Regional differences in carboxylesterase activity between human subcutaneous and omental adipose tissue

Human adipose tissue was shown to contain carboxylesterase activity when measured by methylbutyrate as substrate. The enzyme has the same characteristics as carboxylesterase purified from rat epididymal adipose tissue. Like lipoprotein lipase, carboxylesterase activity was higher in large than in small fat cells. Both cell size and carboxylesterase activity were greater in human subcutaneous than in omental adipose tissue. However, the linear regression lines between the enzyme activity and cell volume in the two tissues were almost superimposable, suggesting that cell size is a determinant of enzyme activity. Although the physiological significance of adipose tissue carboxylesterase must await further clarification, it is possible that the enzyme is related to the hydrolysis of long-chain monoacylglycerols.     

The relationship between the carboxylesterase and monoacylglycerol lipase activities of chicken liver microsomes

The carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) and monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) activities, measured against ethyl butyrate and emulsified monooleoylglycerol respectively, were determined for chicken liver microsomes and highly purified chicken liver carboxylesterase. The activity ratio (ethyl butyrate activity/monooleoylglycerol activity) was approx. 5 for microsomes and approx. 400 for carboxylesterase. Homogenization of microsomes in 0.1 M Tris-HCl buffer (pH 7.92) released all of the ethyl butyrate activity and about half of the monooleoylglycerol activity into a soluble form. Both activities eluted from a Sephadex G-200 column with the same elution volume as that of pure carboxylesterase. This fraction (fraction B) had an activity ratio of approx. 15, an average pI of 5.01 (cf. 4.75 for carboxylesterase), and ran on polyacrylamide gel electrophoresis at pH 8.6 as a number of closely spaced esterase bands with mobilities considerably less than those of the esterase bands present in the carboxylesterase. Fraction B activities against both substrates were completely inhibited by diethyl p-nitrophenyl phosphate and completely precipitated by antibody to carboxylesterase. The remaining half of the monoacylglycerol lipase activity of microsomes was solubilized by treatment with 1.5% (w/v) Triton X-100. This solubilized monoacylglycerol lipase was completely inhibited by diethyl p-nitrophenyl phosphate, showing it to be a serine-dependent enzyme like the carboxylesterases. However, it had no detectable activity against ethyl butyrate, indicating that it is not closely related to the carboxylesterases.     

Human liver carboxylesterase. Properties and comparison with human serum carboxylesterase

Carboxylesterase was obtained from human liver in an electrophoretically homogeneous form. The monomeric molecular weight of the enzyme was 60,000 and the enzyme associated to form trimers. Purified human liver carboxylesterase was compared with human serum carboxylesterase, purified earlier. Serum carboxylesterase hydrolyzed a typical cholinesterase substrate and aryl acylamide, whereas liver carboxylesterase did not hydrolyze these compounds. Both carboxylesterases catalyzed the hydrolysis of short-chain triacylglycerols, such as tributyrin, and medium-chain monoacylglycerols, such as monocaprin, but not the hydrolysis of long-chain triacylglycerols. Serum carboxylesterase activity was inhibited by p-trimethylammoniumanilinium dichloride and neostigmine, whereas liver carboxylesterase activity was not affected by these compounds. Liver and serum carboxylesterase activities were both strongly inhibited by phenylmethylsulfonyl fluoride.     

杀虫剂不同施用方式对烟蚜抗药性发展和羧酸酯酶活力的影响

室内模拟田间杀虫剂施药方式,研究3种杀虫剂连用及其顺序轮用对烟蚜Myzus persicae(Sulzer)抗药性发展和羧酸酯酶活力的影响。结果表明,氧化乐果、灭多威、高效氟氯氰菊酯连续施药9次后,其抗性分别增长73.3,8.9,10.4倍;氧化乐果→灭多威→高效氟氯氰菊酯顺序轮用3次(施药9次)后,氧化乐果抗性指数增长了47.3倍,灭多威抗性指数增长了6.7倍,高效氟氯氰菊酯抗性指数增长了5.0倍。羧酸酯酶活力检测结果表明,3种杀虫剂连用9次后,烟蚜种群的α-NACarE活力分别增长了20.0,24.0和15.6倍,酶活力在0.6(OD600nm/aphid/min)以上的个体比例分别增加了73.4%,87.6%和43.8%;而3种杀虫剂顺序轮用3次(施药9次)后,烟蚜种群的α-NACarE活力增长了10.2倍,酶活力在0.6以上的个体比例增加了4.8%。结果证实杀虫剂轮换施用能延缓烟蚜抗药性的发展和烟蚜种群α-NACarE活力及高活力个体频率的增加。     

