The mechanism of increase in the ATPase activity of sarcoplasmic reticulum vesicles treated with n-alcohols.

Treatment of sarcoplasmic reticulum vesicles with aqueous n-alcohols caused inhibition of calcium uptake and enhancement of ATPase activity. With increasing alcohol concentration, the ATPase activity reached a maximum (in the case of n-butanol, at about 350 mM) and then decreased. The effect of n-butanol was extensively studied. The purified ATPase enzyme and leaky vesicles treated with Triton X-100 or phospholipase A showed high ATPase activity in the absence of n-butanol. With increasing n-butanol concentration, their atpase activities began to decrease above about 250 mM n-butanol, without any enhancement. In the presence of ATP, the turnover rate of calcium after calcium accumulation had reached a steady level was the same as that at the initial uptake. n-Butanol did not affect these rates. Kinetic analyses of these experiments were carried out. The mechanisms of calcium transport and of increase of ATPase activity in the presence of alcohol were interpreted as follows. After calcium accumulation had reached a steady level, fast influx and efflux continued; the influx was coupled with phosphorylated enzyme (E-P) formation and most of the efflux was coupled with rephosphorylation of ATP from ADP and E-P. The observed ATPase activity is the difference between these two reactions. If alcohol molecules make the vesicles leaky, calcium ions will flow out without ATP synthesis and the apparent ATPase activity will increase. The effect of alcohols on sarcoplasmic reticulum vesicles was separated into two actions. The enhancement of ATPase activity was attributed to a leakage of calcium ions from the vesicles, while the decrease of ATPase activity at higher concentrations of alcohols was attributed to denaturation of the ATPase enzyme itself. The two effects were interpreted in terms of equilibrium binding of alcohol molecules to two different sites of the vesicles; leakage and denaturation sites. Similar analysis was carried out for various n-alcohols from methanol to n-heptanol. The apparent free energies of binding of the methylene groups of n-alcohols were evaluated to be -863 cal/mol for the leakage site, and -732 cal/mol for the denaturation site.     

A comparative study of inhibition of acetylcholinesterase,trypsin, neuropathy target esterase,and spleen cell activation by structurally related organophosphorus compounds

Organophosphorus (OP) compounds can bind to and inactivate several target molecules other than acetylcholinesterase (AChE). In the present study, five sets of structurally related organophosphorus compounds were used to evaluate the relationships between organophosphorus binding sites of AChE, neuropathy target esterase (NTE), trypsin, and the target molecule(s) involved in inhibition of splenocyte activation by OP compounds. The concentration of each OP compound required to inhibit enzyme activity or splenocyte activation by concanavalin A by 50% was determined. The pattern of IC₅₀ values indicated that AChE, trypsin, NTE, and the molecule(s) involved in inhibition of splenocyte activation are distinct with regard to patterns of inhibition by OP compounds. However, there was a striking similarity in the patterns of inhibition for trypsin and NTE with substantial differences for only 2 of 20 compounds. This pattern suggests similarity in the active sites of these molecules. There were also similarities in the IC₅₀ patterns for lymphocyte activation and trypsin or NTE activity. However, the correlation was not as strong as between NTE and trypsin, and the data suggested the possibility of multiple target molecules for inhibition of splenocyte activation by OP compounds. More importantly, there was essentially no correlation between the pattern of IC₅₀ values for AChE and splenocyte activation. This strongly suggests that acetylcholine and AChE of the type found in the brain are not important in the regulation of splenocyte activation by concanavalin A.     

Inhibition of choline transport in erythrocytes by n-alkanols

The choline transport system of erythrocytes is reversibly inhibited by ethanol, n-butanol, n-hexanol, n-octanol, and n-decanol, but not by n-dodecanol. Each methylene group in the alkyl chain contributes 560 cal/mol to the free energy of binding at the inhibitory site. Inhibition results from the cooperative binding of two molecules of an alcohol, judging by the Hill coefficient n of 1.7-1.9. The mechanism of inhibition is noncompetitive, and the partition of the carrier between inward-facing and outward-facing forms is unaffected by the alcohols; it follows that the four main carrier forms, the inner and outer free carrier, and the inner and outer carrier-substrate complex, are equally susceptible to inhibition. Hexanol and decanol accelerate the reaction of N-ethylmaleimide with a thiol group in the inner carrier channel, but ethanol and butanol, at concentrations that inhibit transport by 70%, do not. The disproportionate effects on substrate transport and the N-ethylmaleimide reaction are most simply explained as the direct result of binding of alcohol molecules in different regions of the carrier, rather than as the indirect result of a disturbance in the structure of the lipid bilayer induced by the alcohols.     

