Molecular simulation of the effects of alcohols on peptide structure

The effects of alcohols on local protein structure have been simulated using computational approaches and model peptides. Molecular simulations were carried out on a 7-residue peptide created in both an extended conformation and an alpha-helix to explore alcohol-induced changes in peptide structure. It was assumed that alcohols hydrogen bond at peptide carbonyl groups with an optimum geometry and compete with water molecules at these site. Energy minimization of the peptide/alcohol assemblies revealed that alcohols induced a twist in the peptide backbone as a function of (1) the methylene chain length, (2) the hydrogen-bond geometry, (3) halogenation of the molecule, (4) concentration, and (5) the dielectric constant. The rank ordering of the potencies of the alcohols was hexafluoroisopropanol > trifluoroethanol approximately pentanol > butanol > ethanol > methanol. Helix destabilization by cosolvent was measured by examining the hydrogen-bond lengths in peptide structures that resulted from a combination of energy minimization and molecular dynamics simulations. Destabilization was also found to be dependent upon the chemical nature of the alcohol and the hydrogen-bond geometry. The data suggest that alcohols at low concentrations affect protein structure mainly through a combination of hydrogen-bonding and hydrophobic interactions that are influenced by the properties of the solvent.     

CHEMICAL STRUCTURE AND NARCOTIC POTENCY TO GRAIN WEEVILS. ALCOHOLS AND METHYL ESTERS OF FATTY ACIDS

The narcotic and toxic potencies of the first seven homologous alcohols and the methyl esters of the first seven homologous fatty acids have been determined using grain weevils (Calandra granaria). The experiments were designed as balanced incomplete blocks, and Finney's probit plane technique was applied to the results. In both series of compounds the narcotic and toxic potencies expressed in thermodynamic concentrations decreased with increasing number of carbon atoms. The margin between narcotic dose and toxic dose is greater in the alcohol series than in the ester series. The methyl esters of the fatty acids showed a stepwise descent in biological potency, the odd numbered series being more active than the even series. The penetration of these compounds into grain weevils and their biological action are discussed in relation to the coefficients of response to log concentration and log time of exposure.     

Comparative Effects of Aliphatic Compounds on Inhibition of Lettuce Fruit Germination

Germination of lettuce (Lactuca sativa L. cv. Great Lakes) fruitswas inhibited by alcohols, aldehydes and ketones. Inhibitoryactivity increased with increasing length of the carbon chain,being lower for the branched chain isomers. Additional hydroxylgroups decreased inhibitory activity. Allyl alcohol and acro-lcinwere markedly inhibitory but usually unsaturation in the chainhad little effect. Although the main structure-activity correlationwas with lipophilic properties some other structural featuresmodified the action. A comparison was made between these findingsand effects recorded in the literature on membrane perturbationsespecially in connection with mitochondrial activity.     

Influence of some aliphatic alcohols on the metabolism of rat liver slices

The influence of some aliphatic alcohols on oxygen uptake, carbon dioxide production, acid formation and lactate and pyruvate concentrations of rat liver slices was studied. At the concentrations used, none of the alcohols was found to influence oxygen uptake. Of the alcohols that are not oxidized by liver alcohol dehydrogenase, methanol increased carbon dioxide production, propan-2-ol decreased it and 2-methylpropan-2-ol was without influence. All the alcohols that are oxidized by the enzyme strongly decreased carbon dioxide production. The alcohols that are not oxidized had no marked effect on the lactate/pyruvate concentration ratio, whereas the other alcohols strongly increased the ratio. A highly significant correlation was found between the effects of the alcohol on pyruvate concentration and carbon dioxide production. It is assumed that the shift in the redox potential inhibits the function of the tricarboxylic acid cycle of the liver.     

Lipid solubility is not the sole criterion for the inhibition of a Ca2(+)-activated K+ channel by alcohols

The effect of a homologous series of n-alkanols (C2-C8) on the 86Rb+ influx through charybdotoxin sensitive Ca2(+)-activated K+ channels of rat glioma C6 cells was investigated. The lipid solubility of the n-alkanols was not the sole determinant of the inhibitory potency of these substances for ion flux inhibition. 1-Hexanol for example was about 8-times less potent than one would expect on the basis of its lipid solubility. The introduction of a second OH-group in this molecule (giving 1,6-hexanediol) or a structural shift in the OH-group of 1-hexanol from position 1 to 3 strongly increased the potency of the alcohol. The above data cannot be explained by a pure lipid site of action of the alcohols. Therefore it seems likely that direct effects on protein are involved in the inhibitory action of some of the alcohols.     

