Ethanol-acetylcholinesterase-inhibitor interactions: inhibitor hydrophobicity and site specificity dependence.

1. Depending on the hydrophobicity and the site specificity of an inhibitor, striking differences were found in ethanol-acetylcholinesterase (AChE)-inhibitor interactions. 2. AChE used was from electric eel and was purified by affinity chromatography. 3. Ethanol at 10-200 mM reduced the inhibitory ability of tetrabutylammonium bromide (Bu4NBr). 4. The observed reduction might be a result of Bu4NBr inhibition being partially compensated for by an ethanol activation effect. 5. In contrast to Bu4NBr, propidium and edrophonium are not involved in hydrophobic interaction with AChE. 6. Their abilities to inhibit AChE activity were enhanced by ethanol. 7. Such an enhancement could not result from combining individual perturbations from ethanol and propidium or edrophonium, since ethanol itself increased the AChE activity. 8. In the presence of ethanol, propidium which binds to the peripheral site of the enzyme remained as an uncompetitive inhibitor, while edrophonium which binds to the active site was changed from a competitive inhibitor to a mixed one. 9. The effect of ethanol was therefore greater in the inhibitor which is involved with the active-site binding. 10. Fluorescence quenching studies of propidium-bound enzyme and edrophonium-bound enzyme revealed that ethanol in the concentration less than or equal to 400 mM did not cause significant conformational change at both the peripheral and the active sites of the enzyme.     

The effects of ethanol on the structural stability of acetylcholine receptor and the activity of various molecular forms of acetylcholinesterase

The actions of ethanol on the structural stability of acetylcholine receptor (AchR)-enriched membrane vesicles and the activity of various molecular forms of acetylcholinesterase (AchE) were investigated, using the receptor and the enzyme isolated from the electric organ of Torpedo californica. In the presence of ethanol up to 200 mM, the thermogram of AchR-enriched membranes exhibited no significant decrease in the temperature (td) of receptor transition at 57 degrees C, but a decrease in the enthalpy change (delta Hd) indicated a slight ethanol-induced structural perturbation. The presence of 12.5 nmol alpha-bungarotoxin also caused a decrease in delta Hd. A complete loss of the receptor transition was observed at a higher concentration 500 nmol of alpha-bungarotoxin and no recovery of the transition was found with the addition of 200 mM ethanol. The results suggested a noncompetitive interaction of ethanol with the receptor. In the presence of 200-1000 mM ethanol, the activity of two soluble forms of AchE, a higher (117 S) aggregate and a lower (10 S) aggregate was not significantly affected. Comparing the activity of these two aggregates over a wide concentration range of ethanol (200-2000 mM) revealed no obvious difference in the level of ethanol effect between them. However, after removal of ethanol, the higher aggregate form of AchE exhibited a greater recoverability of the activity, suggesting a possible slightly greater structure-functional stability for it. Studies of soluble AchE and membrane-bound AchE showed that the presence of 200 or 600 mM ethanol caused a greater level of inhibition in membrane-bound enzyme than in soluble enzyme, possible due to a disruption of protein-lipid interaction needed to maintain the conformation of membrane-bound AchE. Interestingly, at a much higher concentration of ethanol (2.0 M), membrane-bound AchE became more resistant to ethanol than did the soluble forms of AchE. In this case, the effective concentration of ethanol felt by the enzyme was expected to be less for membrane-bound AchE, owing to ethanol's solubility in lipids.     

Alpha adrenoreceptor mediated alteration of ethanol effects on (Na+ + K+)-ATPase of rat neuronal membranes

Noradrenaline (NA) sensitizes rat brain (Na+ + K+)-ATPase to inhibition by ethanol (EtOH). This effect of NA increases with the degree of enrichment of the enzyme: 0.1 mM NA + 0.05 M EtOH produced 27% inhibition in whole brain homogenates, 40% in 2.5-fold purified P2 fractions, and 45% in 5-fold purified microsomal fractions. The sensitization by NA was prevented by 0.1 microM phentolamine but not by 100 microM propranolol. Adrenaline and phenylephrine also sensitized the enzyme to EtOH inhibition in all of the fractions but isoproterenol did not. For all three alpha agonists the degree of sensitization was concentration dependent and the degree of reversal of this effect varied with the concentration of phentolamine added. These findings suggest that the NA + EtOH interaction is a direct effect on the membrane, probably mediated by an alpha receptor modified perturbation of the membrane microenvironment of the enzyme.     

