Physical and reactive extraction equilibria of penicillin G in a hydrogen-bond acceptor solvent system

Physical and reactive extraction equilibria of penicillin G were investigated experimentally and theoretically in the existence of n-butyl acetate as a hydrogen-bond acceptor solvent. Physical extraction equilibrium experiments were carried out varying the pH of aqueous phase and overall penicillin concentration. We compared the experimental data with the calculated results from four physical extraction equilibrium models suggested here and obtained the most reasonable model. Also, penicillin G was reactively extracted using Amberlite LA-2 in n-butyl acetate. The experimental variables were pH of the aqueous phase, overall amine concentration, and overall penicillin concentration. A combined equilibrium model including our physical extraction equilibrium expression and the reactive extraction equilibrium expression suggested by Reschke and Schügerl was used so as to analyze the current reactive extraction equilibrium system. The calculated results from the reactive extraction equilibrium model were in good agreement with the experimental data.     

红树植物木榄有机溶剂提取物对球形棕囊藻的抑制效应

基于化感作用原理,利用有机溶剂对红树植物木榄(Bruguiera gymnorrhiza)叶片中的活性物质进行连续提取分离,分别得到正己烷相、乙酸乙酯相、正丁醇相和水相提取物。利用这些提取物进行抑藻实验,通过测定球形棕囊藻(Phaeocystis globosa)的细胞密度发现:四种组分提取物对球形棕囊藻均具有显著的化感抑制作用;正己烷相和乙酸乙酯相提取物对球形棕囊藻的抑制效果明显优于正丁醇相和水相提取物的抑制效果。正己烷相提取物和乙酸乙酯相提取物对球形棕囊藻的48hEC₅₀分别为14.90mg/L和12.18mg/L。研究表明,藻细胞初始接种密度影响提取物的抑藻效应,低接种密度时抑制率极高,而随着接种密度的升高抑制率下降;接种密度极高时,提取物不但不会抑制甚至还会促进藻细胞的生长。     

Behavioral determinations of thresholds for n-butyl acetate and nbutyl alcohol in the tiger salamander (Ambystoma tigrinum)

Two groups of tiger salamanders (Ambystoma tigrinum) were conditionedto respond to odorant-air mixtures of n-butyl acetate (8.9 x10^–5M) or n-butyl alcohol (6.7 x l0^–5M). They werethen given tests with various concentrations of the trainingodorants presented using a temporal forced-choice method ofascending limits. Results showed that reliable responses toodorant-air presentations were obtained with concentrationsof n-butyl acetate above 2.4 x l0^–7M and with concentrationsof n-butyl alcohol above 8.5 x 10^–8M. These results arein substantial agreement with previous dectrophysiological findings.     

Presence and Measurement of Imidazoleacetic Acid, a γ-Aminobutyric Acid Agonist, in Rat Brain and Human Cerebrospinal Fluid

Imidazoleacetic acid (IAA) was unequivocally demonstrated in rat brain, human CSF, and human plasma by a gas chromatographic-mass spectrometric method that can reliably quantify as little as 8 pmol, i.e., 1 ng. Owing to tautomerism of the imidazole ring, IAA and [15N, 15N]IAA, the internal standard, each formed two chromatographically distinct isomers after derivatization of the ring nitrogens with either ethyl chloroformate or methyl chloroformate. The isomers of n-butyl(N-ethoxycarbonyl)imidazole acetate and n-butyl(N-methoxycarbonyl)imidazole acetate were identified by analysis with methane chemical ionization and electron impact ionization of molecular and fragment ions. The levels (mean +/- SEM) of free IAA were 140 +/- 14 pmol/g and 2.7 +/- 0.2 pmol/ml in brains of untreated rats and human lumbar CSF, respectively. Mean levels of IAA in brains of anesthetized rats, perfused free of blood, did not differ significantly from mean levels of anesthetized, nonperfused controls or from untreated rats. The source or sources of IAA in brain and CSF are unknown. Because IAA is a potent agonist at gamma-aminobutyrate receptors, it merits examination as a regulator in brain.     

