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Reactivation of immobilized acetyl cholinesterase in an amperometric biosensor for organophosphorus pesticide 固定化乙酰胆碱酯酶在有机磷农药安培传感器中的再激活
Pub Date : 2002-05-20 DOI: 10.1016/S0167-4838(02)00268-6
K.C. Gulla, M.D. Gouda, M.S. Thakur, N.G. Karanth

Biosensors based on acetyl cholinesterase (AChE) inhibition have been known for monitoring of pesticides in food and water samples. However, strong inhibition of the enzyme is a major drawback in practical application of the biosensor which can be overcome by reactivation of the enzyme for repeated use. In the present study, enzyme reactivation by oximes was explored for this purpose. Two oximes viz., 1,1′-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were compared for the reactivation of the immobilized AChE. TMB-4 was found to be a more efficient reactivator under repeated use, retaining more than 60% of initial activity after 11 reuses, whereas in the case of 2-PAM, the activity retention dropped to less than 50% after only 6 reuses. Investigations also showed that reactivation must be effected within 10 min after each analysis to eliminate the ageing effect, which reduces the efficiency of reactivation.

基于乙酰胆碱酯酶(AChE)抑制的生物传感器已被用于监测食品和水样中的农药。然而,酶的强抑制作用是生物传感器实际应用中的一个主要缺点,这可以通过酶的再激活来克服。在本研究中,酶的再活化由肟探讨了这一目的。比较了1,1′-三亚甲基双4-甲酰基溴化吡啶二肟(TMB-4)和吡啶2-醛肟甲氧基(2-PAM)两种肟类对固定化AChE的再活化作用。在重复使用的情况下,TMB-4被发现是一个更有效的再活化剂,在重复使用11次后保留了60%以上的初始活性,而在2-PAM的情况下,重复使用6次后活性保留率下降到50%以下。调查还表明,每次分析后必须在10分钟内进行再活化,以消除老化效应,这降低了再活化的效率。
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引用次数: 62
A new syringopeptin produced by bean strains of Pseudomonas syringae pv. syringae 丁香假单胞菌产一种新的丁香素。两
Pub Date : 2002-05-20 DOI: 10.1016/S0167-4838(02)00283-2
Ingeborg Grgurina , Feliciana Mariotti , Vincenzo Fogliano , Monica Gallo , Andrea Scaloni , Nicola S. Iacobellis , Pietro Lo Cantore , Luisa Mannina , Valeria van Axel Castelli , Maria Luigia Greco , Antonio Graniti

Two strains (B728a and Y37) of the phytopathogenic bacterium Pseudomonas syringae pv. syringae isolated from bean (Phaseolus vulgaris) plants were shown to produce in culture both syringomycin, a lipodepsinonapeptide secreted by the majority of the strains of the bacterium, and a new form of syringopeptin, SP22Phv. The structure of the latter metabolite was elucidated by the combined use of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and chemical procedures. Comparative phytotoxic and antimicrobial assays showed that SP22Phv did not differ substantially from the previously characterized syringopeptin 22 (SP22) as far as toxicity to plants was concerned, but was less active in inhibiting the growth of the test fungi Rhodotorula pilimanae and Geotrichum candidum and of the Gram-positive bacterium Bacillus megaterium.

植物致病菌丁香假单胞菌(Pseudomonas syringae pv) B728a和Y37。从豆(Phaseolus vulgaris)植物中分离的丁香属植物在培养中显示出紫丁香霉素(一种由大多数菌株分泌的脂沉肽)和一种新形式的紫丁香素(SP22Phv)。后一种代谢物的结构由质谱(MS)、核磁共振(NMR)谱和化学方法联合阐明。对比植物毒性和抗菌试验表明,SP22Phv与先前鉴定的syringopeptin 22 (SP22)在植物毒性方面没有太大差异,但对试验真菌红霉菌(Rhodotorula pilimanae)和铁皮土曲菌(Geotrichum candidum)以及革兰氏阳性细菌巨芽孢杆菌的抑制作用较弱。
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引用次数: 38
Mechanistic implications of variable stoichiometries of oxygen consumption during tyrosinase catalyzed oxidation of monophenols and o-diphenols 酪氨酸酶催化单酚和邻二酚氧化过程中氧消耗的可变化学计量学的机制含义
Pub Date : 2002-05-20 DOI: 10.1016/S0167-4838(02)00264-9
Marı́a José Peñalver , Alexander N.P. Hiner , José Neptuno Rodrı́guez-López , Francisco Garcı́a-Cánovas , José Tudela

