Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Purification and preliminary characterization.

A quick, reliable, purification procedure was developed for purifying both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from a single batch of Acinetobacter calcoaceticus N.C.I.B. 8250. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernatant, followed by chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CL-6B and Matrex Gel Red A, and finally gel filtration through a Superose 12 fast-protein-liquid-chromatography column. The enzymes co-purified as far as the Blue Sepharose CL-6B step were separated on the Matrex Gel Red A column. The final preparations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II gave single bands on electrophoresis under non-denaturing conditions or on SDS/polyacrylamide-gel electrophoresis. The enzymes are tetramers, as judged by comparison of their subunit (benzyl alcohol dehydrogenase, 39,700; benzaldehyde dehydrogenase II, 55,000) and native (benzyl alcohol dehydrogenase, 155,000; benzaldehyde dehydrogenase II, 222,500) Mr values, estimated by SDS/polyacrylamide-gel electrophoresis and gel filtration respectively. The optimum pH values for the oxidation reactions were 9.2 for benzyl alcohol dehydrogenase and 9.5 for benzaldehyde dehydrogenase II. The pH optimum for the reduction reaction for benzyl alcohol dehydrogenase was 8.9. The equilibrium constant for oxidation of benzyl alcohol to benzaldehyde by benzyl alcohol dehydrogenase was determined to be 3.08 x 10(-11) M; the ready reversibility of the reaction catalysed by benzyl alcohol dehydrogenase necessitated the development of an assay procedure in which hydrazine was used to trap the benzaldehyde formed by the NAD+-dependent oxidation of benzyl alcohol. The oxidation reaction catalysed by benzaldehyde dehydrogenase II was essentially irreversible. The maximum velocities for the oxidation reactions catalysed by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were 231 and 76 mumol/min per mg of protein respectively; the maximum velocity of the reduction reaction of benzyl alcohol dehydrogenase was 366 mumol/min per mg of protein. The pI values were 5.0 for benzyl alcohol dehydrogenase and 4.6 for benzaldehyde dehydrogenase II. Neither enzyme activity was affected when assayed in the presence of a range of salts. Absorption spectra of the two enzymes showed no evidence that they contain any cofactors such as cytochrome, flavin, or pyrroloquinoline quinone. The kinetic coefficients of the purified enzymes with benzyl alcohol, benzaldehyde, NAD+ and NADH are also presented.     

Effect of benzyl alcohol on lipid bilayers. A comparisons of bilayer systems.

The effect of the small anesthetic molecule, benzyl alcohol, on the structure of various bilayer system has been studied by optical, electrical, and x-ray diffraction techniques. We find that the modifications in bilayer thickness caused by benzyl alcohol differ dramatically for planar (or black lipid) bilayers containing solvent, planar bilayers containing little or no solvent, and vesicular bilayers. Benzyl alcohol increases the thickness of planar bilayers containing n-alkane solvents, yet decreases the thickness of "solvent-free" planar bilayers. The effect of benzyl alcohol on vesicular bilayers below the phase transition temperature also depends on whether solvent is present in the bilayers. Without solvent, gel-state bilayers are reduced in thickness by benzyl alcohol, whereas in the presence of solvent, the thickness is unchanged. Above the phase transition temperature, benzyl alcohol has no measurable effect on vesicular bilayer thickness, whether solvent is present or not. These results indicate that different model membrane systems respond quite differently to a particular anesthetic.     

Benzyl alcohol alters the hemoglobin phenotype of mouse erythroleukemia cells

The hemoglobin minor/hemoglobin major ratio expressed in mouse erythroleukemia (MEL) cells grown in vitro varies according to the differentiation inducer utilized. For example, butyrate and hemin induce higher hemoglobin minor/hemoglobin major ratios than do dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA). Benzyl alcohol in non-toxic concentrations was found to markedly reduce the hemoglobin minor/hemoglobin major ratio and to moderately reduce the total hemoglobin induced by DMSO or HMBA in MEL cells, while only slightly decreasing the ratio induced by hemin or butyrate. The addition of dexamethasone (another and more potent inhibitor of the induction of hemoglobin synthesis than benzyl alcohol) to the media during HMBA induction of differentiation increased the hemoglobin minor/hemoglobin major ratio. This is similar to other "inhibitory" treatments (i.e., treatments that result in sub-optimal hemoglobin production) that have been previously reported. Therefore, although benzyl alcohol and dexamethasone both partly inhibit the induction of total hemoglobin production, they have opposite effects on the induced hemoglobin phenotype: benzyl alcohol decreases the hemoglobin minor/hemoglobin major ratio while dexamethasone increases it. The mechanism(s) of the alteration in the hemoglobin phenotype is unknown as is the mechanism of induction by any of the various inducing agents or of the inhibition of induction by any treatment. However, it appears that if the signal for the induction of hemoglobin minor is sufficiently potent (as it is during butyrate or hemin induction), it cannot be overcome by benzyl alcohol at a "non-toxic" concentration.     