Improved methodology for intracellular enzyme reaction and inhibition kinetics by flow cytometry

Flow cytoenzymology is the determination of enzyme activities or concentrations in single intact cells. Using the flow cytometer built and designed in our laboratory and recent modifications to hardware and software, we have developed an improved dynamic flow cytoenzymological procedure for the assay of cellular enzyme kinetics. The reaction mixture is sampled continuously, and the computer clock incorporates time as a parameter for kinetic determinations. Conditions for cellular esterase analysis were optimized and the rates of hydrolysis of two fluorogenic substrates, fluorescein diacetate (FDA) and 4-methylumbelliferone acetate (MUA), by esterases in EMT6 mouse mammary tumor cells were studied. Reaction kinetics were characterized, and Km values of 19 and 72 microM were obtained for the hydrolysis of FDA and MUA respectively. The kinetics of the cellular efflux of fluorescein were investigated, and a half-life of 7.5 min obtained. Enzyme inhibition kinetics were investigated using the competitive substrates p-nitrophenyl acetate and phenyl acetate, and the carbamoylating agents physostigmine and n-butyl isocyanate. The latter was particularly potent with an I50 of 4.8 X 10(-5) M for FDA hydrolysis compared with 6.5 X 10(-3) M for physostigmine. The I50 of 8.8 X 10(-5) M for n-butyl isocyanate inhibition of MUA hydrolysis was similar to that obtained with FDA as substrate. By monitoring FDA and MUA reactions separately and simultaneously, we showed them to act as competitive substrates. A comparison of flow cytoenzymological and conventional spectrofluorimetric analysis was also made, and differences identified in some cases.     

Production of volatile organic sulfur compounds (VOSCs) by basidiomycetous yeasts

Thirty-seven basidiomycetous yeasts belonging to 30 species of seven genera were grown on media containing l-cysteine or l-methionine as sole nitrogen sources with the objective of evaluating volatile organic sulfur compound (VOSC) production. The headspace of yeast cultures was analyzed by the solid-phase microextraction (SPME) sampling method, and volatile compounds were quantified and identified by GC-MS techniques. Ten strains assimilating L-methionine produced the following VOSCs: 3-(methylthio)-1-propanol, methanethiol, S-methyl thioacetate, dimethyl disulfide, dimethyl trisulfide, allyl methyl sulphide and 4,5-dihydro-3(2H)-thiophenone. Production was <1 mgl(-1) except for 3-(methylthio)-1-propanol of which between 40 and 400 mgl(-1) was synthesized. Higher alcohols (isobutyl alcohol, isoamyl alcohol and active amyl alcohol) and esters (ethyl acetate, ethyl propionate, n-propyl acetate, isobutyl acetate, n-propyl propionate, n-butyl acetate, isoamyl acetate, amyl acetate, isoamyl propionate, amyl propionate and 2-phenylmethyl acetate) were also sporadically produced. This is the first report of VOSCs production by basidiomycetous yeasts. Consequently, basidiomycetous yeasts may be considered an interesting new group of microbial VOSCs producers for the flavor industry.     

基于HPLC-DAD技术对大鼠灌胃八珍汤后尿液样品处理方法的研究

对大鼠口服八珍汤后的尿液样品预处理方法进行多样性考察,以减少尿液样品中内源性杂质干扰,富集来源于八珍汤的化学成分,为八珍汤体内代谢产物的多样性分析奠定基础.分别采用水饱和的正丁醇、乙酸乙酯、SPE小柱固相萃取法对空白尿液和含药尿液进行处理,然后使用高效液相对各样品进行成分分析,通过对比经过各方法优化的尿液色谱图,确定含八珍汤的尿液样品的最佳处理方法,并据此进一步考察确定尿液的最佳收集时间段.通过分析各液相色谱图发现,在大鼠口服八珍汤后4～8h时间段内,采用SPE小柱固相萃取法纯化的尿液样品出峰效果最好,数目最多.从而确定了含八珍汤的大鼠尿液的最优纯化方法为SPE小柱固相萃取法,最佳收集时间段为4～8h.     

