value of –0.96 ± 0.19 for the N-glycosides and –0.05 ± 0.05 for the O-glycosides. The latter, low value of |βlg| is evidence for extensive proton donation to the oxygen at the transition state, even for the departure of acidic aglycones. The dependence of rate on protonation of a group of pKa < 6 required by this idea is indeed observed. (2) Comparison of log kcat values with spontaneous hydrolysis rates indicates that nucleophilic and non-covalent interactions accelerate C–N bond cleavage in the ES complex by a factor of 10(8 + 0.3[pka]N) for glucosyl pyridinium salts where [pKa]N is the pKa of the pyridine. On the assumption that these interactions are similar for O-glucosides, proton donation to the aglycone oxygen atom can be estimated to contribute a rate-enhancement of 10(0.1 + 0.7[pKa]O), where [pKa]O is that of the free phenol. (3)D-Glucono-δ-lactone and 5-amino-5-deoxy-D-gluconolactam are bound 102.8 and 102.2 times, respectively, more tightly than β-D-glucopyranose, because of their analogy to a transition state in which α-deuterium kinetic isotope effects have shown the anomeric carbon atom to have substantial sp2 character. (4) Cationic inhibitors are bound 102.5–103.5 times more tightly than directly comparable neutral ones, if the positive charge resides on the equatorial substituent at C-1, but protonated 2-amino-2-deoxyglucose is bound no more tightly than glucose. This indicates the presence of a negative charge in the El complex near C-1 on the α face of the pyranose ring (an aspartate residue previously identified by covalent labelling) which is partly compensated by a positive charge near C-2.

Cell wall extension in Nitella as influenced by acids and ions.

The giant internode cells of Nitella axillaris exhibit acid-induced growth similar to that found in higher plants. The threshold pH is 4.5, with a maximum at 3.5. The acid growth effect is transient, lasting no more than 32 min. Extensibility measurements of isolated cell walls showed a similar pattern of acid enhancement. Prolonged boiling in water (12 hr) only partially inhibited the acid-induced wall extensibility and actually increased the extensibility at pH 6. It was concluded that physical, rather than enzymatic, processes were responsible for acid-enhanced continuous extension ("creep") in Nitella walls. A complex cation-sensitive mechanism that affects extensibility was also characterized. Among the stimulatory (wall-softening) cations, divalents were generally more effective than monovalents, with magnesium being the most stimulatory. The inhibitory (wall-hardening) cations included divalents and trivalents, aluminum being the most inhibitory. Ionic effects on extensibility were even less sensitive to prolonged boiling in water than acid effects.

The interaction of Crotalus atrox phospholipase A2 with calcium ion and 1-anilinonaphthalene-8-sulfonate.

Phospholipase A2 (EC 3.1.1.4) was isolated from lyophilized Crotalus atrox venom by chromatography on Sephadex G-75, DEAE-cellulose chromatography and isoelectric focussing in a pH gradient, as a homogeneous protein. In confirmation of an earlier report (Hachimori, Y., Wells, M. A., and Hanahan, D. J. (1971) Biochemistry 10, 4084–4089) the enzyme had a molecular weight of 28 850 and contained two tryptophan residues per mole. At neutral pH the enzyme is a dimer of two identical polypeptide chains which dissociate at low pH. The tryptophan residues in the dimer reside in a restricted, hydrophobic environment, which is disrupted on dissociation into monomers. The circular dichroism (CD) spectrum of the enzyme at neutral pH exhibits a characteristic 1Lb tryptophan spectrum in the aromatic region, with peaks of positive ellipticity at 276, 282 and 290 nm, which are lowered and broadened at low pH, and the difference absorption spectra (pH 7.5 vs pH 2.0) exhibit a prominent peak at 290 nm. No change in the CD spectrum in the peptide region was found at low pH, or in the presence or absence of calcium ion, and a rather low helix content (9%) was estimated from such spectra. The enzyme requires calcium ion for activity, and a dissociation constant for the enzyme–calcium complex of about 10−3 M was estimated from kinetic data. Binding of Ca2+ and the inhibitory cations Hg2+ and Zn2+ was accompanied by perturbation of the tryptophanyl CD spectrum characteristic of exposure of tryptophans to the solvent, but there was no change in the absorption spectrum. The enzyme binds 1-anilinonaphthalene-8-sulfonate (ANS). In the presence of 10−3 M calcium ion, only one ANS binding site was found with Kdiss = 11.9 × 10−6 M, while in the absence of calcium a cooperative binding isotherm was observed, and two molecules of ANS appeared to be bound sequentially with Kdiss values of about 11.7 × 10−6 M and 0.14 × 10−6 M, respectively. The ANS binding site in the presence of 10−3 M calcium ion was more hydrophobic than those in the absence of calcium. At 0.02 M calcium ion in the presence of ANS, a further conformational change in the protein occurred, detected by a large increase in the quantum yield of ANS fluorescence. A structural hypothesis is advanced which qualitatively accounts for these observations.

Influence of potassium and calcium chlorides on the anticholine sterase activity of esters of diphenylphosphinic and diphenylthiophosphinic acids

1. The influence of KCl and CaCl2 on the inhibition of the catalytic activity of butyrylcholinesterase (BuCE) and acetylcholinesterase (ACE) by cationic organophosphorus inhibitors with combined action and with a reversible type of inhibition was investigated at 25‡, pH 7.5, and ionic strength (Μ) 0.0044, 0.11, and 0.55. Increasing Μ reduces irreversible and reversible inhibition. 2. In the case of the same increase in Μ, the influence of KCl on the values of the inhibitor constants, as a rule, was substantially greater than the influence of CaCl2; ACE proved to be more sensitive to a change in Μ than BuCE. The presence of specific salt effects in the reactions of cholinesterases was demonstrated.