Acetylcholinesterase from oat Seedlings. I. Preliminary biochemical characterization of the enzyme

The activity of acetylcholinesterase (AChE) isolated from coleoptiles of etiolated oat seedlings is strongly inhibited by neostigmine and less so by eserine. The optimum of the enzyme activity occurs at pH 7.2 and a temperature of + 36 °C. The enzyme Michaelis constant is 280 μM. Choline within the range of concentration from 0.001 to 10 mM does not affect the enzyme activity. Calcium ions at 5 mM concentration cause inhibition, while magnesium and manganese ions do not affect the enzyme activity. AChE isolated from oat seedlings differs in a number of properties from AChE occurring in the tissues of other plants. This research was supported in part by grant CPBP 05.02.4.07.     

Calpain Inhibition Prevents Ethanol-Induced Alterations in Spinal Motoneurons

Long-term exposure of ethanol (EtOH) alters the structure and function in brain and spinal cord. The present study addresses the mechanisms of EtOH-induced damaging effects on spinal motoneurons in vitro. Altered morphology and biochemical changes of such damage were demonstrated by in situ Wright staining and DNA ladder assay. EtOH at low to moderate (25–50 mM) concentrations induced damaging effects in the motoneuronal scaffold which involved activation of proteases like μ-calpain and caspase-3. Caspase-8 was seen only at higher (100 mM) EtOH concentration. Further, pretreatment with calpeptin, a potent calpain inhibitor, confirmed the involvement of active proteases in EtOH-induced damage to motoneurons. The lysosomal enzyme cathepsin D was also elevated in the motoneurons by EtOH, and this effect was significantly attenuated by inhibitor treatment. Overall, EtOH exposure rendered spinal motoneurons vulnerable to damage, and calpeptin provided protection, suggesting a critical role of calpain activation in EtOH-induced alterations in spinal motoneurons.     

Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase

Fatty acyl ethyl esters, previously identified in our laboratory as metabolites of ethanol in human and rabbit myocardium, arise from an esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A. This study was designed to isolate and purify the enzyme(s) in rabbit myocardium that catalyze(s) this reaction. Enzyme activity in homogenates of rabbit myocardium, as assayed by the rate of synthesis of ethyl [14C]oleate from 0.4 mM [14C]oleic acid and 0.2 M ethanol, was 31 nmol/(g.h), and all of it was recovered in the 48400g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elution with a NaCl gradient (0-0.25 M) separated two enzyme activities accounting for 13 and 87% of recovered synthase activity. The major enzyme activity was then purified over 5000-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti-albumin affinity chromatographies with an overall yield of 40%. Up to 45 micrograms of enzyme was present per g of myocardium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single polypeptide with Mr 26 000, and gel permeation chromatography under nondenaturing conditions indicated a Mr of 50 000 for the active enzyme. Kinetic analyses using the purified enzyme indicated that greatest rates of ethyl ester synthesis were observed with unsaturated octadecanoic fatty acid substrates [Vmax = 1.9 and 1.5 nmol/(mg.s) for linoleate and oleate, respectively], with lesser rates associated with palmitate, stearate, and arachidonate substrates [0.14, 0.03, and 0.35 nmol/(mg.s), respectively].(ABSTRACT TRUNCATED AT 250 WORDS)     

A neutral phospholipase D activity from rat brain synaptic plasma membranes. Identification and partial characterization