两个单点突变对昆虫羧酸酯酶降解马拉硫磷的影响

马拉硫磷是一种高效低毒的有机磷杀虫剂, 分子量大且结构特殊, 广泛用于农业害虫的防治。羧酸酯酶突变是昆虫对有机磷类杀虫剂产生代谢抗性的重要机制之一。本实验室前期已从棉蚜Aphis gossypii、 褐飞虱Nilaparvata lugens、 斜纹夜蛾Spodoptera litura、 家蚕Bombyx mori、 异色瓢虫Harmonia axyridis、 赤拟谷盗Tribolium castaneum和西方蜜蜂Apis mellifera中各克隆了一个非特异性羧酸酯酶基因, 通过体外定点突变构建了G/A151D和W271L两种突变体, 并进行了原核细胞表达和纯化。本实验在体外测定了这7种昆虫野生型和两种突变型羧酸酯酶对马拉硫磷的降解。结果显示： 棉蚜、 西方蜜蜂、 斜纹夜蛾、 赤拟谷盗的野生型羧酸酯酶能够降解马拉硫磷, 两个突变并不能提高它们的降解活性, 而家蚕、 异色瓢虫和褐飞虱的野生型羧酸酯酶不能降解马拉硫磷, G/A151D和/或W271L突变能使这些酯酶获得马拉硫磷羧酸酯酶（MCE）的活性, 有可能使这些昆虫对马拉硫磷产生抗性。不同物种的MCE活性相差较大, 斜纹夜蛾的MCE活性最高, 其k_cat/K_m值为1.8~1.9 L/μmol·min, 其次是赤拟谷盗, 其K_cat/K_m值为0.87~0.95 L/μmol·min, 其他昆虫的MCE活性相对较低, 相差可高达10倍。     

Structural requirements for the inhibition of human monocyte carboxylesterase by organophosphorus compounds

Human blood monocyte carboxylesterase (CBE) is inhibited by a variety of organophosphorus compounds including arylphosphates and arylphosphites and some alkylphosphites. Triphenyl phosphate and triphenyl phosphite with K_i values of 8 × 10⁻⁹ M and 4.8 × 10⁻⁸ M, respectively, are the most potent inhibitors of this enzyme evaluated by this study. The arylphosphates vary in their capacity to inhibit carboxylesterase activity. Diphenyl phosphate with its strong negative charge is not a potent inhibitor (K_i = 1 × 10⁻⁴ M), whereas if its negative charge is neutralized, as in diphenyl methyl phosphate, its capacity to inhibit carboxylesterase is significantly increased. Compounds with increased bulk, such as trinaphthyl phosphate, only inhibit the enzyme at concentrations of 10⁻⁵ M or greater. Arylphosphites have inhibitory capacities similar to the arylphosphates. Alkylphosphites (tributyl phosphite/triethyl phosphite) inhibit carboxylesterase activity, whereas alkylphosphates (tributyl phosphate/triethyl phosphate) have no inhibitory effect. Arylphosphines and arylphosphine oxides do not inhibit carboxylesterase activity. This study demonstrates that organophosphates and organophosphites are relatively effective inhibitors of human monocyte CBE activity with the exception of the alkylphosphates which have no inhibitory activity. We conclude that molecular bulk and charge have a significant role in determining the potency of organophosphorus inhibitors of monocyte CBE. The observed variations in the degree of esterase inhibition by organophosphorus compounds as well as the differences in the pathological expression of neuropathic disorders associated with such chemicals suggest that different esterase enzymes derived from the family of esterase genes may mediate the different neuropathies observed with organophosphorus exposures. Such data also provide the rationale for the kinetic analyses of esterases and the design of non-toxic organophosphorus compounds with low or no monocyte CBE inhibitory capacity to reduce the potential of these commonly used chemicals for human toxicity.     

