Enzyme-mediated individual nanoparticle release assay

Numerous methods have been developed to measure the presence of macromolecular species in a sample; however, the number of methods that detect functional activity or modulators of that activity is more limited. To address this limitation, an approach was developed that uses the optical detection of nanoparticles as a measure of enzyme activity. Nanoparticles are increasingly being used as biological labels in static binding assays; here, we describe their use in a release assay format, where the enzyme-mediated liberation of individual nanoparticles from a surface is measured. A double-stranded fragment of DNA is used as the initial tether to bind the nanoparticles to a solid surface. The nanoparticle spatial distribution and number are determined using dark-field optical microscopy and digital image capture. Site-specific cleavage of the DNA tether results in nanoparticle release. The methodology and validation of this approach for measuring enzyme-mediated, individual DNA cleavage events, rapidly, with high specificity, and in real-time are described. This approach was used to detect and discriminate between nonmethylated and methylated DNA, and demonstrates a novel platform for high-throughput screening of modulators of enzyme activity.     

酶分子设计常用策略和进展

酶分子的生物学功能很大程度上是由其三维空间结构和所处溶剂环境共同决定的。因此,优化酶分子的结构性质以及探索其性质最优的溶剂环境是改善酶分子功能以及进行理性设计的一个可行途径。从实际应用的角度来看,分子设计方法可以为酶工程提供一种有效的解决方案。目前,酶分子设计有两个重要的研究方向,包括提高酶分子的催化活力和优化其稳定性。同时,对酶分子设计方法的研究也有助于对蛋白质生物学机理的探索。在近些年的学术界酶分子设计案例中,生物信息学方法得到广泛的应用。本文系统地总结基于生物信息学的酶分子设计方法的背景、策略和一些经典案例。     

Freeze-fracture cytochemistry: a new method combining immunocytochemistry and enzyme cytochemistry on replicas.

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphates activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.     

Covalent immobilization of avidin on glassy carbon electrodes as the basis for multivalent biosensors.

One of the most crucial steps for the successful construction of a biosensor is the appropriate and reproducible coupling of the biological part (e.g. enzyme, antibody) to the inorganic moiety of the device (e.g. electrode, microchip). In this paper three methods of immobilization of avidin to a glassy carbon electrode are described. Depending on the type of immobilization, avidin may lose its biological activity as determined by an enzyme immunoassay, using biotinylated reagents. If avidin is covalently bound to the glassy carbon electrode via the bridge molecule 4.4'-diaminodiphenylamine, the biological activity is retained. About 1.5 pmol of avidin can be bound to the electrode (3 mm in diameter), resulting in a nearly complete monolayer of protein.     

Determination of enzymatic activity by capillary electrophoresis

Enzymes are biological catalysts that play an important role in biochemical reactions necessary for normal growth, maturation and reproduction through whole live world. Their accurate quantitation in biological samples is important in many fields of biochemistry, not only in routine biochemistry and in fundamental research, but also in clinical and pharmacological research and diagnosis. Since the direct measurement of enzymes by masses is impossible, they must be quantified by their catalytic activities. Many different methods have been applied for this purpose so far. Although photometric methods are undoubtedly the most frequently used, separation methods will further gain their position in this field. The article reviews different possibilities for the assay of enzymatic activity by means of capillary electrophoresis (CE). Both the off-line and on-line enzyme assays based on CE are discussed.     

Prediction of enzyme family classes

Classes of newly found enzyme sequences are usually determined either by biochemical analysis of eukaryotic and prokaryotic genomes or by microarray chips. These experimental methods are both time-consuming and costly. With the explosion of protein sequences entering into databanks, it is highly desirable to explore the feasibility of selectively classifying newly found enzyme sequences into their respective enzyme classes by means of an automated method. This is indeed important because knowing which family or subfamily an enzyme belongs to may help deduce its catalytic mechanism and specificity, giving clues to the relevant biological function. In this study, a bioinformatical analysis was conducted for 2640 oxidoreductases classified into 16 subclasses according to the different types of substrates they act on during the catalytic process. Although it is an extremely complicated problem and might involve the knowledge of 3-dimensional structure as well as many other physical chemistry factors, some quite promising results have been obtained indicating that the family or subfamily of an enzyme is predictable to a considerable degree by means of sequence-based approach alone if a good training dataset can be established.     

A rapid method for measuring guanylate cyclase activity in mammary tissue.

A simple and rapid method for measuring guanylate cyclase activity in broken cell preparations of biological tissues is described. This method employs the rate of conversion of [32P]GTP to [32P]cyclic-GMP. The product of this reaction is isolated by ion-exchange chromatography and by a ZnSO4-Ba(OH)2 precipitation at pH 5.7. Using this method, about 30-50 samples can be assayed for guanylate cyclase activity during a 5-6 hr period. The characteristics of this enzyme in the mammary gland were found to be similar to those described for other tissues using different methods for measuring guanylate cyclase activity.     