Rapid activation of phospholipase D (PLD) in response to cell stimulation was recently demonstrated in many systems, raising the hypothesis that PLD participates in transduction of extracellular signals across the plasma membrane. In the present study, we describe the identification of a neutral PLD activity in purified rat brain synaptic plasma membranes, and the in vitro conditions required to assay its catalytic activity with exogenous [3H]phosphatidylcholine as substrate. Production of [3H]phosphatidic acid, the natural lipid product of PLD and of [3H]phosphatidylethanol, catalyzed by PLD in the presence of ethanol via transphosphatidylation, were measured. The synaptic membrane PLD exhibited its highest activity at pH 7.2 and was thus defined as a neutral PLD. Enzyme activity was absolutely dependent on the presence of sodium oleate and was strongly activated by Mg2+ ions (at 1 mM). Ca2+ at concentrations up to 0.25 mM was as stimulatory as Mg2+, but at 2 mM it completely inhibited enzyme activity. Mg2+ extended the linear phase of PLD activity from 2 to 15 min, suggesting that it may stabilize the enzyme under our assay conditions. The production of [3H]phosphatidylethanol was a saturable function of ethanol concentration. Production of [3H] phosphatidic acid was inversely related to the concentration of ethanol and to the accumulation of phosphatidylethanol, indicating that the two phospholipids are indeed produced by the competing hydrolase and transferase activities of the same enzyme. beta,beta-Dimethylglutaric acid, utilized previously as a buffer in studies of rat brain PLD, inhibited enzyme activity at neutral pH but not at acidic pH. The properties of the neutral synaptic membrane PLD and its relationships with other in vitro, acid, and neutral PLD activities, as well as with the signal-dependent PLD detected in intact cells, are discussed.     

Interaction of 3-Alkylpyridinium Polymers from the Sea Sponge Reniera sarai with Insect Acetylcholinesterase

3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS–PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS–PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.     

Interaction of 3-Alkylpyridinium Polymers from the Sea Sponge Reniera sarai with Insect Acetylcholinesterase

3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS–PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS–PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.     

Differential effects of ethanol on membrane-bound and soluble acetylcholinesterase from sarcoplasmic reticulum membranes

The action of ethanol on the activity of membrane-bound and soluble acetylcholinesterase (AChE) in sarcoplasmic reticulum of skeletal muscle has been studied. Treatment of membranes with 2.5–12.5% v/v ethanol produced a slight stimulation of the AChE activity and inhibition at higher concentration. The enzyme remained associated with the membranes after these treatments. The enzyme solubilized with Triton X-100 was inhibited by ethanol in a time-independent manner. Isolated 16 S (A₁₂), 10.5 S (G₄) and 4.5 S (G₁) forms of AChE were inhibited by ethanol to a similar extent. Samples were reversibly inhibited by ethanol, up to 12.5% v/v, and irreversibly at higher concentrations. Kinetic studies performed with isolated forms in the presence of 5–12.5% v/v ethanol showed that the solvent behaved as a competitive inhibitor of the asymmetric form but as a mixed inhibitor of the tetrameric and monomeric forms. The results show that the solvent interacts with active and/or regulatory sites of AChE from muscle microsomes.     

Purification of a 51 kDa endo-β-N-acetylglucosaminidase from Staphylococcus aureus

A bacteriolytic enzyme obtained from the culture fluid of Staphylococcus aureus FDA 209P was purified to homogeneity utilizing dye-ligand affinity column chromatography, hydrophobic interaction high pressure liquid chromatography (HPLC) and hydroxyapatite HPLC. Subsequent characterizations indicated that the purified enzyme acted as endo-beta-N-acetylglucosaminidase. The molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was 51,000 and the isoelectric point was higher than 10. The optimum pH for the enzyme activity on whole cells of Micrococcus luteus as a substrate was 8.0. Some heavy metal cations (Cu2+ and Zn2+) inhibited the enzyme activity at a concentration of 0.1 mM and others (Ba2+, Mg2+ and Co2+) showed a stimulating effect at a concentration of 1 mM.     