羧酸酯酶A2在大肠杆菌中的表达及特征分析

本研究采用RT-PCR法从抗性蚊虫（Culex quinquefasciatus)中克隆了羧酸酯酶A2的全长cDNA,对其进行了序列测定。并构建了融合表达质粒pET-ESTA2,转化大肠杆菌BL21后,在异丙基硫代半乳糖苷（IPTG）的诱导下,使羧酸酯酶A2在大肠杆菌内得到表达,表达产物经亲和层析纯化获得了1条带的重组蛋白,与报道的从蚊虫体内纯化的羧酸酯酶相比,从表达产物中纯化的羧酸酯酶Km与其一致,但从表达产物纯化的羧酸酯酶的Vm比蚊虫中纯化的羧酸酯酶的Vm高,表明用亲和层析纯化的羧酸酯酶A2比从蚊虫中提取的纯度高,羧酸酯酶A2表达纯化及特征分析为其应用奠定了基础。     

Interaction of dialkyl(alpha-carbomethoxy-beta,beta,beta-trifluoro- ethyl) phosphates with mammalian esterases

The interaction of dialkyl (alpha-carbometoxy-beta,beta,beta-trifluoroethyl) phosphates (RO)2P(O) . OCH(CF3)COOMe (R = Me, Et, Pr, Pri, Bu, Bui, Am, Hex) (I-VIII) with human erythrocyte acetylcholinesterase, horse serum butyrylcholinesterase, pig liver carboxylesterase was studied and acute toxicity in mice was estimated. Compounds (I)-(VIII) were not hydrolyzed by carboxylesterase, slowly and irreversibly inhibited acetylcholinesterase (kII = 10(2)-10(4) M-1 X min-1) and more efficiently inhibited butyrylcholinesterase and carboxylesterase (kII = 10(3)-10(7) M-1 X min-1). The structure--antienzymatic activity relationships were investigated. With increasing of hydrophobicity of alkoxy groups, antienzymatic activity to butyrylcholinesterase and carboxylesterase ("sites of loss") rises equally and more significantly, than antiacetylcholinesterase activity (delta lg kII 1.0 and 2.4 for R = CH3 and C5H11 resp.). Branching at the alpha-position of alkoxy groups leads to sharp reducing of acetylcholinesterase and butyrylcholinesterase inhibition constants, the carboxylesterase inhibition mechanism becoming reversible. Multiple regression analysis (the Kubinyi model) showed that influence of steric hindrances is revealed at the phosphorylation stage. It was found that phosphates (I)-(VIII) possess low acute toxicity in mice (900-2000 mg/kg). The toxicity of this homologous series appears to be independent of the hydrophobicity. Role of esterases in toxicological effect of compounds (I)-(VIII) is discussed.     

Analysis of lipid hydrolytic activity by esterase on blotting membrane followed by separation using non-denaturing two-dimensional gel electrophoresis

After separation by microscale non-denaturing two-dimensional gel electrophoresis (2DE) and transferring to a blotting membrane, major proteins are detected by a staining of direct blue 71 in a neutral solution. The carboxylesterase on the membrane hydrolyzes phosphatidylcholine after the spot of carboxylesterase is excised from the membrane, and incubated with phosphatidylcholine. Lipids of human serum proteins and the purified human high density lipoprotein (HDL) are removed by enzymatic hydrolysis when human serum proteins and the purified HDL are respectively incubated with the spot of carboxylesterase on the membrane. These results indicate that carboxylesterase on the membrane hydrolyzes not only lipids such as phosphatidylcholine but also lipids of lipoproteins such as HDL after separation by the 2DE, transferring to the membrane and staining without impairing the activity. These results also indicate that a micro-immobilized enzyme reactor on the membrane can be produced when biological enzymes are separated by microscale 2DE, transferred to the membrane and stained without impairing their activities.     

马桑内酯对粘虫体内蛋白质和消化酶的影响

用马桑内酯分别对粘虫采用注射和饲喂处理,48 h后测定粘虫不同部位消化酶活性及蛋白质含量,以探讨马桑有效成分的作用及其杀虫机理.结果显示:(1)注射羟基马桑毒素处理后粘虫组织中蛋白质含量(与丙酮处理比较)均降低,皮组织降低幅度最大为29.47%,饲喂处理后各组织中蛋白质含量均升高,其中皮组织的升高幅度最大为56.87%;但该毒素对粘虫体内蛋白酶和羧酸酯酶的影响不明显.(2)注射与饲喂马桑亭、马桑宁,粘虫体内蛋白质含量均明显比对照处理升高,蛋白酶活性增强,其中饲喂马桑亭处理的粘虫血淋巴中蛋白质含量升高最大为511.49%,蛋白酶活性增强幅度最大为4 640.26%.马桑亭和马桑宁均可使粘虫组织中羧酸酯酶活性降低,其中饲喂马桑宁处理降低幅度最大为82.94%.结果表明:羟基马桑毒素对粘虫体内蛋白质代谢的影响与用药方式有关,马桑亭和马桑宁处理后的粘虫体内蛋白质含量增高、蛋白酶活性增强及酯酶活性降低均达到显著水平.     