The stoichiometry of oxygen consumption during tyrosinase-catalyzed oxidation of an o-diphenol (4-tert-butylcatechol, TBC) and a monophenol (4-tert-butylphenol, TBP) has been determined. At high [substrate]/[enzyme] ratios, in the case of o-diphenols, the stoichiometry of the enzyme-catalyzed reaction was always 1 O2/2 o-diphenols, although if the o-quinone product was unstable, the apparent stoichiometry could tend to 1 O2/1 o-diphenol due to regeneration of an o-diphenol in a side reaction. In the case of monophenols, the stoichiometry could be 1 O2/1 monophenol or 1.5 O2/1 monophenol depending if the o-quinone product was stable or unstable, respectively. However, at low [substrate]/[enzyme] ratios, the oxygen/substrate stoichiometry could, even in the case where stable products are formed, be lower than 1 O2/2 substrates for o-diphenols or higher than 1 O2/1 substrate for monophenols. These data supported the mechanism proposed by Rodrı́guez-López et al. [J. Biol. Chem. 267 (1992) 3801–3810], in which, during hydroxylation of monophenols, tyrosinase first transformed monophenol to o-diphenol and then either catalyzed a further oxidation to form o-quinone or released it into the reaction medium. In this second case, subsequent oxidation of the o-diphenol resulted in additional oxygen consumption.

测定了酪氨酸酶催化氧化邻二酚(4-叔丁基儿茶酚,TBC)和单酚(4-叔丁基酚,TBP)过程中耗氧量的化学计量学。在高[底物]/[酶]比的情况下,邻二酚的酶催化反应的化学计量量始终为1 O2/2邻二酚,尽管如果邻醌产物不稳定,由于邻二酚在副反应中再生,表观化学计量量可能倾向于1 O2/1邻二酚。在单酚的情况下,根据邻醌产物是稳定的还是不稳定的,化学计量可以分别为1 O2/1单酚或1.5 O2/1单酚。然而,在低[底物]/[酶]比率下,即使在形成稳定产物的情况下,氧/底物化学计量也可能低于邻二酚的1 O2/2底物,或高于单酚的1 O2/1底物。这些数据支持了rodriguez guez-López等人提出的机制[J]。医学杂志。化学,267(1992)3801-3810],其中,在单酚羟基化过程中,酪氨酸酶首先将单酚转化为邻二酚,然后催化进一步氧化形成邻醌或将其释放到反应介质中。在第二种情况下,随后的邻二酚氧化导致额外的氧气消耗。
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引用次数: 16
Solvent-dependent precipitation of prion protein 朊病毒蛋白的溶剂依赖性沉淀
Pub Date : 2002-05-20 DOI: 10.1016/S0167-4838(02)00282-0
Kang Cai , Jeanette L.C. Miller , Christopher J. Stenland , Kevin J. Gilligan , Randal C. Hartwell , Jarrett C. Terry , Rosemary B. Evans-Storms , Richard Rubenstein , Stephen R. Petteway Jr. , Douglas C. Lee

The misfolded isoform of the prion protein (PrPSc) possesses many unusual physiochemical properties. Previously, we and others reported on the differential partitioning of PrPSc from plasma derived therapeutic proteins during their purification processes. To understand the driving force behind these partitioning differences, we investigated the effects of various solvent conditions on the precipitation of PrPSc. In a physiological buffer, PrPSc remained in the supernatant after low speed centrifugation. At pH 5, PrPSc precipitation was nearly complete regardless of the salt content. PrPSc could also be precipitated at pH 8 by adding ethanol, but this precipitation was salt dependent. Based on these observations, an empirical mathematical model was constructed in which the PrPSc precipitation trends were fully described as a function of solvent pH, salt, and ethanol concentration. This model consistently predicted PrPSc partitioning during cold ethanol precipitation steps used in plasma protein purification processes, as shown by experimentally determined distributions of PrPSc and transmissible spongiform encephalopathy (TSE) infectivity. These results indicate that pH, salt, and ethanol content are the major solvent factors determining the precipitation of the infectious PrPSc in these processes and may provide a useful tool for assessing the differential partitioning of PrPSc in a given solvent environment.