Gas chromatographic–mass spectrometric identification and quantitation of benzyl alcohol in serum after derivatization with perfluorooctanoyl chloride: a new derivative

Benzyl alcohol is commonly used as an antibacterial agent in a variety of pharmaceutical formulations. Several fatalities in neonates have been linked to benzyl alcohol poisoning. Most methods for measuring benzyl alcohol concentrations in serum utilize direct extraction followed by high-performance liquid chromatography. We describe here a novel derivatization of benzyl alcohol using perfluorooctanoyl chloride after extraction from human serum for analysis by gas chromatography–mass spectrometry (GC–MS). The derivative was eluted at a significantly higher temperature respective to underivatized molecule and the method was free from interferences from more volatile components in serum and hemolyzed specimens. Another advantage of this derivatization technique is the conversion of low-molecular-mass benzyl alcohol (M_r 108) to a high-molecular-mass derivative (M_r 504). The positive identification of benzyl alcohol can be achieved by observing a distinct molecular ion at m/z 504 as well as the base peak at m/z 91. Quantitation of benzyl alcohol in human serum can easily be achieved by using 3,4-dimethylphenol as an internal standard. The within run and between run precisions (using serum standard of benzyl alcohol: 25 mg/l) were 2.7% (mean=24.1, S.D.=0.66 mg/l, n=8) and 4.2% (mean=24.3, S.D.=1.03 mg/l, n=8), respectively. The assay was linear for the serum benzyl alcohol concentrations of 2 mg/l to 200 mg/l and the detection limit was 0.1 mg/l. We observed no carry-over (memory effect) problem in our assay as when 2 μl ethyl acetate was injected into the GC–MS system after analyzing serum specimens containing 200 mg/l of benzyl alcohol, we observed no peak for either benzyl alcohol or the internal standard in the total ion chromatogram.     

Antagonism of Benzodiazepine Binding in Brain by Antilirium, Benzyl Alcohol, and Physostigmine

Abstract: Antilirium®, which contains eserine (physostigmine) and benzyl alcohol, is effective in reversing diazepam-induced sleep in man and is capable in vitro of inhibiting the binding of labeled benzodiazepine to both rat and human brain homogenates in a dose-dependent manner. We have examined the constituents of Antilirium and report that both benzyl alcohol and eserine inhibit [³HI-diazepam binding to rat brain in a dose-dependent manner. A major portion of the inhibition of binding found with Antilirium is accounted for by benzyl alcohol. Scatchard analysis of inhibition of benzodiazepine binding by benzyl alcohol reveals loss of binding sites and change in equilibrium dissociation constant. Methanol, ethanol, and butanol did not inhibit benzodiazepine binding. The inhibition by benzyl alcohol may be specific since there was no inhibition of labeled ligand binding to the γ-aminobutyric acid, opiate, muscarinic acetylcholine, or β-adrenergic receptors. The other constituent, eserine, is a competitive inhibitor. While eserine is a more potent inhibitor at the benzodiazepine receptor than is benzyl alcohol, it is also much less specific. Eserine inhibited binding of labeled ligand to the γ-aminobutyric acid, opiate, and muscarinic acetylcholine receptors. The inhibition of benzodiazepine binding to brain in vitro by Antilirium and its constituents, eserine and benzyl alcohol, may be the explanation, at least in part, for the reversal by Antilirium of diazepam-induced narcosis in viva, without postulating a cholinergic mechanism for the in vivo effect.     

Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Treatment of human platelets with concentrations of benzyl alcohol up to 50 mM augmented adenylate cyclase activity when it was assayed in the basal state and when stimulated by prostaglandin E1 (PGE1), isoprenaline or NaF. Benzyl alcohol antagonized the stimulatory effect exerted on the catalytic unit of adenylate cyclase by the diterpene forskolin. Benzyl alcohol did not modify the magnitude of the inhibitory response when the catalytic unit of adenylate cyclase was inhibited by using either low concentrations of guanosine 5'-[beta gamma-imido]triphosphate, which acts selectively on the inhibitory guanine nucleotide-regulatory protein Gi, or during alpha 2-adrenoceptor occupancy, by using adrenaline (+ propranolol). Some 34% of the potent inhibitory action of adrenaline on PGE1-stimulated adenylate cyclase was obliterated in a dose-dependent fashion (concn. giving 50% inhibition = 12.5 mM) by benzyl alcohol, with the residual inhibitory action being apparently resistant to the action of benzyl alcohol at concentrations up to 50 mM. Treatment of membranes with benzyl alcohol did not lead to the release of either the alpha-subunit of Gi or G-protein subunits. The alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase was abolished when assays were performed in the presence of Mn2+ rather than Mg2+ and, under such conditions, dose-effect curves for the action of benzyl alcohol on PGE1-stimulated adenylate cyclase activity were similar whether or not adrenaline (+propranolol) was present. We suggest that (i) alpha 2-adrenoceptor- and Gi-mediated inhibition of PGE1-stimulated adenylate cyclase may have two components, one of which is sensitive to inhibition by benzyl alcohol, and (ii) the Gi-mediated inhibition of forskolin-stimulated adenylate cyclase exhibits predominantly the benzyl alcohol-insensitive component.     

The molecular organisation of bimolecular lipid membranes. The effect of benzyl alcohol on the structure

The separate effects of benzyl alcohol on the hydrocarbon and polar-head region capacitances and conductances of phosphatidylcholine bimolecular lipid membranes were obtained from measurements of the very low frequency impedance dispersion. It was found that the conductance of the hydrocarbon region (and, to a lesser extent, the polar-head region) increased progressively with increasing concentrations of benzyl alcohol in the external solution. The polar-head capacitance did not show a systematic dependence on the concentration of benzyl alcohol.At low concentrations of benzyl alcohol (7.5 μM) the capacitance of the hydrocarbon region was not significantly affected by the alcohol. At high concentrations (? 7.5 mM) of benzyl alcohol, however, the capacitance of this region was reduced by ≈25%. This is interpreted in terms of an increase in the thickness of this region caused by a straightening of the otherwise kinked, folded (across neighbouring molecules) and perhaps even partially interdigitated hydrocarbon tails of the phosphatidylcholine molecules. This effect of benzyl alcohol is probably closely related also to the apparent increase in the fluidity of the membrane. The effect of benzyl alcohol on the thickness of the hydrocarbon region of the membrane provides a ready insight into its mode of action as a local anaesthetic.     

In-situ recovery of butanol during fermentation

End product inhibition can be reduced by the in situ removal of inhibitory fermentation products as they form. Extractive fermentation, in which an immiscible organic solvent is added to the fermentor in order to extract inhibitory products, was applied to the acetone-butanol fermentation. Six solvents or solvent mixtures were tested in batch extractive fermentations: kerosene, 30 wt% tetradecanol in kerosene, 50 wt% dodecanol in kerosene, oleyl alcohol, 50 wt% oleyl alcohol in a decane fraction and 50 wt% oleyl alcohol in benzyl benzoate. The best results were obtained with oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate. In normal batch fermentation of Clostridium acetobutylicum, glucose consumption is limited to about 80 kg/m³ due to the accumulation of butanol in the broth. In extractive fermentation using oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate, over 100 kg/m³ of glucose can be fermented. Removal of butanol from the broth as it formed also increased the rate of butanol production. Maximum volumetric butanol productivity was increased by as much as 60% in extractive fermentation compared to batch fermentation. Butanol productivities obtained in extractive fermentation compare favorably with other in situ product removal fermentations.     

Benzyl alcohol inhibits <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate receptor-mediated neurotoxicity and calcium accumulation in cultured rat cortical neurons

The purpose of this study is to examine whether benzyl alcohol affects N-methyl-D-aspartate (NMDA) receptor in cortical cells. Benzyl alcohol (0.5–2 mM) inhibited NMDA-induced cytotoxicity. The protective effect of benzyl alcohol on NMDA-induced toxicity disappeared by washing cells with buffer to remove benzyl alcohol. Benzyl alcohol reduced NMDA receptor-mediated calcium accumulation, indicating that benzyl alcohol inhibits NMDA receptor activity.     