四种类型杀虫剂对麦长管蚜的温度效应及其与主要解毒酶的关系

为了明确杀虫剂毒力受温度的影响及其程度,本文测定了4大类8种药剂在10～25℃下对麦长管蚜的毒力;并测定了麦长管蚜Sitobion avenae(Fabricius)在不同温度下2个解毒酶和1个靶标酶的活性差异。结果表明,高效氯氰菊酯对麦长管蚜表现不规则负温度系数,啶虫脒表现不规则正温度系数,高效氟氯氰菊酯对麦长管蚜的毒力不受温度影响,其他药剂(辛硫磷、毒死蜱、灭多威、丁硫克百威、吡虫啉)均表现为明显的正温度系数效应,以有机磷类杀虫剂表现最为明显,毒死蜱温度系数高达57.70。酶活性实验表明:麦长管蚜在高温下GST活性增强,羧酸酯酶和乙酰胆碱酯酶活性降低。它们的变化规律表明:GST活性与负温度系数密切相关,正温度系数与羧酸酯酶活性和靶标酶乙酰胆碱酯酶活性有关。     

Kinetic properties of human pancreatic carboxylesterase

Fractionation of pancreatic juice by heparin-Sepharose and cholate-Sepharose affinity chromatography indicated that pancreatic carboxylesterase can be separated from pancreatic lipase with the former retained and the latter unretained by both columns. The chromatographic behavior of pancreatic carboxylesterase was found to be similar to that of human milk bile salt-activated lipase. The partially purified pancreatic carboxylesterase had a specific activity of 30 mumol/min per mg protein when assayed with p-nitrophenyl acetate. The reaction mechanism of human pancreatic carboxylesterase was studied using p-nitrophenyl acetate as substrate and taurocholate as activator. The reaction of the enzyme was found to follow a rapid-equilibrium random mechanism. Because of the presence of basal activity, the role of taurocholate can be considered as a non-essential activator and the dissociation constant for the enzyme-taurocholate binary complex was determined to be 0.20 mM. The activation effect of taurocholate consists in increasing the affinity of the enzyme to the substrate (5.6-fold) and in increasing the Vmax (2.3-fold). Based on the kinetic property of human pancreatic carboxylesterase and human milk bile salt-activated lipase with p-nitrophenyl acetate, cholesterol oleate and triolein as substrate, we conclude that they share common substrate specificity but show minor differences in kinetic parameters. Fluorescence studies indicated that both enzymes showed a decreased intrinsic tryptophanyl fluorescence upon incubation with taurocholate. This indicates that bile salt caused a conformational change of the enzymes, with a resultant decreased hydrophobicity in the microenvironment of tryptophan residues.     

Carboxylesterase activities during the establishment of the procambium and procortex in embryos of pisum sativum L.

A cytochemical and electrophoretic analysis of alpha-naphthyl and naphthol AS-D acetate esterases from the radicles of pea seeds germinated for 24, 36 and 48 h permitted the detection of the loss of activity from the procortex with time after germination, but a stability of this activity in the prostele. Inhibitor studies showed the procambial enzyme as a single band of carboxylesterase activity the remainder being acetylesterase activity. The procambial carboxylesterase which is present in the more mature root apex corresponds to that of the 48-h embryo (separating at Rf 0·74).     

应用酶标仪动力学方法监测棉蚜的抗药性

用酶标仪动力学测定法对3个抗性水平不同的棉蚜品系（R1、R2和R3）和1个敏感品系（S）的羧酸酯酶进行了研究,S、R1、R2和R3品系对α-乙酸萘酯（α-NA）的平均比活力分别为57．10、1171．69、1236．14和3293．00μmol·mgpro－1·min-1（分光光度计终点测定法）和38．24、85．27、198．14和762．25mOD·min－1·aphid-1（酶标仪动力学法）。终点测定法结果显示出不同品系间最大相差达60倍;酶标仪动力学测定法研究表明,4个棉蚜品系羧酸酶活性与其抗药性程度显著相关。通过对这两种方法的比较,酶动力学方法的测定结果更可靠。     

Carboxylesterases (EC 3.1.1). The source of variations in substrate specificity and properties of pig liver carboxylesterase.

During the final stage of purification of pig liver carboxylesterase on CM-Sephadex, several enzyme activities are present in addition to the pig liver carboxylesterase reported from this laboratory (Horgan et al., 1969a). Variation in substrate specificity and specific activity of the material isolated from CM-Sephadex has been analysed with four substrates - methyl, ethyl and phenyl butyrates and butyrycholine. The sensitivity of various fractions to paraoxon, eserine and phenylmethanesulfonyl fluoride is also examined. The results support the conclusion that the variability of pig liver carboxylesterase reported from other laboratories is due to the heterogeneity here defined.     