钙离子对江浙蝮蛇毒酸性磷脂酶A_2结构与功能的影响

钙离子在江浙蝮蛇毒酸性磷脂酶Ａ_２中的作用是多方面的。它不仅能够引起酸性磷脂酶Ａ_２在溶液中构象的变化,而且对该酶活性有较大的影响。Ｃａ~（２＋）为酶活力所必需,当Ｃａ~（２＋）浓度达到０．０６ｍｍｏｌ／Ｌ时,酶表现出很高的活力;Ｃａ~（２＋）浓度超过０．５ｍｍｏｌ／Ｌ时,催化反应出现一个明显的延滞期。化学修饰表明Ｈｉｓ_（４７）在表现活力方面起重要作用,Ｃａ~（２＋）的存在可降低其修饰反应的速度,提示这是由于Ｃａ~（２＋）引起构象发生变化而造成的。     

A structural motif of acetylcholinesterase that promotes amyloid beta-peptide fibril formation

Acetylcholinesterase (AChE) has been found to be associated with the core of senile plaques. We have shown that AChE interacts with the amyloid beta-peptide (Abeta) and promotes amyloid fibril formation by a hydrophobic environment close to the peripheral anionic binding site (PAS) of the enzyme. Here we present evidence for the structural motif of AChE involved in this interaction. First, we modeled the docking of Abeta onto the structure of Torpedo californica AChE, and identified four potential sites for AChE-Abeta complex formation. One of these, Site I, spans a major hydrophobic sequence exposed on the surface of AChE, which had been previously shown to interact with liposomes [Shin et al. (1996) Protein Sci. 5, 42-51]. Second, we examined several AChE-derived peptides and found that a synthetic 35-residue peptide corresponding to the above hydrophobic sequence was able to promote amyloid formation. We also studied the ability to promote amyloid formation of two synthetic 24-residue peptides derived from the sequence of a Omega-loop, which has been suggested as an AChE-Abeta interacting motif. Kinetic analyses indicate that only the 35-residue hydrophobic peptide mimics the effect of intact AChE on amyloid formation. Moreover, RP-HPLC analysis revealed that the 35-residue peptide was incorporated into the growing Abeta-fibrils. Finally, fluorescence binding studies showed that this peptide binds Abeta with a K(d) = 184 microM, independent of salt concentration, indicating that the interaction is primarily hydrophobic. Our results indicate that the homologous human AChE motif is capable of accelerating Abeta fibrillogenesis.     

A novel hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, as a probe of the K+ occlusion center of Na+/K(+)-ATPase

A hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), was examined as a probe of the K+ occlusion center of Na+/K(+)-ATPase. Treatment of the enzyme with AU-1421 at 37 degrees C and pH 7.0 produced irreversible inactivation of the enzyme. This inactivation was prevented, with simple competitive kinetics, by K+ or its congeners in the order of Tl+ greater than Rb+ greater than NH+4 greater than Cs+. The concentrations of these cations required for the protection, were consistent with the affinities for transport and ATPase activity. The apparent binding constant for K+ was calculated to be 0.03 mM, from the competition with AU-1421. This protection was cancelled by a high concentration of ATP or ADP. A high concentration of Na+ (Kd = 6.5-6.9 mM), as a substitute for K+, also prevented the inactivation by AU-1421. Thus, the enzyme was protected from AU-1421 when the occlusion center was occupied by a monovalent cation, irrespective of the enzyme conformation, E1 (Na(+)-bound form) or E2 (K(+)-bound form). On the other hand, the enzyme was most sensitive to AU-1421 in the presence of low concentration of Na+ (0.4-0.8 mM) or a high concentration of ATP. Tris, imidazole or choline, which favors the E1 state, also accelerated the inactivation by AU-1421. These suggest that AU-1421 reacts with the occlusion center through the E1 state.     