Effects of benzyl alcohol on enzyme activities and D-glucose transport in kidney brush-border membranes

Addition of increasing amounts of benzyl alcohol progressively reduced the steady-state anisotropies of diphenylhexatriene and trimethylammoniumdiphenylhexatriene in brush-border membranes from rat kidney. The decrease in order of membrane lipids, equivalent for 50 mM benzyl alcohol to that produced by a rise in temperature of approx. 6 degrees C, had no effect on the activities of alkaline phosphatase or gamma-glutamyltranspeptidase. On the other hand, benzyl alcohol markedly inhibited the D-glucose uptakes measured in the presence of a 100 mM sodium gradient. For concentrations less than 30 mM, benzyl alcohol reduced the Jmax without significant effects on Km, 22Na+ uptake or the vesicular volume of brush-border preparations. Comparable results were obtained substituting octanol for benzyl alcohol. Our data strongly suggest that, at constant temperature, the D-glucose carrier present in renal brush-border membranes is extremely sensitive to variations in membrane physical state.     

Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two enzymes of the xylene degradative pathway encoded by the plasmid TOL of a Gram-negative bacterium Pseudomonas putida, were purified and characterized. Benzyl alcohol dehydrogenase catalyses the oxidation of benzyl alcohol to benzaldehyde with the concomitant reduction of NAD+; the reaction is reversible. Benzaldehyde dehydrogenase catalyses the oxidation of benzaldehyde to benzoic acid with the concomitant reduction of NAD+; the reaction is irreversible. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase also catalyse the oxidation of many substituted benzyl alcohols and benzaldehydes, respectively, though they were not capable of oxidizing aliphatic alcohols and aldehydes. The apparent Km value of benzyl alcohol dehydrogenase for benzyl alcohol was 220 microM, while that of benzaldehyde dehydrogenase for benzaldehyde was 460 microM. Neither enzyme contained a prosthetic group such as FAD or FMN, and both enzymes were inactivated by SH-blocking agents such as N-ethylmaleimide. Both enzymes were dimers of identical subunits; the monomer of benzyl alcohol dehydrogenase has a mass of 42 kDa whereas that of the monomer of benzaldehyde dehydrogenase was 57 kDa. Both enzymes transfer hydride to the pro-R side of the prochiral C4 of the pyridine ring of NAD+.     

Deuterium isotope effects on toluene metabolism. Product release as a rate-limiting step in cytochrome P-450 catalysis

Liver microsomes from phenobarbital-induced rats oxidize toluene to a mixture of benzyl alcohol plus o-, m- and p-cresol (ca. 69:31). Stepwise deuteration of the methyl group causes stepwise decreases in the yield of benzyl alcohol relative to cresols (ca. 24:76 for toluene-d3). For benzyl alcohol formation from toluene-d3 DV = 1.92 and D(V/K) = 3.53. Surprisingly, however, stepwise deuteration induces stepwise increases in total oxidation, giving rise to an inverse isotope effect overall (DV = 0.67 for toluene-d3). Throughout the series (i.e. d0, d1, d2, d3) the ratios of cresol isomers remain constant. These results are interpreted in terms of product release for benzyl alcohol being slower than release of cresols (or their epoxide precursors), and slow enough to be partially rate-limiting in turnover. Thus metabolic switching to cresol formation causes a net acceleration of turnover.     

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)     

Temperature dependence of benzyl alcohol- and 8-anilinonaphthalene-1-sulfonate-induced aggregation of recombinant human interleukin-1 receptor antagonist