错误折叠的朊病毒蛋白(PrPSc)具有许多不寻常的物理化学性质。之前,我们和其他人报道了血浆来源的治疗蛋白在纯化过程中对PrPSc的差异分配。为了了解这些分配差异背后的驱动力,我们研究了不同溶剂条件对PrPSc沉淀的影响。在生理缓冲液中,低速离心后PrPSc保留在上清液中。在pH为5时,无论含盐量如何,PrPSc的沉淀几乎完全完成。在pH为8的条件下,加入乙醇也能使PrPSc析出,但这种析出与盐有关。基于这些观测结果,构建了一个经验数学模型,其中PrPSc的沉淀趋势完全描述为溶剂pH、盐和乙醇浓度的函数。该模型一致地预测了血浆蛋白纯化过程中使用的冷乙醇沉淀步骤中PrPSc的分配,如实验确定的PrPSc分布和传染性海绵状脑病(TSE)传染性所示。这些结果表明,pH、盐和乙醇含量是决定这些过程中感染性PrPSc沉淀的主要溶剂因素,并可能为评估PrPSc在给定溶剂环境下的差异分配提供有用的工具。
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引用次数: 38
The distinctiveness of ATP:citrate lyase from Aspergillus nidulans 细粒曲霉柠檬酸裂解酶的特性
Pub Date : 2002-05-20 DOI: 10.1016/S0167-4838(02)00276-5
Ian P. Adams, Stephen Dack, F.Mark Dickinson, Colin Ratledge

ATP:citrate lyase (ACL), an important enzyme in lipid synthesis, has been purified from Aspergillus nidulans to a specific activity of 19.6 μmol min−1 mg−1, almost twice that of any other purified ACL and shown to be distinct from any previously purified ACL. The enzyme is a 371±31 kDa hexamer of 3α, 3β proteins, unlike the 4α tetramer found in rats or yeasts. The molecular weights of the α and β protein subunits were determined by SDS-PAGE to be 70 and 55 kDa.

ACL in A. nidulans (unlike Aspergillus niger) appears to be regulated by the carbon source present in the media. In crude extracts, it was found at high activity (88 μmol min−1 mg protein−1) in glucose-grown cells but only at low activity (10 μmol min−1 mg protein−1) in acetate-grown cells.

ATP:柠檬酸裂解酶(ATP:citrate lyase, ACL)是一种重要的脂质合成酶,其比活性为19.6 μmol min - 1 mg - 1,几乎是其他纯化的ACL的两倍,与以往纯化的ACL不同。该酶是371±31 kDa的3α, 3β蛋白六聚体,不同于在大鼠或酵母中发现的4α四聚体。SDS-PAGE测定α和β蛋白亚基分子量分别为70和55 kDa。A. nidulans的ACL(与黑曲霉不同)似乎受到培养基中碳源的调节。粗提物在葡萄糖培养的细胞中具有高活性(88 μmol min−1 mg protein−1),而在醋酸盐培养的细胞中只有低活性(10 μmol min−1 mg protein−1)。
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引用次数: 24
Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism 谷胱甘肽转移酶A1-1 c端区芳香族残基影响催化机制中速率决定步骤
Pub Date : 2002-05-20 DOI: 10.1016/S0167-4838(02)00286-8
Lisa O. Nilsson, Maryam Edalat, Pär L. Pettersson, Bengt Mannervik

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (α9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the α9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters Kcat and Kcat/Km for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the α9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Δ5-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on Kcat/Km. The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.