Linking number anomalies in DNA under conditions close to condensation

Changes in linking number and the apparent winding angle of pBR322 DNA have been evaluated in mixed ethanol-water solvents containing either Na or Mg as the major counterion contributing to the electrostatic shielding of the duplex. The average number of superhelical turns (tau) produced in the standard electrophoresis buffer (Tris-borate-EDTA, pH 8.0) by the transfer of DNA, relaxed in 200 mM NaCl, 10 mM NaH2PO4/Na2HPO4, and 2 mM EDTA, pH 7, by calf thymus topoisomerase or ligated in 6.6 mM MgCl2, 1 mM KCl, 1 mM ATP, 1 mM dithiothreitol, and 66 mM Tris, pH 7.6, by T4 ligase, was determined as a function of the EtOH concentration. At low enzyme concentrations, the tau values became increasingly more positive in the presence of both cations as the ethanol concentration increased, indicating that the duplex structure was overwound in the ethanol solvents. Winding angle changes between 0 and 20% ethanol, calculated from these values of tau, exhibited the same correlations with CD spectral properties as had been previously observed for 100% aqueous systems containing monovalent cations [Kilkuskie, R., Wood, N., Shinn, R., Ringquist, S., & Hanlon, S. (1988) Biochemistry 27, 4377-4386]. The results at higher concentrations of ethanol (25-30%), however, were anomalous for the Mg-ligase system. The anomalies increased with higher ethanol, ligase, or Mg concentration. Gel run under these conditions showed enhanced concentrations of slow-moving components, indicative of ligation of intermolecular associated DNA species. At a 10-fold higher level of ligase, ethanol appeared to unwind the duplex, confirming the results of Lee, Mizusawa, and Kakefuda [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2838-2842]. All of these anomalies occur under solvent conditions which are close to conditions which produce a heterogeneous dispersion of sedimenting species in ultracentrifugal experiments and compact rodlike structures, visualized by electron microscopy. The circular dichroism spectra at the onset of the formation of these structures show the characteristics of a chirally packed array of DNA duplexes. The reversal of the trend of the ethanol effect on linking number at higher enzyme and Mg(II) concentrations can be most easily explained by the promotion of the condensation phenomenon by either the ligase or a contaminating factor in the preparation. We suggest that the anomalies in the linking number and winding angle values are due to either ligation of chirally bent DNA species or a change in the helical period as the linear DNA adapts to the conformation required for collapse.(ABSTRACT TRUNCATED AT 400 WORDS)     

Environmental impact of mosquito pesticides: influence of temefos on the brain acetylcholinesterase of killifish.

Temefos and six of its metabolites were tested for their capacity to inhibit the in vitro activity of brain acetylcholinesterase (AChE) of Fundulus heteroclitus. While temefos was not inhibitory at levels up to 10.7 mM in brain homogenate samples, its metabolites were active within the range of 2 X 10(-4)mM to 1.26 mM in causing a 50% reduction in the enzyme activity. Exposure of F. heteroclitus to temefos under laboratory conditions caused a reduction in AChE activity, which was proportional to the pesticide concentration and the exposure period. Visible symptoms of organophosphate poisoning were apparent only after the AChE inhibition reached 80%. F. heteroclitus and Cyprinidon variegatus exposed to 10 biweekly applications of temefos granules in the field showed no inhibition of brain AChE. However, exposure of F. heteroclitus to biweekly applications (four) of temefos emulsion caused a reduction in the enzyme (50%), but only in the pre-third application samples. A gradual increase in brain AChE occurred both in F. heteroclitus and C. variegatus as the season progressed from April to October.     

A facile purification procedure of phospholipase D from cabbage and its characterization.

Phospholipase D (PLD), an enzyme predestined for the preparation of new phospholipids, was isolated from cabbage and purified in a highly efficient way by using a combination of hydrophobic chromatography and a specific calcium effect. In the presence of calcium ions (50mM), PLD is bound from the crude enzyme solution to Octyl-Sepharose and subsequently selectively eluted by removing the calcium ions. The obtained enzyme is electrophoretically pure (95%), its molecular mass and isoelectric point were determined to be 87,000 Da and 4.7, respectively. The purified enzyme was kinetically characterized by use of mixed phosphatidylcholine-SDS micelles as well as the short-chain lecithins 1,2-dihexanoyl- and 1,2-diheptanoyl-sn-glycero-3-phosphocholine as substrates. A hyperbolic upsilon/[S]-characteristic was obtained for the mixed micellar system, whereas the upsilon/[S] curves of the short-chain lecithins reflect the dependence of velocity on the physical state of the substrate. A small velocity increase was observed up to a critical substrate concentration near the critical micelle concentration, from where the velocity increases hyperbolically.     