The critical role played by temperature in ligand-induced protein aggregation was investigated. Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and the ligands benzyl alcohol and 8-anilinonaphthalene-1-sulfonate (ANS) were used. We investigated aggregation kinetics and the conformation and cysteine reactivity of rhIL-1ra in buffer alone or in the presence of 0.9% (w/v) benzyl alcohol or 4.2 or 21 mM ANS at 25 and 37 degrees C. In buffer, protein aggregation was not detected at 25 degrees C but occurred at 37 degrees C. At 25 degrees C, neither benzyl alcohol nor 4.2 mM ANS enhanced aggregation. However, at 37 degrees C, both compounds greatly accelerated protein aggregation. With 21 mM ANS, rhIL-1ra aggregation was accelerated at both temperatures, but the effect was more pronounced at 37 degrees C than at 25 degrees C. Increasing the temperature from 25 to 37 degrees C caused a minor perturbation in the tertiary structure of rhIL-1ra in buffer but no detectable alteration in secondary structure. Benzyl alcohol enhanced the tertiary structural perturbation at 37 degrees C, but the secondary structure was not affected by the ligand. The reactivity of buried free cysteines of rhIL-1ra was enhanced by benzyl alcohol at 37 degrees C but not at 25 degrees C, consistent with the structural results. Isothermal titration calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra was hydrophobic and that the degree of hydrophobic interactions increased with temperature. At 25 degrees C, the interaction of ANS with rhIL-1ra was electrostatic, but at 37 degrees C, both electrostatic and hydrophobic interactions were important. Taken together, our results support the conclusion that benzyl alcohol and ANS interact hydrophobically with partially unfolded aggregation-prone protein molecules, resulting in temperature-dependent increases in their levels and acceleration of protein aggregation.     

Benzyl alcohol increases membrane fluidity and modulates cyclic AMP synthesis in intact renal epithelial cells

To evaluate a possible modulation by membrane fluidity of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of the neutral local anesthetic, benzyl alcohol, on membrane fluidity and on basal and stimulated intracellular cAMP content in intact MDCK cells. Benzyl alcohol induced a dose-dependent decrease of lipid order which was measured by steady-state fluorescence anisotropy using trimethylammonium-diphenylhexatriene and propionyl-diphenylhexatriene as fluorescent probes. Benzyl alcohol induced a 2-fold increase in basal cAMP content, likely as a consequence of increased prostaglandin synthesis since this effect was abolished by indomethacin. The effect of benzyl alcohol on stimulated cAMP synthesis depended on the nature of the ligand: 10 mM benzyl alcohol increased significantly the stimulatory effect of prostaglandin E2, glucagon and forskolin but not of vasopressin. At higher concentrations (40 mM), benzyl alcohol did not affect significantly the glucagon-stimulated cAMP content, while it inhibited significantly the prostaglandin E2-, forskolin- and vasopressin-stimulated cAMP synthesis. The 40 mM benzyl alcohol-induced inhibition was reversed by 1 mM Mn2+, which is known to block the inhibitory GTP-binding protein Ni. These results suggest that: (i) the various components of the adenylate cyclase-cAMP system and their coupling are affected differently by changes in membrane fluidity, which might reflect differences in their lipid environment, (ii) changes in membrane fluidity can modulate responses of renal tubular cells to hormones, and thus tubular functions.     

The nature of the changes in liver mitochondrial function induced by glucagon treatment of rats. The effects of intramitochondrial volume,aging and benzyl alcohol

(1) The effects of changes in the intramitochondrial volume, benzyl alcohol treatment and calcium-induced mitochondrial aging on the behaviour of liver mitochondria from control and glucagon-treated rats are reported. (2) The stimulatory effects of glucagon on mitochondrial respiration, pyruvate metabolism and citrulline synthesis could be mimicked by hypo-osmotic treatment of control mitochondria and reversed by calcium-induced aging of mitochondria or by treatment with 20 mM benzyl alcohol. Hypo-osmotic treatment increased the matrix volume whilst aging but not benzyl alcohol decreased this parameter. (3) Liver mitochondria from glucagon and adrenaline-treated rats were shown to be less susceptible to damage by exposure to calcium than control mitochondria and frequently showed slightly (15%) elevated intramitochondrial volumes. (4) Aging, benzyl alcohol and hypo-osmotic media increased the susceptibility of mitochondria to damage caused by exposure to calcium. (5) Glucagon-treated mitochondria were less leaky to adenine nucleotides than control mitochondria. (6) These results suggest that glucagon may exert its action on a wide variety of mitochondrial parameters through a change in the disposition of the inner mitochondrial membrane, possibly by stabilisation against endogenous phospholipase A₂ activity. This effect may be mimicked by an increase in the matrix volume or reversed by calcium-dependent mitochondrial aging.     