Inhibition of forskolin-activated adenylate cyclase by ethanol and other solvents

Ethanol and a variety of solvents are known to activate basal and Gpp(NH)p- and hormone-stimulated adenylate cyclase. We report here that ethanol and other solvents inhibit the activation of adenylate cyclase by forskolin. In the presence of 10 microM forskolin, 2% ethanol gives about 20% inhibition and 5% ethanol gives 40% inhibition of enzyme activity. Analysis of ethanol inhibition at several forskolin concentrations suggests that inhibition is competitive versus forskolin. Thus the effect of ethanol is greater at low forskolin concentrations and minimal at high concentrations. In addition to ethanol, inhibition of forskolin activation was observed with acetone, n-butanol, t-butanol, dimethyl formamide, dioxane, methanol and n-propanol. Dimethyl sulfoxide was inhibitory only at high concentrations (10%). Since some solvent is needed to prepare forskolin solutions and to maintain solubility at higher concentrations, the inhibitory effects reported here are an important consideration in studies employing forskolin activation. To minimize solvent inhibition we recommend that dimethyl sulfoxide be used to prepare forskolin solutions. At concentrations of 5% and less, dimethyl sulfoxide gives little if any inhibition of forskolin activation and causes only small increases in basal activity.     

PROPERTIES OF THE EXTERNAL ACETYLCHOLINESTERASE IN GUINEA-PIG IRIS

Abstract— The acetylcholinesterase (AChE) of intact iris, the so-called external AChE, differs in several respects from the AChE in an homogenate of iris, called the total AChE. Maximum enzyme activities of the external and total AChE were obtained with an ACh concentration of 10 and 1.3 m m , respectively. The total AChE exhibited substrate inhibition at high substrate concentrations, whereas the external enzyme did not exhibit substrate inhibition in the range studied. The external AChE activity, when measured at 1.3 m m -ACh. accounted only for 12% of the total enzyme activity. After irreversible inhibition of AChE with diisopropylfluorophosphate (DFP) or methylisocyclopentylfluorophosphate (soman) the external AChE recovered to almost normal values after 48 h, whereas only 30% of the total AChE recovered during this period. Pupillographic studies after inhibition with DFP demonstrated that pupillary diameter had reached normal size after 24 h.
Destruction of the cholinergic input to iris reduced the total AChE activity by 40%, but did not alter the external AChE activity nor its rate of recovery after DFP inhibition. The specific activities of total AChE and total choline acetyltransferase were significantly higher in the sphincter than in the dilator muscle. After such denervation of iris the greatest reduction in total AChE and choline acetyltransferase were found in the sphincter region. After treatment with DFP the total AChE was inhibited to the same extent and recovered at the same rate in both regions.
After extraction of AChE from iris with various salt solutions and detergents, the particulate enzyme recovered faster than the soluble enzyme from DFP inhibition.     

Changes of Acetylcholinesterase Activity in Brain Areas and Liver of Sucrose- and Ethanol-Fed Rats

The effects of chronic ethanol or sucrose administration to rats on acetylcholinesterase from brain and liver were investigated. Membrane-bound and soluble acetylcholinesterase activities were determined in fractions prepared by centrifugation. The thermal stability and the effects of temperature and different types of alcohols on acetylcholinesterase activity were also studied. Membrane-bound acetylcholinesterase activity increased (p < 0.01) in the liver after chronic ethanol administration, whereas no differences among groups in the encephalic areas, except in the brain stem soluble form, were found. Membrane-bound acetylcholinesterase from the ethanol- and sucrose-treated groups was more stable at the different temperatures assayed between 10 and 50°C than that corresponding to the control group. Non-linear Arrhenius plots were obtained with preparations of membrane-bound acetylcholinesterase from rat liver, with discontinuities at 30°C (control or sucrose groups) or 34–35°C (alcohol group). Assays made with membrane-bound or soluble enzyme from brain showed linear Arrhenius plots in all groups studied. The inhibitory effects of increasing concentrations of ethanol, n-propanol and n-butanol on acetylcholinesterase preparations from forebrain, cerebellum, brain stem and liver of the three experimental groups (control, sucrose-fed and ethanol-fed) were very similar. However, n-butanol displayed a biphasic action on particulate or soluble preparations of rat forebrain. n-butanol inhibited (competitive inhibition) at higher concentrations (250–500 mM), while at lower concentrations (10–25 mM), the alcohol inhibited at low substrate concentrations but activated at high substrate concentration. These results suggest that the liver is more affected by ethanol than the brain. Moreover, the lipid composition of membranes is probably modified by ethanol or sucrose ingestion and this would affect membrane fluidity and consecuently the behaviour of acetylcholinesterase.     