Modulation of FAD-dependent monooxygenase activity from aromatic compounds-degrading Stenotrophomonas maltophilia strain KB2

The purpose of this study was purification and characterization of phenol monooxygenase from Stenotrophomonas maltophilia strain KB2, enzyme that catabolises phenol and its derivatives through the initial hydroxylation to catechols. The enzyme requires NADH and FAD as a cofactors for activity, catalyses hydroxylation of a wide range of monocyclic phenols, aromatic acids and dihydroxylated derivatives of benzene except for catechol. High activity of this monooxygenase was observed in cell extract of strain KB2 grown on phenol, 2-methylphenol, 3-metylphenol or 4-methylphenol. Ionic surfactants as well as cytochrome P450 inhibitors or 1,4-dioxane, acetone and n-butyl acetate inhibited the enzyme activity, while non-ionic surfactants, chloroethane, ethylbenzene, ethyl acetate, cyclohexane, and benzene enhanced it. These results indicate that the phenol monooxygenase from Stenotrophomonas maltophilia strain KB2 holds great potential for bioremediation.     

A chemical-modification approach to the olfactory code. Studies with a thiol-specific reagent

The effects of thiol-specific reagents on the amplitude of the electro-olfactogram (E.O.G.) responses elicited from frog olfactory mucosa by pulses of odorant vapours was studied. The impermeant thiol-specific reagent mersalyl [(3-{[2-(carboxymethoxy)-benzoyl]amino}-2-methoxypropyl)hydroxymercury monosodium salt] brings about a rapid decrease in the E.O.G. signal obtained with the odorant pentyl acetate. The extent of the decrease is proportional to the concentration of the mersalyl applied and the effect of the reagent is partially but incompletely reversed by treatment of the labelled mucosa with dithiothreitol. The sites labelled by mersalyl can be protected by pretreating the mucosa with a dilute solution of the odorant pentyl acetate and leaving the solution in contact with the tissue after the addition of mersalyl. When the protecting odorant is washed out of the tissue, the original E.O.G. amplitude is regained. Pentyl acetate applied to the mucosa protected the E.O.G. response to vapour pulses of the following odorants from the effects of mersalyl: n-butyric acid, n-butyl acetate, phenylacetaldehyde and cineole (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane). The pentyl acetate applied to the mucosa failed to protect the E.O.G. response to vapour pulses of the following odorants from the effects of mersalyl: butan-1-ol, benzyl acetate, nitrobenzene, beta-ionone and linalyl acetate. The significance of the differential protection effects for the odour-quality-coding mechanism in the olfactory primary neurons is discussed. It is suggested that the olfactory code at this level of the olfactory system may be elucidated by chemical-modification methods.     