人谷胱甘肽转移酶A1-1 (GST A1-1)具有一个柔性的c端片段,当谷胱甘肽与小的亲电底物如1-氯-2,4-二硝基苯(CDNB)结合时,形成螺旋状(α9)关闭活性位点。在没有活性位点配体的情况下,c端段不固定在一个位置,在晶体结构中无法检测到。α9螺旋上的一个关键残基是Phe 220,它可以与酶结合的谷胱甘肽和第二底物相互作用,并可能引导反应物进入过渡态。结果表明,Phe 220突变为Ala和Thr会降低GST A1-1的催化效率。另一个残基Phe 222的突变导致活性进一步下降。反应介质中粘原的存在降低了野生型GST A1-1催化CDNB偶联的动力学参数Kcat和Kcat/Km,符合产物释放是底物饱和酶限速的观点。突变导致两个动力学参数的粘度依赖性降低,表明α9-螺旋的运动与野生型GST A1-1的催化作用有关。与替代底物Δ5-androstene-3,17-二酮(AD)的异构化反应以类似的方式受到粘原的影响。突变对Kcat/Km的影响表明,与CDNB偶联反应一样,异构化反应的过渡态能被Phe 220降低。结果表明,动态c端段的Phe 220和Phe 222对取代反应和异构化反应的催化机制中的速率决定步骤都有影响。
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引用次数: 35
Thermodynamic properties of nucleotide-free EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors of its GTPase activity 来自嗜热热菌的无核苷酸EF-Tu在GTPase活性的低分子量效应物存在下的热力学性质
Pub Date : 2002-05-20 DOI: 10.1016/S0167-4838(02)00270-4
Erik Sedlák , Gabriel Žoldák , Marián Antalı́k , Mathias Sprinzl

The thermal transition of elongation factor EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors was studied by differential scanning calorimetry. The effectors of GTPase activity used were the antibiotic kirromycin and the cations Li+, Na+, K+ and NH4+ in the chloride form. The temperature of thermal denaturation and the cooperativity of the transition of nucleotide-free EF-Tu (EF-Tuf) in the presence of kirromycin are comparable with those of the EF-Tu·guanosine-5′-[β,γ-imido]triphosphate (GppNHp) form, indicating similar conformational states. Increased concentrations of Na+ and K+ stabilized EF-Tuf in a manner similar to GppNHp. NH4+ decreased the transition temperature of EF-Tuf and Li+ decreased both the temperature and the calorimetric enthalpy of the thermal transition of EF-Tuf. In the presence of salts, binding of kirromycin had a stabilizing effect on EF-Tuf. Correlation between the GTPase activity and thermodynamic characteristics of EF-Tuf induced by kirromycin in the absence or presence of the cations is discussed.

采用差示扫描量热法研究了在低分子量效应剂存在下,嗜热菌中延伸因子EF-Tu的热转变。GTPase活性的影响因子是抗生素kirromycin和氯离子Li+、Na+、K+和NH4+。无核苷酸EF-Tu (EF-Tuf)在克罗霉素存在下的热变性温度和转变的协同性与EF-Tu·鸟苷-5′-[β,γ-亚胺]三磷酸(GppNHp)形式相当,表明相似的构象状态。Na+和K+浓度的增加以类似于GppNHp的方式稳定EF-Tuf。NH4+降低了EF-Tuf的转变温度,Li+降低了EF-Tuf的温度和热焓。在盐的存在下,克罗霉素的结合对EF-Tuf有稳定作用。讨论了在不存在或不存在这些阳离子的情况下,由克罗霉素诱导的EF-Tuf的GTPase活性与热力学特性的关系。
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引用次数: 5
Cleavage specificity of the subtilisin-like protease C1 from soybean 大豆枯草杆菌样蛋白酶C1的裂解特异性
Pub Date : 2002-04-29 DOI: 10.1016/S0167-4838(02)00228-5
Patrick M. Boyd , Neel Barnaby, Anna Tan-Wilson, Karl A. Wilson

The cleavage specificity of protease C1, isolated from soybean (Glycine max (L.) Merrill) seedling cotyledons, was examined using oligopeptide substrates in an HPLC based assay. A series of peptides based on the sequence Ac-KVEKEESEEGE-NH2 was used, mimicking a natural cleavage site of protease C1 in the α subunit of the storage protein β-conglycinin. A study of substrate peptides truncated from either the N- or C-terminus indicates that the minimal requirements for cleavage by protease C2 are three residues N-terminal to the cleaved bond, and two residues C-terminal (i.e. P3-P2′). The maximal rate of cleavage is reached with substrates containing four to five residues N-terminal to the cleaved bond and four residues C-terminal (i.e. P4 or P5 to P4′). The importance of Glu residues at the P1, P1′, and P4 positions was examined using a series of substituted nonapeptides (P5-P4′) with a base sequence of Ac-KVEKEESEE-NH2. At the P1 position, the relative ranking, based on kcat/Km, was E>Q>K>A>D>F>S. Substitutions at the P1′ position yield the ranking E≅Q>A>S>D>K>F, while those at P4′ had less effect on kcat/Km, yielding the ranking F≅S≅E≅D>K>A≅Q. These data show that protease C1 prefers to cleave at Glu-Glu and Glu-Gln bonds, and that the nature of the P4′ position is less important. The fact that there is specificity in the cleavage of the oligopeptides suggests that the more limited specific cleavage of the α and α′ subunits of β-conglycinin by protease C1 is due to a combination of the sequence cleavage specificity of the protease and the accessibility of appropriate scissile peptide bonds on the surface of the substrate protein.