Effect of environment on allosteric properties of acetylcholinesterase]

In the presence of organophosphorus inhibitors (OPI) AChE inhibition is initiated at a lower concentration of ACh; the plot reaction rate versus substrate concentration shows two maxima with a distinct minimum between them. It was shown that extremely mild conditions (short-term heating up to 50 degrees C; acidic or alkaline pH shift by 0.5 units; high concentrations of bivalent cations; erythrocyte storage) which do not affect substrate inhibition, remove this effect. The data obtained suggest that OPI effect is not directed to the site of AChE responsible for enzyme inhibition by ACh excess ("substrate inhibition site"), but to some other area. This results in a change in the conformation of the substrate inhibition site and a pronounced inhibition of the AChE activity takes place at lower substrate concentration.     

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

The neuropeptide TRH has a minor effect on the enzymatic activity of acetylcholinesterase in vitro

The neuropeptide thyrotropin-releasing hormone (TRH) elicits a variety of physiological effects of which some are due to cholinergic mechanisms. TRH modulates in vivo the effects of compounds affecting acetylcholinesterase (AChE). In the present study the in vitro effects of TRH on the activity of AChE were explored. TRH has no effect at physiologically relevant concentrations. At unphysiologically high concentrations (>5 mM) a slight inhibition was found. This was noticed also when the enzyme was exposed to the amide-free tripeptide analog p-Glu-His-Pro. We conclude that any cholinergic effect of TRH observed in vivo is unlikely to be due to a direct interaction of the peptide with AChE.     

Redox potential dependence of photophosphorylation and electron transfer in continuous illumination of Rhodopseudomonas sphaeroides chromatophores

The rate of ethanol elimination in fed and fasted rats can be predicted based on the liver content of alcohol dehydrogenase (EC 1.1.1.1), the steady-state rate equation, and the concentrations of substrates and products in liver during ethanol metabolism. The specific activity, kinetic constants, and multiplicity of enzyme forms are similar in fed and fasted rats, although the liver content of alcohol dehydrogenase falls 40% with fasting. The two major forms of the enzyme were separated and found to have very similar kinetic properties. The rat alcohol dehydrogenase is subject to substrate inhibition by ethanol at concentrations above 10 mM and follows a Theorell-Chance mechanism. The steady-state rate equation for this mechanism predicts that the in vivo activity of the enzyme is limited by NADH product inhibition at low ethanol concentrations and by both NADH inhibition and substrate inhibition at high ethanol concentrations. When the steady-state rate equation and the measured concentrations of substrates and products in freeze-clamped liver of fed and fasted rats metabolizing alcohol are employed to calculate alcohol oxidation rates, the values agree very well with the actual rates of ethanol elimination determined in vivo.     

Triphasic effects of short chain n-alcohols on synaptic membrane transport of choline and of gamma-aminobutyric acid

n-Alcohols, when added in increasing concentrations, had an unusual triphasic effect on the uptake of choline and of gamma-aminobutyric acid by isolated synaptosomes. There was slight inhibition of these uptakes at low n-alcohol concentrations, followed by a sharp peak of uptake enhancement, and then greater inhibition. The n-alcohol concentrations required for these effects were proportional to published n-alcohol membrane/buffer partition coefficients, with the peaks of uptake enhancement occurring at 60 mM n-propanol, 20 mM n-butanol and 7.5 mM n-pentanol. Synaptosomal membrane potential, as estimated from synaptosomal accumulation of the permeant cation [3H]tetraphenylphosphonium, was not affected by n-alcohols in the concentrations used in this study, suggesting that neither the inhibitory or enhancing effects of these n-alcohols were attributable to changes in trans-synaptosomal membrane ion gradients. The inhibiting and enhancing effects of n-alcohols could be reproduced in determinations of gamma-aminobutyric acid uptake by isolated synaptic plasma membranes, suggesting that the observed effects are due to a direct action of the n-alcohols on the synaptosomal plasma membrane. These effects may be attributable to a change in membrane binding of these alcohols from the membrane core to the membrane surface as alcohol concentration is increased.     