Biodegradation of volatile organic compounds by five fungal species

Five fungal species, Cladosporium resinae (ATCC 34066), Cladosporium sphaerospermum (ATCC 200384), Exophiala lecanii-corni (CBS 102400), Mucor rouxii (ATCC 44260), and Phanerochaete chrysosporium (ATCC 24725), were tested for their ability to degrade nine compounds commonly found in industrial off-gas emissions. Fungal cultures inoculated on ceramic support media were provided with volatile organic compounds (VOCs) via the vapor phase as their sole carbon and energy sources. Compounds tested included aromatic hydrocarbons (benzene, ethylbenzene, toluene, and styrene), ketones (methyl ethyl ketone, methyl isobutyl ketone, and methyl propyl ketone), and organic acids ( n-butyl acetate, ethyl 3-ethoxypropionate). Experiments were conducted using three pH values ranging from 3.5 to 6.5. Fungal ability to degrade each VOC was determined by observing the presence or absence of visible growth on the ceramic support medium during a 30-day test period. Results indicate that E. lecanii-corni and C. sphaerospermum can readily utilize each of the nine VOCs as a sole carbon and energy source. P. chrysosporium was able to degrade all VOCs tested except for styrene under the conditions imposed. C. resinae was able to degrade both organic acids, all of the ketones, and some of the aromatic compounds (ethylbenzene and toluene); however, it was not able to grow utilizing benzene or styrene under the conditions tested. With the VOCs tested, M. rouxiiproduced visible growth only when supplied with n-butyl acetate or ethyl 3-ethoxypropionate. Maximum growth for most fungi was observed at a pH of approximately 5.0. The experimental protocol utilized in these studies is a useful tool for assessing the ability of different fungal species to degrade gas-phase VOCs under conditions expected in a biofilter application.     

General occurrence of binding to acetylcholinesterase-substrate complex in noncompetitive inhibition and in inhibition by substrate

To assess the relative importance of binding to enzyme-substrate complex (E.S) and to acetylenzyme (EA), noncompetitive inhibition has been studied in hydrolysis by acetylcholinesterase (AcChE) of cationic and uncharged substrates - acetylcholine (AcCh), 3,3-dimethylbutyl acetate, n-butyl acetate, 2-(methylammonio)ethyl acetate, 2- (N,N-diethyl-N-n-butylammonio)ethyl acetate (DEBAAc) and 2-(methylsulfonyl)ethyl acetate. For the N-trimethyl quaternary ions related to AcCh, tetramethylammonium ion, choline and choline ethyl ether, noncompetitive inhibition (Ki(nonc) is more favorable with the slower substrates than with AcCh, i.e., when E.S greater than EA, and is attributed to formation of enzyme-substrate-inhibitor complexes, E.S.I'. Noncompetitive inhibition by tetraethyl-, tert-butyl- and isopropylammonium ions, and acetamidocholine and its lower dimethyl analogue, is also attributed to E.S.I' complexes. Peripheral binding of these inhibitors decreases acylation more than deacylation. Some tertiary dimethylamonio ions have more favorable Ki(nonc) values with AcCh, decreasing deacylation more than acylation. The substrate DEBAAc is a more effective noncompetitive than competitive inhibitor in hydrolysis of AcCh, indicating that it binds more strongly in a peripheral site than in the active site of the free enzyme. In its hydrolysis by AcChE, it acts as its own noncompetitive inhibitor, by this non-productive binding. Formation of E.S.I' complexes is a general characteristic of hydrolysis by AcChE and decrease in rates at high concentrations of AcCh and related substrates is attributed to peripheral regulatory site binding, formation of E.S.S' complexes, rather than to binding to the acetylenzyme.     

鹅膏菌抑真菌活性及其活性物质的提取工艺优化(英文)

通过鹅膏菌抑菌活性物质对杨树烂皮病病原菌金黄壳囊孢菌的菌丝生长及其孢子抑制效果的研究,分析鹅膏菌的抗真菌活性。将培养15 d的鹅膏菌液体发酵物分成培养液和菌丝体两部分,然后分别将培养液、菌丝体提取物和发酵液本身对金黄壳囊孢菌菌丝生长和孢子萌发抑制效果进行检测。结果表明:培养液对金黄壳囊孢菌菌丝生长的抑制率最高,为68.90%;三者对孢子萌发都有明显的抑制效果,即在处理后10 d孢子都没有萌发。菌丝体在超声波、加热和浸泡的条件下,利用乙醇,丙酮、乙酸乙酯和正丁醇进行提取,其提取物用于病原菌的抑制效果检测,结果表明:用乙醇和丙酮提取时,超声波、浸泡及加热处理,对金黄壳囊孢菌的抑制效果都与对照有显著差异(a=0.01),而正丁醇的提取物的抑制效果较差,与对照没有显著差异。通过不同提取物对金黄壳囊孢菌菌丝生长和孢子萌发的抑制效果进行分析,并对抑菌活性物质提取工艺进行筛选,得到抑菌活性物质提取的最佳方法为摇床培养,乙醇是最适提取溶剂,处理方式(超声波、浸泡或者加热)相互间差异不显著。     