Ethanol Reduces Norepinephrine-Stimulated Melatonin Synthesis in Rat Pinealocytes

In this study, the in vitro effects of ethanol on norepinephrine-stimulated cyclic AMP (cAMP), N-acetyltransferase (NAT), and melatonin (MT) production were examined in dispersed rat pinealocytes. Cellular cAMP content was determined 15 min after treatment; whereas NAT activity and MT release in the medium were determined 4.5 h after treatment. It was found that ethanol less than or equal to 200 mM had no effect on norepinephrine-stimulated cAMP response, whereas 25 mM ethanol resulted in a significant inhibition of norepinephrine-stimulated NAT and MT levels. Furthermore, ethanol was equally effective in inhibiting the dibutyryl cAMP-stimulated NAT and MT levels. The inhibitory action of ethanol was not due to a direct effect or a delay in the onset of NAT activity. When alcohols with different chain lengths were used, it was found that their inhibitory potencies were related to their chain lengths with butanol greater than propanol greater than ethanol greater than methanol. Taken together, these findings indicate that (1) ethanol has an inhibitory action on norepinephrine-stimulated MT synthesis, (2) one site of ethanol action is distal to cAMP elevation, and (3) the inhibitory effect of ethanol on pineal MT synthesis appears to be secondary to its hydrophobic action.     

Inhibition of the aminopeptidase from Aeromonas proteolytica by aliphatic alcohols. Characterization of the hydrophobic substrate recognition site.

Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols, even up to concentrations of 20% (v/v). All of the alcohols studied were competitive inhibitors of AAP with K(i) values between 860 and 0.98 mM. The clear trend in the data was that as the carbon chain length increases from one to four, the K(i) values increase. Branching of the carbon chains also increases the K(i) values, but large bulky groups, such as that found in tert-butyl alcohol, do not inhibit AAP as well as leucine analogues, such as 3-methyl-1-butanol. The competitive nature of the inhibition indicates that the substrate and each alcohol studied are mutually exclusive due to binding at the same site on the enzyme. On the basis of EPR and electronic absorption data for Co(II)-substituted AAP, none of the alcohols studied binds to the dinuclear metallo-active site of AAP. Thus, reaction of the inhibitory alcohols with the catalytic metal ions cannot constitute the mechanism of inhibition. Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the binding of substrate. Thus, the first step in peptide hydrolysis is the recognition of the N-terminal amino acid side chain by the hydrophobic pocket adjacent to the dinuclear active site of AAP.     

Synthesis and antimycobacterial activity of ferrocenyl ethambutol analogues and ferrocenyl diamines

A new series of ferrocenyl diamino alcohols and diamines were synthesized and their inhibitory potencies were probed with Mycobacterium tuberculosis. Interestingly, ferrocenyl diamines 6a and b display significant activities against M. tuberculosis H37Rv.     

How aliphatic alcohols and pH affect reactivity of horse blood serum cholinesterase at its interaction with organophosphorus inhibitors

There was studied action of aliphatic alcohols (ethanol, propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-butanol), and pH on various kinds of serum cholinesterase. At inhibition of the cholinesterase hydrolytic activity under effect of alcohols the key role was played not by the total number of carbon atoms in the alcohol molecule, but by the “efficient length” of the carbohydrate chain. The fact that the presence of alcohols did not affect parameters of reversible inhibition of cholinesterase by onium ions tetramethylammonium and choline allows suggesting the absence of action of solvents on specific sorption of acetylcholine in the enzyme active center. With aid of two sets of hydrophobic organophosphorus inhibitors (OPI) (12 compounds), we have managed to estimate both the degree and the character itself of serum cholinesterase.     

ESR analysis with long-chain alkyl spin labels in bovine blood platelets. Relationship between the increase in membrane fluidity by alcohols and phenolic compounds and their inhibitory effects on aggregation

Four spin-labeled probes (5-doxylstearic acid (5-NS), its methyl ester (5-NMS), 16-doxylmethylstearate (16-NMS) and 4-(N,N-dimethyl-N-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-ox yl (CAT-15)) were used to monitor membrane fluidity change in bovine platelets induced by three alkyl alcohols, benzyl alcohol and two phenolic compounds. The relationship between the increase in membrane fluidity induced by these compounds and their inhibitory effects on platelet aggregation was observed. Experiments with the four probes showed that n-hexyl alcohol induced decreases in the order parameter of 5-NS and apparent rotational correlation times of the other probes at the same minimal alcohol concentration. The decreases were observed in the concentration range that inhibited aggregation. n-Amyl alcohol and n-butyl alcohol decreased the values of the parameters of the above mentioned only at higher concentrations that were dependent on their hydrophobicities. Like alkyl alcohols, benzyl alcohol and phenolic compounds decreased the values of the parameters in the concentration ranges in which these compounds inhibited platelet aggregation. The concentration of these compounds causing 50% inhibition of platelet aggregation, the IC50 values, and data on 5-NS-labeled platelets indicated that they inhibited aggregation and decreased the value of the order parameter at lower concentrations relative to their Poct values in comparison to the effective concentrations of alcohols. Phenolic compounds also decreased the values of the apparent rotational correlation times of 5-NMS and 16-NMS. These results indicate that the inhibition of platelet aggregation by alcohols and phenolic compounds is due to membrane perturbation in wide range in depths within the lipid bilayer.     