Design, synthesis and evaluation of isaindigotone derivatives as dual inhibitors for acetylcholinesterase and amyloid beta aggregation

A series of isaindigotone derivatives and analogues were designed, synthesized and evaluated as dual inhibitors of cholinesterases (ChEs) and self-induced β-amyloid (Aβ) aggregation. The synthetic compounds had IC(50) values at micro or nano molar range for cholinesterase inhibition, and some compounds exhibited strong inhibitory activity for AChE and high selectivity for AChE over BuChE, which were much better than the isaindigotone derivatives previously reported by our group. Most of these compounds showed higher self-induced Aβ aggregation inhibitory activity than a reference compound curcumin. The structure-activity relationship studies revealed that the derivatives with higher inhibition activity on AChE also showed higher selectivity for AChE over BuChE. Compound 6c exhibiting excellent inhibition for both AChE and self-induced Aβ aggregation was further studied using CD, EM, molecular docking and kinetics.     

THE INFLUENCE OF IONIC STRENGTH ON THE INTERACTION OF QUATERNARY DRUGS WITH MAMMALIAN ACETYLCHOLINESTERASE IN RELATION TO POSSIBLE REGULATORY EFFECTS

Abstract— The effects of inorganic salts, gallamine triethiodide and (+)-tubocurarine chloride on mammalian acetylcholinesterase (AChE) were examined. The results were obtained mainly from soluble erythrocyte AChE; particle-bound and detergent-solubilized rat brain enzymes were also used. Three aspects of AChE were examined, namely direct effects on activity, the recovery of activity of the diethylphosphorylated enzyme and thirdly the aggregation of the enzyme at low ionic strength as shown by chromatography on columns of Sepharose 6B. The action of gallamine on AChE was controlled by the substrate concentration and the ionic strength of the medium. Both inhibition and activation by gallamine could be observed, depending on the particular conditions used. All effects of gallamine disappeared when the ionic strength was raised to 015. The action of gallamine closely resembled the result of increasing ionic strength by adding NaCl, for in both cases the apparent affinity of AChE for substrate decreased and concomitantly the maximum velocity of hydrolysis increased. The phosphorylated enzyme recovered activity more rapidly when gallamine or tubocurarine were present, or when the ionic strength was increased. Aggregation of all enzyme forms was observed at low ionic strength; an increase to I = 015 dissociated AChE to the single molecular form. It was concluded that the mammalian enzymes closely resembled electric eel AChE. The possible methods by which regulation of AChE activity could occur are discussed in relation to these results.     

Effects of temperature and benzyl alcohol on the structure and adenylate cyclase activity of plasma membranes from bovine adrenal cortex

Adenylate cyclase activation by corticotropin (ACTH), fluoride and forskolin was studied as a function of membrane structure in plasma membranes from bovine adrenal cortex. The composition of these membranes was characterized by a very low cholesterol and sphingomyelin content and a high protein content. The fluorescent probes 1,6-diphenylhexa-1,3,5-triene (DPH) and a cationic analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) were, respectively, used to probe the hydrophobic and polar head regions of the bilayer. When both probes were embedded either in the plasma membranes or in liposomes obtained from their lipid extracts, they exhibited lifetime heterogeneity, and in terms of the order parameter S, hindered motion. Under all the experimental conditions tested, S was higher for TMA-DPH than for DPH but both S values decreased linearly with temperature within the range of 10 to 40 degrees C, in the plasma membranes and the liposomes. This indicated the absence of lipid phase transition and phase separation. Addition to the membranes of up to 100 mM benzyl alcohol at 20 degrees C also resulted in a linear decrease in S values. Membrane perturbations by temperature changes or benzyl alcohol treatment made it possible to distinguish between the characteristics of adenylate cyclase activation with each of the three effectors used. Linear Arrhenius plots showed that when adenylate cyclase activity was stimulated by forskolin or NaF, the activation energy was similar (70 kJ.mol-1). Fluidification of the membrane with benzyl alcohol concentrations of up to 100 mM at 12 or 24 degrees C produced a linear decrease in the forskolin-stimulated activity, that led to its inhibition by 50%. By contrast, NaF stabilized adenylate cyclase activity against the perturbations induced by benzyl alcohol at both temperatures. In the presence of ACTH, biphasic Arrhenius plots were characterized by a well-defined break at 18 degrees C, which shifted at 12.5 degrees C in the presence of 40 mM benzyl alcohol. These plots suggested that ACTH-sensitive adenylate cyclase exists in two different states. This hypothesis was supported by the striking difference in the effects of benzyl alcohol perturbation when experiments were performed below and above the break temperature. The present results are consistent with the possibility that clusters of ACTH receptors form in the membrane as a function of temperature and/or lipid phase fluidity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Substrate activation in acetylcholinesterase induced by low pH or mutation in the pi-cation subsite

Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the pi-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the pi-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.     

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.     