The effect of benzyl alcohol and cholesterol on the acyl chain order and alkane solubility of bimolecular phosphatidylcholine membranes

An investigation was made of the effects of cholesterol and benzyl alcohol on the partitioning of n-alkanes between lipid bilayer membranes and bulk lipid/alkane solutions (in the torus). Bilayers were formed from solutions containing alkanes of different chain lengths, together with phosphatidylcholine and cholesterol in varying proportions. The partitioning of the alkanes was determined from measurements of the very low frequency (1 Hz) capacitance of the membranes. Perturbation of the internal membrane structure by the inclusion of cholesterol and benzyl alcohol produced very significant changes in the n-alkane partition coefficient, cholesterol causing a decrease and benzyl alcohol an increase in the alkane partitioning into the bilayer. A correlation exists between the effects of these compounds on the alkane partitioning and their effect on the segmental chain order of the acyl chains in the bilayer and this correlation is consistent with a statistical-mechanical model of the lipid/alkane bilayers in the liquid crystalline state. The perturbation by cholesterol and benzyl alcohol of the internal structure of membranes bears on the conflicting reports of the effects of these substances on artificial lipid bilayers and could also be relevant to their known physiological effects.     

Purification and Properties of Benzyl Alcohol Oxidase from Botrytis cinerea

A benzyl alcohol oxidase (BAO) was purified to homogeneity from Botrytis cinerea. The enzyme was found to have a molecular mass of 214 kD with a trimeric structure, and optimal pH and temperature of 5.0 and 30°C, respectively. The enzyme activity was not sensitive to metal ions or to metal ion chelators, while thiol blocking reagents strongly inhibited BAO activity. Sulfur dioxide irreversibly inhibited the enzyme activity and the inhibitory effect of ethanol was weak and reversible. Benzyl alcohol was the most effective alcohol substrate for BAO. Para or meta monosubstituted benzyl alcohol with methyl or methoxy groups were good substrates. BAO also oxidized cinnamyl alcohol, furfuryl alcohol, and some terpenic alcohols· with an alkenyl group near the reactive carbinol. Secondary alcohol, methanol and phenol were not substrates. Product inhibition studies suggested that benzaldehyde and benzyl alcohol were bound at different places to the active site. O₂ was the only electron acceptor identified and Botrytis cinerea benzyl alcohol oxidase was classified .as EC 1.1.3.7 according to stoichiometrical studies. We discuss the metabolic role of BAO in the Botrytis cinerea-grape host-parasite relationship.     

Acid-base catalysis in the yeast alcohol dehydrogenase reaction.

The effect of pH on steady state kinetic parameters for the yeast alcohol dehydrogenase-catalyzed reduction of aldehydes and oxidation of alcohols has been studied. The oxidation of p-CH3 benzyl alcohol-1,1-h2 and -1,1-d2 by NAD+ was found to be characterized by large deuterium isotope effects (kH/kD = 4.1 plus or minus 0.1) between pH 7.5 and 9.5, indicating a rate-limiting hydride trahsfer step in this pH range; a plot of kCAT versus pH could be fit to a theoretical titration curve, pK = 8.25, where kCAT increases with increasing pH. The Michaelis constnat for p-CH3 benzyl alcohol was independent of pH. The reduction of p-CH3 benzaldehyde by NADH and reduced nicotinamide adenine dinucleotide with deuterium in the 4-A position (NADD) cound not be studied below pH 8.5 due to substrate inhibition; however, between pH 8.5 and 9.5, kCAT was found to decrease with increasing pH and to be characterized by significant isotope effects (kH/kD = 3.3 plus or minus 0.3). In the case of acetaldehyde reduction by NADH and NADD, isotope effects were found to be small and exxentially invariant (kH/kD = 2.O plus or minus 0.4) between pH 7.2 and 9.5, suggesting a partially rate-limiting hydride transger step for this substrate; a plot of kCAT/K'b (where K'b is the Michaelis constant for acetaldehyde) versus pH could be fit to a titration curve, pK = 8.25. The titration curve for acetaldehyde reduction has the same pK but is opposite in direction to that observed for p-CH3 benzyl alcohol oxidation. The data presented in this paper indicate a dependence on different enzyme forms for aldehyde reduction and alcohol oxidation and are consistent with a single active site side chain, pK = 8.25, which functions in acid-base catalysis of the hydride transfer step.