大豆(Glycine max (L.))蛋白酶C1的裂解特异性用高效液相色谱法检测了美林(Merrill)幼苗子叶的寡肽底物。使用了一系列基于Ac-KVEKEESEEGE-NH2序列的肽,模拟了储存蛋白β-甘氨酸α亚基中蛋白酶C1的天然裂解位点。对从N端或c端截断的底物肽的研究表明,蛋白酶C2切割的最低要求是在被切割的键的N端有三个残基,c端有两个残基(即P3-P2 ')。当底物含有4到5个残基n端到被劈裂的键和4个残基c端(即P4或P5到P4 ')时,达到最大的裂解速率。通过一系列以Ac-KVEKEESEE-NH2为碱基序列的取代非肽(P5-P4 ')来检测P1, P1 '和P4位置上Glu残基的重要性。在P1位置,基于kcat/Km的相对排名是E>Q>K>A>D>F>S。P1′位置的替换得到E = Q>A>S>D>K>F,而P4′位置的替换对kcat/Km的影响较小,得到F = S = E = D>K>A = Q。这些数据表明,蛋白酶C1更倾向于在Glu-Glu和Glu-Gln键上切割,而P4 '位置的性质不太重要。寡肽的切割具有特异性,这表明蛋白酶C1对β-甘氨酸α和α′亚基的特异性较有限的切割是由于蛋白酶的序列切割特异性和底物蛋白表面适当的可剪切肽键的可及性的结合。
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引用次数: 29
Inhibition of yeast lipase (CRL1) and cholesterol esterase (CRL3) by 6-chloro-2-pyrones: comparison with porcine cholesterol esterase 6-氯-2-吡咯酮对酵母脂肪酶(CRL1)和胆固醇酯酶(CRL3)的抑制作用:与猪胆固醇酯酶的比较
Pub Date : 2002-04-29 DOI: 10.1016/S0167-4838(01)00304-1
Mary Stoddard Hatch , William M Brown , Jason A Deck , Lucy A Hunsaker , Lorraine M Deck , David L Vander Jagt

Previously, it was demonstrated that pancreatic cholesterol esterase is selectively inhibited by 6-chloro-2-pyrones with cyclic aliphatic substituents in the 3-position. Inhibition is reversible and is competitive with substrate. Pancreatic cholesterol esterase is a potential target for treatment of hypercholesterolemia. In the present study, yeast cholesterol esterase from Candida cylindracea (also called C. rugosa CRL3) was compared to porcine pancreatic cholesterol esterase for inhibition by a series of 3-alkyl- or 5-alkyl-6-chloro-2-pyrones. In addition, CRL3 was compared with the related yeast lipase CRL1. Inhibition of CRL3 by substituted 6-chloro-2-pyrones was competitive with binding of the substrate p-nitrophenyl butyrate. Inhibition constants ranged from 0.2 μM to >90 μM. Small changes in the alkyl group had profound effects on binding. The pattern of inhibition of CRL3 is quite distinct from that observed with porcine cholesterol esterase. Molecular modeling studies suggest that the orientation of binding of these inhibitors at the active site of CRL3 can vary but that the pyrone ring consistently occupies a position close to the active site serine. CRL1 is highly homologous to CRL3. Nevertheless, patterns of inhibition of CRL1 by substituted 6-chloro-2-pyrones differ markedly from patterns observed with CRL3. The substituted 6-chloro-2-pyrones are slowly hydrolyzed in the presence of CRL1 and are pseudosubstrates of CRL3, but are simple reversible inhibitors of pancreatic cholesterol esterase