家蚕肠道环境对外源纤维素酶活力稳定性影响的研究

纤维素酶对家蚕消化桑叶纤维素起重要生理作用,本试验研究了不同温度、pH对外源纤维素酶活力的影响及纤维素酶的热稳定性与pH稳定性,同时在模拟家蚕肠道环境条件下,研究了纤维素酶活力的稳定性。结果表明：所选的外源纤维素酶在家蚕肠液中的最适催化温度为30℃左右,最适pH为8．0左右;酶在家蚕体温范围内具有较好的稳定性,在pH8．0-10．0范围内酶活力较稳定;模拟家蚕肠道环境条件下酶活力稳定性实验表明,在80min时间内,纤维素酶能够保持较高的活力而发挥生物活性。     

堆肥化过程中生物酶活性的研究进展

在土壤酶学基础上发展起来的堆肥酶活研究能够从生化方面更深入地反映堆肥产品的腐熟程度、有机物质的降解以及重金属、芳香族化合物等有害物质的转化情况。文章系统总结了堆肥中酶活变化与原料成分、添加剂、微生物种类和数量、有机质降解的关系,不同酶活性之间的相关性以及堆肥中酶动力学的研究成果。提出筛选少数几个彼此独立的综合性酶指标,利用多元回归分析建立堆肥化与酶活的动态关系。同时提出堆肥中酶学发展的新思路,酶活检测技术的改进以及堆肥化中酶系统影响因素的探讨。     

Recent development of highly sensitive protease assay methods: Signal amplification through enzyme cascades

Proteases are involved in almost all biological processes, and therefore, aberrant activity of many of these enzymes is an important indicator of disease. Various methods have been developed to analyze protease activity, among which, protease assays based on resonance energy transfer are currently used most widely. However, quantitative methods with relatively higher sensitivity are needed, especially for disease diagnosis at early stages. One of the strategies to achieve higher sensitivity is to implement signal amplification of the protease activity. In this review, we briefly summarize the protease assay methods based on resonance energy transfer, and then elaborate the efforts to develop sensitive protease assays through signal amplification by using enzyme cascades.     

Metabolism of oxidized linoleic acid: distribution of activity for the enzymatic oxidation of 13-hydroxyoctadecadienoic acid to 13-oxooctadecadienoic acid in rat tissues

Oxidation products of linoleic acid, including hydroperoxy- and hydroxyoctadecadienoic acids have been shown to possess biological activities in a number of different systems. In this work we describe an enzymatic activity which catalyzes the conversion of 13-hydroxyoctadecadienoic acid to a 2,4-dienone product, 13-oxooctadecadienoic acid. The enzyme activity is widely distributed, with the highest activity in the colon and the liver. The distribution of activity among various tissues is distinct from other dehydrogenases known to use oxygenated unsaturated fatty acids as substrates. This enzyme may play a key role in the metabolism of 13-hydroxyoctadecadienoic acid in epithelial tissues.     

Testing alternative captive breeding strategies with the subsequent release into the wild

We used the housefly (Musca domestica L.) as an experimental model to compare two strategies for the captive breeding of an endangered species: a strategy to minimize inbreeding and balance founder contributions (termed “MAI” for “maximum avoidance of inbreeding”) versus a scheme to select against less fit individuals (disregarding relatedness). By balancing the initial founder contributions, the MAI protocol was analogous to methods for minimizing kinship. In both breeding strategies, the population growth rate was limited to a maximum increase of 50% per generation. Five replicate populations, each starting with five male–female pairs, were subjected to five generations of captive breeding. Six generations of simulated “release into the wild” allowed ad lib breeding with less restrictive population growth potential, in either a benign or stressful environment (i.e., constant or variable temperature). Population size, fecundity, and fertility were assayed throughout the experiment, with juvenile‐to‐adult survival assayed in the second phase of the project. Allozyme assays determined the resultant inbreeding coefficients from the captive breeding schemes. The MAI breeding scheme resulted in significantly lower inbreeding coefficients and higher fitness, with qualitatively reduced extinction potential, most notable in the stressful environment. Spontaneous fitness rebounds suggested that the MAI strategy facilitated some form of purging of inbreeding depression effects. Importantly, the advantages of the MAI strategy were difficult to detect during the captive breeding phase, suggesting that the long‐term advantages of the MAI approach could be underestimated in actual breeding programs. We concur with the common recommendation of maximum avoidance of inbreeding at least for systems with low reproductive potential. Zoo Biol 0:1–18, 2005. © 2005 Wiley‐Liss, Inc.     

Novel Biocatalysts by Identification and Design

Enzymes produced from bacteria and eukaryotic organisms are presently being used for a large variety of different biotechnological applications. The rapidly increasing demand for enzymes which are active towards novel and often non-natural substrates has triggered the development of novel molecular biological methods of enzyme isolation and design. The metagenome approach is a cultivation-independent method which allows the direct cloning and expression of environmental DNA thereby providing access to a wealth of so-far unknown biocatalysts. Additionally, newly identified or existing biocatalysts can be further optimized by different methods of directed evolution. Here, the principle of the metagenome approach is outlined and a strategy is presented for the optimization of a bacterial lipase using a combination of rational design and directed evolution.     