Tissue Carboxylesterases and Chlorpyrifos Toxicity in the Developing Rat

Young animals are more sensitive than adults to the neurotoxic effects of some organophosphorus insecticides. Many investigators attribute this difference in sensitivity to the immaturity of the detoxification capacity of preweanling rats. Chlorpyrifos [O,O-diethylO-(3,5,6-trichloro-2-pyridyl)phosphorothionate] is an organophosphorus insecticide that demonstrates considerable age-related sensitivity. The carboxylesterases are a group of related enzymes that detoxify organophosphorus insecticides by stoichiometrically binding these molecules before they can inhibit acetylcholinesterase. This study presents in vitro and in vivo evidence demonstrating that the carboxylesterases are critical for explaining the age-related sensitivity of chlorpyrifos. The data show that the fetal rat and the postnatal day 17 (PND17) rat pup have fewer molecules of carboxylesterase (less activity), less sensitive molecules of carboxylesterase, and a larger proportion of chlorpyrifos-insensitive molecules of carboxylesterase. An in vitro mixing experiment, using adult striatum as a source of acetylcholinesterase and liver homogenates as a source of carboxylesterase, demonstrates that the adult liver carboxylesterases are superior to the PND17 liver carboxylesterases for detoxifying chlorpyrifos. In the in vivo experiments the time course profiles of carboxylesterase and cholinesterase activity following a maximum tolerated dose of chlorpyrifos also suggest that the carboxylesterases of the PND17 rat were less capable of detoxifying chlorpyrifos. Carboxylesterase activity in the preweanling rat was not as severely inhibited as in the adult, but decrements in cholinesterase activity as a result of chlorpyrifos treatment were comparable. These in vitro and in vivo findings support the previously proffered postulate that the carboxylesterases are critical for determining the age-related sensitivity of chlorpyrifos. In addition, these detailed experiments allow us to propose that the detoxification potential of these enzymes is multifaceted, and depends on the (1) amount of activity (i.e., number of molecules), (2) affinity for the insecticide or metabolite, and (3) amount of carboxylesterase activity that is refractory to inhibition by the insecticide or metabolite.     

运用α-氰代酯荧光底物检测抗性棉蚜羧酸酯酶代谢活性

为了研究抗性和敏感棉蚜Aphis gossypii品系对菊酯类药剂代谢的差异, 本实验合成了溴氰菊酯和高效氯氰菊酯报告荧光底物, 应用这两种底物水解后生成具有荧光化合物的特性,测定了不同品系棉蚜羧酸酯酶的代谢活性。结果表明: 氧化乐果棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为10.0和3.4 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为4.0和2.4 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的2.9和1.7倍; 溴氰菊酯棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为7.6和6.2 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为9.3和5.2 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的1.2和1.8倍。这种衍生的报告荧光底物能够用来检测抗性棉蚜羧酸酯酶的水解活性, 表明羧酸酯酶可能参与棉蚜对溴氰菊酯和氧化乐果抗性的形成。     

Regulatory studies of L-glutamate dehydrogenase from Trypanosoma cruzi epimastigotes

Carboxylesterase activity corresponding to types A and B has been demonstrated in intact T. cruzi epimastigotes as shown by the hydrolysis of several esters of p-nitrophenol and the effect of suitable inhibitors. The in situ carboxylesterase activity was described by the Michaelis Menten kinetic approach. The apparent Vmax for the acetate and butyrate esters were 66.5 and 165.3 nmol hydrolysed per min and mg of protein respectively. An Arrhenius plot of the temperature dependent activity showed two sharp linear regions with a transition temperature of 31.6 degrees C. and energies of activation of 6.2 and 14.1 kcal/mol. The in situ carboxylesterase activity was inhibited 26% by paraoxon and 56% by N-ethylmaleimide, but not by p-chloromercuribenzoate.     

Comparative analysis of esterase and paraoxonase activities of different serum albumin species

Enzymatic activities of three types of serum albumin—rat, bovine and human—were analyzed comparatively using a mathematical model. Kinetic and equilibrium constants of carboxylesterase and paraoxonase activities of albumin in Sudlow’s sites I and II were determined. The effects of specific ligands, ibuprofen and warfarin, on enzyme kinetics in these sites were studied. Ibuprofen was found to have an inhibitory effect both on carboxylesterase and paraoxonase albumin activities, whereas warfarin specifically inhibited only carboxylesterase albumin activity.