Fluorescence-tagged monolignols: synthesis, and application to studying in vitro lignification

Fluorescence-tagged coniferyl alcohols, coniferyl alcohol γ-coupled by ethylenediamine spacers to dimethylaminocoumarin or nitrobenzofuran fluorophores, were tested as photoprobes to study the oxidase-mediated polymerization of monolignols. The fluorescent coniferyl alcohol derivatives readily underwent peroxidase-catalyzed in vitro copolymerization with coniferyl alcohol to yield fluorescent dehydrogenation polymers, the backbone polymers of which were structurally indistinguishable from polymers formed solely from coniferyl alcohol. To illustrate the use of the photoprobes, we successfully monitored in real time the complexation of coniferyl alcohol with horseradish apoperoxidase by Fo?rster resonance energy transfer (FRET) using the protein-tryptophan near the active site and a dimethylaminocoumarin moiety as donor and acceptor fluorophores. Furthermore, mixtures of fluorescence-tagged and normal coniferyl alcohols readily diffused into isolated maize cell walls and reacted with wall-bound peroxidases to form in muro artificial lignins that could be visualized by fluorescence microscopy. Thus we anticipate that fluorescence-tagged monolignols will be useful for in vitro and in vivo studies of cell wall lignification.     

不同提取方法对桂花精油品质的影响

用超临界流体萃取法(SCFE)、可食用石油醚和酒精抽提蒸馏三种不同的抽提方法,提取了咸宁桂花品种——银星的花精油;气质联用(GC-MS)分析不同方法提取精油的香气成分及其相对含量。结果表明:SCFE提取的精油得率最高(0.19%),紫罗兰酮、醇类的相对含量高达36.99%;石油醚浸提结果次之(0.13%),酒精抽提法不可取(0.07%)。     

Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase.

Reductive methylation of lysine residues activates liver alcohol dehydrogenase in the oxidation of primary alcohols, but decreases the activity of the enzyme towards secondary alcohols. The modification also desensitizes the dehydrogenase to substrate inhibition at high alcohol concentrations. Steady-state kinetic studies of methylated liver alcohol dehydrogenase over a wide range of alcohol concentrations suggest that alcohol oxidation proceeds via a random addition of coenzyme and substrate with a pathway for the formation of the productive enzyme-NADH-alcohol complex. To facilitate the analyses of the effects of methylation on liver alcohol dehydrogenase and factors affecting them, new operational kinetic parameters to describe the results at high substrate concentration were introduced. The changes in the dehydrogenase activity on alkylation were found to be associated with changes in the maximum velocities that are affected by the hydrophobicity of alkyl groups introduced at lysine residues. The desensitization of alkylated liver alcohol dehydrogenase to substrate inhibition is identified with a decrease in inhibitory Michaelis constants for alcohols and this is favoured by the steric effects of substituents at the lysine residues.     

The Effect of Alcohols on Growth and Lipase Formation by Staphylococcus aureus

S ummary . The growth of Staphylococcus aureus 111 was inhibited by aliphatic alcohols. The extent of inhibition by primary n -alcohols increased with the elongation of the chain. Secondary alcohols were less inhibitory than the corresponding primary alcohols. The inhibitory effect was influenced by the distance of the hydroxyl group from the end of the carbon chain. When the alcohols were added to the medium in sub-inhibitory amount, enzyme synthesis by the bacteria was decreased, but there was no demonstrable inhibition of enzyme already formed.     

Phase separation in phosphatidylcholine bilayers as a predictor of inhibition of blood platelet aggregation by amantadines

The ability of eleven amantadine derivatives to induce phase separation in dipalmitoyl phosphatidylcholine bilayers was studied by differential scanning calorimetry. The relative potency varied with the shape and size of the hydrocarbon cage. These agents also markedly inhibited blood platelet aggregation. The relative potencies of these compounds to induce phase separation showed a significant correlation ($\text{r = 0.70}$) with their platelet inhibitory activity suggesting that their pharmacologic action may be at the level of the platelet membrane. The effective concentration of the parent component amantadine is similar to its pharmacologic concentration suggesting its use as an anti-platelet drug.     