Kinetic characteristics of n-butyl alcohol and iso-butyl alcohol in a composite bead air biofilter

Kinetic characteristics of n-butyl alcohol and iso-butyl alcohol in a composite bead biofilter were investigated. The microbial growth rate of n-butyl alcohol was greater than that of iso-butyl alcohol in the average inlet concentration range of 50-300 ppm. The microbial growth rate was inhibited at higher inlet concentration, and the inhibitive effect in the concentration range of 50-150 ppm was more pronounced than that in the concentration range of 150-300 ppm. The degree of inhibitive effect for n-butyl alcohol was more sensitive than that for iso-butyl alcohol in the concentration range of 50-150 ppm. The zero-order kinetic with the diffusion rate limitation could be regarded as the most adequate biochemical reaction model. The biodegradation rate of n-butyl alcohol was greater than that of iso-butyl alcohol in the average inlet concentration range of 50-300 ppm. The biochemical reaction rate was also inhibited at higher inlet concentration, and the inhibitive effect for iso-butyl alcohol was more pronounced than that for n-butyl alcohol. The factor of the chemical structure of compound was more predominant in the microbial growth and biochemical reaction processes. The maximum elimination capacity of n-butyl alcohol and iso-butyl alcohol were 55.7 and 34.8 g C h(-1)m(-3) bed volume, respectively. The compound with no side group in the main chain would be easier biodegraded by the microbial.     

Synthesis of (R)-β-nitro alcohols catalyzed by R-selective hydroxynitrile lyase from Arabidopsis thaliana in the aqueous-organic biphasic system

Both enantiomers of β-nitro alcohols are versatile chiral building blocks. However, their synthesis using enzymes as catalysts has received little attention, with the exception of (S)-β-nitro alcohols produced in a reaction catalyzed by an S-selective hydroxynitrile lyase (HNL) from Hevea brasiliensis (HbHNL). An R-selective HNL containing an α/β-hydrolase fold from the noncyanogenic plant Arabidopsis thaliana (AtHNL) accepts nitromethane (MeNO?) as a donor in a reaction with aromatic aldehydes to yield (R)-β-nitro alcohols (Henry reaction; nitro aldol reaction). This reaction proceeded in an aqueous-organic biphasic system. The organic solvent giving the highest enantioselectivity was n-butyl acetate (AcOBu) with an optimum aqueous phase content of 50% (v/v). This is the first example of the R-HNL-catalyzed synthesis of (R)-β-nitro alcohols.     

Kinetic resolution of ligand binding to the alpha and beta chains within human hemoglobin.

Nitric oxide has been used as a chain-specific, spin label of unliganded heme groups present in kinetic mixtures of human hemoglobin and n-butyl isocyanide. In these experiments, deoxyhemoglobin was reacted with n-butyl isocyanide for a controlled time and then mixed rapidly with a high concentration of nitric oxide to fill residual, unoccupied heme sites. The final mixture was frozen immediately after formation to prevent any displacement of bound isonitrile. The EPR spectrum of the frozen sample was resolved into alpha and beta nitric oxide components; these reflect the relative proportions of alpha- and beta-heme sites which were unoccupied by n-butyl isocyanide. Individual time courses for the alpha and beta subunits were obtained by varying the time between the formation of the isonitrile/hemoglobin mixture and its reaction with nitric oxide. At pH 7.0 only the beta chain time course exhibits an initial rapid phase; the alpha chain time course is monophasic, exhibiting almost, exponential behavior. This result shows unequivocally that the beta-hemes within deoxyhemoglobin react much more rapidly with n-butyl isocyanide than the alpha hemes.     