Novel Biocatalysts by Identification and Design

Enzymes produced from bacteria and eukaryotic organisms are presently being used for a large variety of different biotechnological applications. The rapidly increasing demand for enzymes which are active towards novel and often non-natural substrates has triggered the development of novel molecular biological methods of enzyme isolation and design. The metagenome approach is a cultivation-independent method which allows the direct cloning and expression of environmental DNA thereby providing access to a wealth of so-far unknown biocatalysts. Additionally, newly identified or existing biocatalysts can be further optimized by different methods of directed evolution. Here, the principle of the metagenome approach is outlined and a strategy is presented for the optimization of a bacterial lipase using a combination of rational design and directed evolution.     

Evolutionally guided enzyme design

A combination of "rational" and "irrational" strategies for the creation of enzymes with novel properties is proving to be a powerful concept in the field of enzyme engineering. Guided by principles of physical organic chemistry, rational design strategies are used to identify suitable target enzymes and to choose appropriate molecular biological methods for engineering purposes. In contrast, irrational (or random) strategies are centered around the biological paradigm of stochastic molecular evolution. As illustrated in this review, such a hybrid approach is particularly useful for the design of new modular enzymes. (c) 1996 John Wiley & Sons, Inc.     

青脆枝化学成分及其活性研究进展

喜树碱是已发现的唯一专门通过抑制拓扑异构酶Ⅰ发挥细胞毒性的天然植物成分,具有广谱抗肿瘤活性。青脆枝Nothapodytes nimmoniana含有多种喜树碱类化合物及其他活性成分,其中喜树碱、9-甲氧基喜树碱和羟基喜树碱含量最为丰富,是重要的药用植物。文中综述青脆枝主要化学成分、生物活性及其提取方法,并对青脆枝的应用前景作展望。     

Probing the potential of platinum(II) complexes for the inhibition of thiol-dependent enzymatic activity

The synthesis and biological evaluation of platinum(II) amine complexes designed to act as inhibitors of the human cysteine protease cathepsin B, a thiol-dependent enzyme, is described. The complexes, composed of a cathepsin targeting ligand and a platinum(II) moiety with varying degrees of reactivity towards nucleophiles were characterized by physical-analytical methods and a proof of principle was illustrated in a model reaction. In biological tests for inhibitory activity against cathepsin B the presented compounds did not show significant inhibitory activity.     

Ultrafast dynamics-driven biomolecular recognition where fast activities dictate slow events

In general, biological macromolecules require significant dynamical freedom to carry out their different functions, including signal transduction, metabolism, catalysis and gene regulation. Effectors (ligands, DNA and external milieu, etc) are considered to function in a purely dynamical manner by selectively stabilizing a specific dynamical state, thereby regulating biological function. In particular, proteins in presence of these effectors can exist in several dynamical states with distinct binding or enzymatic activity. Here, we have reviewed the efficacy of ultrafast fluorescence spectroscopy to monitor the dynamical flexibility of various proteins in presence of different effectors leading to their biological activity. Recent studies demonstrate the potency of a combined approach involving picosecond-resolved Förster resonance energy transfer, polarisation-gated fluorescence and time-dependent stokes shift for the exploration of ultrafast dynamics in biomolecular recognition of various protein molecules. The allosteric protein–protein recognition following differential protein–DNA interaction is shown to be a consequence of some ultrafast segmental motions at the C-terminal of Gal repressor protein dimer with DNA operator sequences O_E and O_I. Differential ultrafast dynamics at the C-terminal of λ-repressor protein with two different operator DNA sequences for the protein–protein interaction with different strengths is also reviewed. We have also systemically briefed the study on the role of ultrafast dynamics of water molecules on the functionality of enzyme proteins α-chymotrypsin and deoxyribonuclease I. The studies on the essential ultrafast dynamics at the active site of the enzyme α-chymotrypsin by using an anthraniloyl fluorescent extrinsic probe covalently attached to the serine-195 residue for the enzymatic activity at homeothermic condition has also been reviewed. Finally, we have highlighted the evidence that a photoinduced dynamical event dictates the molecular recognition of a photochromic ligand, dihydroindolizine with the serine protease α-chymotrypsin and with a liposome (L-α-phosphatidylcholine).     

Effect of destruction of Escherichia coli cells on the catalytic ability of catalase

The possibility for investigation of catalase (CAT) activity under the conditions of intact E. coli cells was estimated. This approach is based on the possibility of hydrogen peroxide freely cross biological membranes. CAT activity of native cells had a broad maximum between pH values 4.5 and 7.5. Desintegration of cells by freezing--thawing and ultrasonication indicated that there were two CAT activity peaks at pH values about 3.5 and 7.0. Activity of CAT with acid pH-optimum decreased at cell desintegration, but one with neutral pH-optimum was rather stable under this procedure. The enzyme in native conditions was less sensitive to the inhibition by high concentrations of hydrogen peroxide than its counterpart from destroyed cells. Activity of CAT in native and desintegrated cell preparations had different sensitivity to heating and inhibition by reduced glutathione, but it was inhibited by azide similarly. Difference in the CAT properties of native and desintegrated bacteria preparations may be explained by different possibility to penetrate cell membrane by reagents and/or by possible modification of the enzyme properties at destruction of native microenvironment.