Structural insights into substrate traffic and inhibition in acetylcholinesterase

Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.     

Effect of methamidophos on the growth rate and esterase activity of the common carp Cyprinus carpio L.

The acute toxicity of methamidophos and three other compounds to fingerlings of the common carp Cyprinus carpio L. was determined. The 96-h LC₅₀ values were 68 mg/litre for methamidophos, 1.7 mg/litre for carbaryl, 0.21 mg/litre for lindane and 50 mg/litre for diquat.The acetylcholinesterase (AChE) and carboxylesterase (CarE) activities of the brain and liver were monitored over six weeks in fish poisoned with sublethal doses of methamidophos. Brain CarE was more sensitive than AChE but for the liver the reverse was true. In either case the degree of enzyme inhibition increased with increasing insecticide concentrations in the water.After exposure to methamidophos at 20 mg/litre for 48h liver AChE and CarE recovered faster than those of the brain. For both organs CarE recovered faster than AChE.At sublethal doses methamidophos affected the growth rate of the fish but no direct relationship between growth and insecticide concentrations could be established.     

Sperm motility inhibiting activity of a phytosterol from Alstonia macrophylla Wall ex A. DC. leaf extract: a tribal medicine

The role of methanolic extract and n-butanol fraction of A. macrophylla leaves was investigated on the forward motility of goat spermatozoa. The methanol extract (600 micro/g/ml) and one n-butanol fraction (Fraction A; 100 microg/ml) showed marked inhibition of sperm forward motility, tested by microscopic and spectrophotometric methods. Approximately, 50-60% of the spermatozoa lost their motility when treated with 600 microg/ml of methanol extract or 100 microg/ml of Fraction A. The Fraction A at 400 microg/ml concentration showed complete inhibition of sperm forward motility at 0 min. The inhibitory activity increased with the increasing concentrations of the fraction. The motility inhibitory activity of the Fraction A was stable to heat treatment at 100 degrees C for 2 min. The compound showed high inhibitory effect in the pH range 6.7-7.6. Fraction A also showed high efficacy for inhibiting human sperm motility, assessed by the microscopic method. The phytochemical analysis of methanolic extract of A. macrophylla leaves revealed the presence of sterols, triterpene, flavonoid, alkaloid, tannin and reducing sugar, while the Fraction A contains beta-sitosterol, a common phytosterol. The results demonstrate that Fraction A (beta-sitosterol) is a potent inhibitor of sperm motility and thus it has the potential to serve as a vaginal contraceptive.     

In vitro effects of polyvinylpyrrolidone and sucrose on the acetylcholinesterase,succinate dehydrogenase and lactate dehydrogenase activities in the brain

Summary The supernatant prepared from the brain tissue homogenate incubated in vitro in the presence of PVP or sucrose exhibits a decrease of AChE, SDH as well as of LDH activity. A 0.75% PVP solution inhibits AChE activity by 30%, LDH activity is inhibited by 35% and SDH activity by 40%. A two hours lasting effect of a 7.5% PVP solution at 3° C on enzymatic preparations induces in AChE 20% inhibition of its activity, in LDH an inhibition of 44% and in SDH the inhibition of its activity amounts to 74%. 1 M Sucrose inhibits AChE activity by 34%, LDH activity by 41% and SDH activity is inhibited by 31%. After two hours lasting effect of 1.4 M sucrose at 3° C on the supernatant the AChE activity is inhibited by 22% and that of LDH by 30%. The SDH activity was after a two hours lasting effect of 1 M sucrose at 3° C inhibited by 34%. The inhibition of activity of the above mentioned enzymes localized in brain cortex preparations was compared with the inhibition of activity of the isolated serum cholinesterase. 0.25 M Sucrose inhibited the activity of this enzyme by 25% and 0.75% PVP by 45%. A two hours lasting effect of 7.5% PVP or 1 M sucrose at 3° C on the cholinesterase induced a 40% and 22% inhibition respectively. After double washing of the brain cortical minced tissue, prepared in a 7.5% PVP containing solution, AChE activity was constant. By triple washing of the brain cortical crude mitochondrial fraction, exposed for two hours at 3° C to the effect of 1 M sucrose, SDH activity was also constant.Abbreviations AChE acetylcholinesterase (EC 3.1.1.7.) - INT 2(p-iodophenyl)3-p-nitrophenyl-5-phenyl tetrazolium chloride - LDH lactate dehydrogenase (EC 1.1.1.27.) - PMS phenazine methosulfate - PVP polyvinylpyrrolidone - SDH succinate dehydrogenase (EC 1.3.99.1.)