Biodegradation kinetic behaviors of n-butyl alcohol and sec-butyl alcohol in a composite bead biofilter

Biodegradation kinetic behaviors of n-butyl alcohol and sec-butyl alcohol in a composite bead biofilter were investigated. The microbial growth rate of n-butyl alcohol was greater than that of sec-butyl alcohol in the inlet concentration range of 50–300 ppm. The microbial growth rate was inhibited at higher inlet concentration, and the inhibitive effect in the concentration range of 50–150 ppm was more pronounced than that in the concentration range of 150–300 ppm. The degree of inhibitive effect for n-butyl alcohol was more sensitive than that for sec-butyl alcohol in the concentration range of 50–150 ppm. The zero-order kinetic with the diffusion rate limitation could be regarded as the most adequate biochemical reaction model. For the biochemical reaction process, the biochemical reaction rate coefficient of n-butyl alcohol was greater than that of sec-butyl alcohol in the inlet concentration range of 50–300 ppm. The biochemical reaction rate coefficient was decreased with increasing inlet concentration. The inhibitive effect for sec-butyl alcohol was more pronounced than that for n-butyl alcohol. The factor of the chemical structure of compound was more predominant in the microbial growth and biochemical reaction processes. The maximum elimination capacity of n-butyl alcohol and sec-butyl alcohol were 55.7 and 20.9 g C h^?1 m^?3 bed volume, respectively. The primary alcohol was easily biodegraded by the microbial.     

Lipase-catalyzed transesterification in several reaction systems: An application of room temperature lonic liquids for bi-phasic production ofn-butyl acetate

Organic solvents are widely used in biotransformation systems. There are many efforts to reduce the consumption of organic solvents because of their toxicity to the environment and human health. In recent years, several groups have started to explore novel organic solvents called room temperature ionic liquids in order to substitute conventional organic solvents. In this work, lipase-catalyzed transesterification in several uni-and bi-phasic systems was studied. Two representative hydrophobic ionic liquids based on 1-butyl-3-methylimidazolum coupled with hexafluorophosphate ([BMIM][PF₆]) and bis[(trifluoromethylsulfonyl) imide] ([BMIM] [Tf₂N]) were employed as reaction media for the transesterification ofn-butanol. The commercial lipase, Novozym 435, was used for the transesterification reaction with vinyl acetate as an acyl donor, The conversion yield was increased around 10% in a water/[BMIM][Tf₂N], bi-phasic system compared with that in a water/hexane system. A higher distribution of substrates into the water phase is believed to enhance the conversion yield in a water/[BMIM][Tf₂N] system. Partion coefficients of the substrates in the water/[BMIM][Tf₂N] bi-phasic system were higher than three times that found in the water/hexane system, while n-butyl acetate showed a similar distribution in both systems. Thus, RTILs appear to be a promising substitute of organic solvents in some biotransformation systems.     

Assessment of hepatoprotective potential of Radix Fici Hirtae on alcohol-induced liver injury in Kunming mice

ObjectiveThe objective of the present study was to investigate the hepatoprotective role of Radix Fici Hirtae on acute alcohol-induced liver injury in mice.MethodsThe component of Radix Fici Hirtae was extracted using petroleum ether, chloroform, ethyl acetate and n-butanol and divided into three dose groups of high, medium and low according to the clinical man's normal dose of the 50 g crude drug/d (0.83 g/kg body weight). Saline in concentration of 10 mg/mL, 5 mg/mL and 2.5 mg/mL and a dose of mouse lavage (0.2 mL/10 g mouse body weight) were added to the solution. Histopathlogical analysis of liver was performed. Finally, liver protection was validated by examining the effect of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AKP), and lactate dehydrogenase (LDH) on the hepatic function of mice in alcohol-induced liver injury model.ResultsExcept for group with saturated n-butyl alcohol, for the rest of the groups, pathological changes of hepatic lipid and inflammatory cells infiltration were alleviated and liver sinus was normal. As compared to model group, the concentrations of AST, ALT, AKP and LDH in chloroform groups and ethyl acetate groups were significantly decreased.ConclusionsExtracts of Radix Fici Hirtae are effective for the prevention of alcohol-induced hepatic damage in mice. The results revealed that extracts of Radix Fici Hirtae could be used as hepatoprotective agent.     