先前的研究表明,胰腺胆固醇酯酶被3位环脂肪取代基的6-氯-2-吡咯选择性抑制。抑制作用是可逆的,并与底物竞争。胰胆固醇酯酶是治疗高胆固醇血症的潜在靶点。在本研究中,比较了来自圆柱假丝酵母的酵母胆固醇酯酶(也称为C. rugosa CRL3)与猪胰腺胆固醇酯酶被一系列3-烷基或5-烷基-6-氯-2-吡咯酮抑制的情况。此外,还将CRL3与相关酵母脂肪酶CRL1进行了比较。取代的6-氯-2-吡咯酮对CRL3的抑制作用与底物对硝基苯丁酸酯的结合具有竞争性。抑制常数范围为0.2 μM ~ 90 μM。烷基的微小变化对结合有深远的影响。CRL3的抑制模式与猪胆固醇酯酶的抑制模式截然不同。分子模拟研究表明,这些抑制剂在CRL3活性位点的结合方向可以改变,但吡酮环始终占据靠近活性位点丝氨酸的位置。CRL1与CRL3高度同源。然而,取代的6-氯-2-吡咯酮对CRL1的抑制模式与CRL3观察到的模式明显不同。取代的6-氯-2-吡酮在CRL1存在下缓慢水解,是CRL3的假底物,但是胰腺胆固醇酯酶的简单可逆抑制剂
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引用次数: 20
Enhanced response to antibody binding in engineered β-galactosidase enzymatic sensors 工程β-半乳糖苷酶传感器对抗体结合的增强反应
Pub Date : 2002-04-29 DOI: 10.1016/S0167-4838(02)00226-1
Jordi X Feliu , Neus Ferrer-Miralles , Esther Blanco , Daniel Cazorla , Francisco Sobrino , Antonio Villaverde

Peptide display on solvent-exposed surfaces of engineered enzymes allows them to respond to anti-peptide antibodies by detectable changes in their enzymatic activity, offering a new principle for biosensor development. In this work, we show that multiple peptide insertion in the vicinity of the Escherichia coli β-galactosidase active site dramatically increases the enzyme responsiveness to specific anti-peptide antibodies. The modified enzymes HD7872A and HT7278CA, carrying eight and 12 copies respectively of a foot-and-mouth disease peptide per enzyme molecule, show antibody-mediated activation factors higher than those previously observed in the first generation enzymatic sensors, for HT7278CA being close to 400%. The analysis of the signal transduction process with multiple inserted proteins strongly suggests a new, non-exclusive mechanism of enzymatic regulation in which the target proteins might be stabilised by the bound antibody, extending the enzyme half-life and consequently enhancing the signal–background ratio. In addition, the tested sensors are differently responsive to sera from immune farm animals, depending on the antigenic similarity between the B-cell epitopes in the immunising virus and those in the peptide used as sensing element on the enzyme surface. Altogether, these results point out the utility of these enzymatic biosensors for a simple diagnosis of foot-and-mouth disease in an extremely fast homogeneous assay.

肽显示在工程酶的溶剂暴露表面上,使它们能够通过可检测的酶活性变化对抗肽抗体作出反应,为生物传感器的开发提供了新的原理。在这项工作中,我们发现在大肠杆菌β-半乳糖苷酶活性位点附近插入多个肽可以显著提高酶对特定抗肽抗体的反应性。修饰酶HD7872A和HT7278CA,每个酶分子分别携带8个和12个口蹄疫肽拷贝,显示出抗体介导的激活因子高于先前在第一代酶传感器中观察到的,其中HT7278CA接近400%。对多个插入蛋白的信号转导过程的分析强烈提示了一种新的、非排他的酶调节机制,在这种机制中,结合的抗体可能会稳定靶蛋白,延长酶的半衰期,从而提高信号背景比。此外,测试的传感器对来自免疫农场动物的血清的反应不同,这取决于免疫病毒中的b细胞表位与酶表面用作传感元件的肽中的b细胞表位之间的抗原相似性。总之,这些结果指出了这些酶生物传感器在一个极快的同质分析中对口蹄疫的简单诊断的效用。
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引用次数: 23
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Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
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