Effect of alcohols on the activity of bacterial agmatinase

Aliphatic alcohols inhibit the activity of bacterial agmatinase (E. C. 3.5.3.11). A correlation is demonstrated between the inhibition constant and the length of alcohol carbohydrate radical, and the presence of polar groups has decreased the inhibitory effect. The importance of hydrophobic interactions in the formation of agmatinase-substrate and agmatinase-inhibitor complexes is suggested. The increment of free binding energy within the homologous alcohol row is practically constant (0.5-0.7 kcal/mole). Comparison of deltaF amines and their respective alcohols makes possible to evaluate the contribution of electrostatic interaction in the formation of the enzyme-inhibitor complex. A correlation is observed between solubility of alcohols and their inhibitory effect.     

α-adrenergic inhibition of cyclic AMP accumulation in hamster adipocytes similarity of receptor with α-2 adrenergic receptors

The present communication shows the effects of several α-adrenergic agonists and antagonists on cyclic AMP levels in hamster epididymal adipocytes. In response to ACTH (30 mU/ml) in combination with 1-methyl-3-isobutylxanthine (0.10 mM) or adenosine deaminase (1.0 μg/ml), cyclic AMP levels increased to a maximum by 10 min and this level was maintained for another 20 min. Elevated cyclic AMP levels were partially suppressed by the α-adrenergic agents clonidine, methoxamine, methyl norepinephrine and phenylephrine. The lowest effective concentration of each of these agonists required to suppress cyclic AMP levels was 10 nM clonidine; 3 μM methoxamine; 10 μM methyl norepinephrine; 10 μM phenylephrine. Clonidine and methoxamine suppressed cyclic AMP levels by nearly 65% while phenylephrine and methyl norepinephrine caused only a 30% decline. Studies of the relative potencies of α-adrenergic blocking drugs on prevention of the inhibitory effect of clonidine on cyclic AMP levels disclosed that phentolamine and yohimbine were more potent blockers of clonidine action than phenoxybenzamine and prazosin. The rank order of potencies of agonists at causing suppression of cyclic AMP levels and the rank order of potencies of antagonists of clonidine action suggest similarity of the α-adrenergic receptors present on hamster adipocytes, which affect cyclic AMP accumulation to α-2 adrenergic receptors.     

Inhibitory effects of alcohols on thermolysin activity as examined using a fluorescent substrate

Alcohols inhibit the thermolysin-catalyzed hydrolysis of N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH(2) and decrease the NaCl-induced activation of thermolysin in a concentration-dependent manner [K. Inouye et al. (1997) J. Biochem. 122, 358-364]. In this study, the inhibitory effects of alcohols on thermolysin activity were examined in detail using 10 different alcohols and a fluorescent substrate, (7-methoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2). The inhibition by all alcohols examined is completely reversible, and thermolysin activity is recovered by dilution. The inhibitor constants (K(i)) are in the range of 35-430 mM, and the order of the inhibitory effect is 1-pentanol, 1-propanol, 2-butanol, 2-methyl-1-propanol > 1-butanol > 2-propanol > ethanol, tert-amyl alcohol > tert-butyl alcohol > methanol. Linear and secondary alcohols whose mains chains consist of more than 3 carbons inhibit thermolysin effectively. Thermolysin activity is decreased by decreasing the dielectric constant, D, of the reaction medium containing the alcohol, and the decrease depending on the D value was almost the same manner for all alcohols except methanol, tert-butyl alcohol, and tert-amyl alcohol. Alcohols may inhibit thermolysin activity both by binding to the active site, most possibly to the S1' subsite, of thermolysin and by altering the electrostatic and hydrophobic environment around the thermolysin molecule.     

Mechanisms of aliphatic alcohols oxidation by enzymatic systems of the liver]

The main pathways of aliphatic alcohols oxidation in human and mammalian liver, i.e. dehydration of alcohols by cytosolic alcohol dehydrogenases and oxidation in the presence of microsomal enzymatic system, catalase and hydrogen peroxide are described. A special emphasis is laid upon the interaction of alcohols with terminal oxidase of the microsomal hydroxylating system, i.e. cytochrome P-450. The relative role of these three oxidative pathways in alcohol conversions is evaluated.     

Interaction between flavins and alcohols

The effect of alcohols on the spectral properties of riboflavin derivatives in non-polar solvent was studied by various spectroscopic methods in order to support the view point that alcohol may directly interact with the isoalloxazine moiety of FAD and enhance the catalytic activity of D-amino acid oxidase (DAAO). The most likely association complex between alcohol and riboflavin is 1 : 1 stoichiometric complex through the 3-N imino and the 2-C carbonyl groups of the isoalloxazine ring and the hydroxyl group of alcohols. It appears that methanol has a larger association constant than any other alcohols, and the association constant decreases with the increase in carbon number and with the steric requirement of the alkyl group of alcohols.