苦瓜叶提取物对亚洲玉米螟的生物活性及对斜纹夜蛾卵巢细胞的毒力

【目的】为研究苦瓜(Momordica charantia L.)叶正丁醇萃取物对亚洲玉米螟Ostrinina furnacalis(Güenée)生长发育及生殖力的影响,并探讨其作用机理。【方法】采用饲料混药法测定了苦瓜叶正丁醇萃取物对亚洲玉米螟幼虫存活率、发育历期和雌成虫产卵量的影响。【结果】用0.025%、0.1%和0.2%浓度的苦瓜叶正丁醇萃取物混合饲料饲喂亚洲玉米螟2龄幼虫3 d后,幼虫存活率明显降低,校正死亡率分别为27.43%、39.23%和54.93%,且发育历期明显延长,雌成虫产卵量与对照相比分别下降了12.63%、22.74%和37.27%。进一步的研究结果表明,苦瓜叶正丁醇萃取物对斜纹夜蛾卵巢细胞(SL)的形态、增殖和生长密度有明显的抑制作用。处理后的SL细胞皱缩变圆,细胞核偏移甚至消失,胞质外泄。处理24、48和72 h后,对SL细胞增殖的IC50值分别为180.96、158.54和122.87μg/mL;处理6~72 h后,SL细胞相对生长密度随药剂浓度和处理时间的增加而明显下降;用125μg/mL浓度处理SL细胞2、3、4和5 d后,SL细胞对葡萄糖吸收的抑制率分别为7.66%、13.97%、19.45%和27.30%。【结论】苦瓜叶正丁醇萃取物对SL细胞表现出的较强毒杀作用可能是其导致亚洲玉米螟生殖能力降低的主要作用机制之一。     

The dual effects of alcohols on the kinetic properties of guinea pig liver cytosolic beta-glucosidase

This report demonstrates the effect of primary alcohols on the kinetic properties of guinea pig liver cytosolic beta-glucosidase. Lineweaver-Burk analyses of the kinetic data revealed a biphasic response; at low concentrations the alcohols increased the Vmax 5--7-fold while at higher concentrations they caused a purely competitive type of inhibition. For example, with n-butyl alcohol, increasing the alcohol's concentration in the assay medium from 0 to 0.14 M (0-1% (v/v)) resulted in a progressive increase in Vmax to a value 7-fold above the basal level without affecting the Km. However, between 0.14 and 0.54 M (1 and 4% (v/v)) n-butyl alcohol, the Km for 4-methylumbelliferyl-beta-D-glucopyranoside increased significantly from 0.14 to 0.93 mM. In contrast to n-butyl alcohol or isobutyl alcohol, which are potent activators, structurally related compounds like sec-butyl alcohol, tert-butyl alcohol, butylurea, and butanesulfonic acid did not stimulate the activity of the cytosolic beta-glucosidase. In the concentration range where activation was observed, conventional secondary replots of 1/delta slope versus 1/[alcohol] yielded perfect straight lines, demonstrating that binding of a single molecule of alcohol to the beta-glucosidase was responsible for the initial phase of activation. Furthermore, the glycohydrolase displayed a propensity to bind the longer chain alcohols, as reflected by the KA (binding constant) values of 555, 146, 34.1, and 7.47 mM for ethanol, n-propyl alcohol, n-butyl alcohol, 1-pentanol, respectively. This phenomenon of nonessential activation by alcohols has led us to speculate on the presence of a physiologic activator for the beta-glucosidase in mammalian tissues which contain this enzyme.