The effect of galactose metabolic disorders on rat brain acetylcholinesterase activity

To evaluate whether in classical galactosemia galactose (Gal), galactose-1-phosphate (Gal-1-P) and galactitol (Galtol) affect brain acetylcholinesterase (AChE) activity, various concentrations (1-16 mM) of these compounds were preincubated with brain homogenates of suckling rats as well as with pure eel Electroforus electricus AChE at 37 degrees C for 1 h. Initially, Galtol (up to 2.0 mM) increased (25%) AChE activity which decreased. thereafter, reaching the control value in high Galtol concentrations. Gal-1-P decreased gradually the enzyme activity reaching a plateau (38%), when incubated with 8-16 mM. However, when the usually found 2 mM of Galtol and 2 mM of Gal-1-P, concentrations in galactosemia were added in the incubation mixture simultaneously, brain AChE was stimulated (16%). Galtol or Gal-1-P modulated brain AChE as well as enzyme activity of E.electricus in the same way. Gal, Glucose (Glu) and glucose-1-phosphate (Glu-1-P) had no effect on AChE activity. It is suggested that Galtol as well as Gal-1-P can affect acetylcholine degradation acting directly on AChE molecule. Consequently the direct action of these substances on the enzyme might explain the brain cholinergic dysfunction in untreated galactosemia patients.     

Effects of reconstitution of membrane-bound lipoprotein lipase and dispersity of substrate on its reaction characteristics

Reaction characteristics of a membrane-bound lipoprotein lipase acting on a hydrophobic substrate were investigated in aggregated structures—lipid bilayers of liposomes and mixed micelles of Triton X-100. The enzyme activity was enhanced with increases in Triton X-100 and phospholipid concentrations in micellar and liposomal structures. This higher activity was found to be due to both the solubilization state of the hydrophobic substrate and the hydrophobic interactions of the enzyme with either phospholipid or Triton X-100 molecules as a result of its incorporation into the aggregated systems. The enzyme reconstituted into lipid bilayers of liposomes prepared from 15 mM DMPC in the presence of 0.05% Triton X-100 showed a further 1.5-fold higher activity in comparison with the activity without reconstitution in micelles of 1.0% Triton X-100. These results indicate the necessity of the bilayer structure to retain the membrane-bound enzyme in an active conformation.     

Ethanol effects on voltage-dependent membrane conductances: comparative sensitivity of channel populations inAplysia neurons

The study of ethanol (EtOH) action is interesting because of its clinical relevance and for the insights it provides into structure-function relationships of excitable membranes. This paper describes the concentration dependencies of various parameters of four currents in Aplysia cells. ICa is the most sensitive of the currents studied. There was a significant reduction of ICa at concentrations of 50 mM EtOH. At low concentrations, the reduction of amplitude was the primary effect of ethanol, with the kinetics and voltage dependency of activation not affected. INa and IA were also affected, but at EtOH levels higher than those which altered ICa. The primary effect of EtOH on INa was a reduction in its amplitude, although the time to peak current flow was increased by EtOH. The effects of EtOH on IA were cell specific and, for the purposes of this paper, we examined the giant metacerebral cell (MCC). In MCC, the primary effect of EtOH on IA was an increase in the time course of inactivation. The time to peak IA was also increased by high concentrations of EtOH, but its amplitude was unaffected even at high concentrations. The delayed rectifier current, IK, was the most EtOH resistant of the currents examined. High EtOH concentrations augmented the amplitude of IK, although even at 600 mM concentrations, the percentage change was only 30%. Our results indicate that the calcium channel is very susceptible to the influence of ethanol and is a serious candidate to be the primary target of EtOH action in the nervous system. The differential sensitivity of voltage-dependent currents and individual components of a given current suggests further experiments to probe the relationship between membrane structure and channel function in excitable membranes.