Characterization of bacterial L-(-)-tyrosine decarboxylase by isoelectric focusing and gel chromatography.

The purification of L-(-)-tyrosine apodecarboxylase (TAD) (E.C. 4.1.1.25), obtained from extracts of cells of Streptococcus faecalis, has been investigated by means of preparative isoelectric focusing, molecular sieve chromatography and hydrophobic interaction chromatography. Isoelectric focusing demonstrated two separate fractions possessing enzyme activity that had pI values of 4.5 and ca. 3.2. In the chromatographic methods, however, the activity was obtained in a single peak. It was found that hydrophobic interaction chromatography on phenyl-Sepharose was particularly suitable for purification purposes. The enzyme is very firmly bound to octyl-Sepharose CL-4B but retains most of its activity even in the bound state.     

Synthesis, dihydrofolate reductase inhibition, anti-proliferative testing, and saturation transfer difference ¹H-NMR study of some new 2-substituted-4,6-diaminopyrimidine derivatives

A series of 2-substituted-4,6-diaminipyrimidine derivatives were synthesized and evaluated for their dihydrofolate reductase (DHFR) inhibitory activity. Saturation transfer difference (STD) (1)H-NMR experiments were used to probe the binding characteristics of the compounds with human DHFR enzyme. The most potent molecules, 12 and 15, in enzyme assay study showed the best results in STD experiments indicating their intimate interaction with the receptor. The docking studies were followed to explain the structural basis for the observed interaction between the ligands and DHFR. All the compounds were also assayed in vitro for their growth inhibitory activity on MCF-7, HepG2, SKHep1, and Hela tumor cell lines. Compounds 16, 17, and 22 demonstrated the most potent in vitro anti-proliferative activity among the others.     

核酸酶P1活性中心金属离子与氯化钴(Ⅱ)相互作用

核酸酶P1是一种重要的DNA与RNA水解酶。本文利用ICP、VIS、NMR、酶活性测定等分析方法，首次拓展研究了核酸酶P1与CoCl₂的直接相互作用。结果发现：核酸酶P1活性中心Zn(Ⅱ)离子可被外加CoCl₂中的Co(Ⅱ)部分取代，而Co(Ⅱ)进入酶的活性中心，生成相应的酶衍生物“Co(Ⅱ)-P1”，并影响了酶的催化活性。与此同时还获得了Co(Ⅱ)进入核酸酶P1活性中心Zn2位上的酶衍生物 ¹H NMR谱。     

Candida rugosa lipase Lip1–polyethyleneglycol interaction and the relation with its partition in aqueous two-phase systems

The interaction between a lipase from Candida rugosa (Lip1) and polyethyleneglycols of different molecular masses was studied using fluorescence and circular dichroism approaches in order to be applied to the analysis of the enzyme partition mechanism in aqueous two-phase systems of polyethyleneglycol–potassium phosphate. The decrease of the partition coefficients with the polyethyleneglycol molecular mass showed that the enzyme partition is driven by the excluded volume effect and not by the enzyme–polymer interaction. The polymer did not affect the secondary and tertiary structure of the enzyme nor its biological activity. The lipase from Candida rugosa lyophilizate was partitioned in favour of the polyethyleneglycol rich phase; PEG 2000 being the system which showed the better enzyme recovery (78.26%) with a purification factor of 2.3. This method could be applied as a first step to isolate the enzyme from a culture medium with good recovery and without modifying the enzymatic capacity and the molecular structure.     

反胶团相转移法提纯酵母脂肪酶

反胶团相转移法是80年代兴起的一种新型分离技术，它利用表面活性剂分子在有机溶剂中自发形成的反向胶团（反胶团），在一定条件下将水溶性蛋白质分子增溶进反胶团的极性核（水池）中，再创造条件将蛋白质抽提至另一水相，实现蛋白质的相转移，达到分离和提纯蛋白质的目的[1]．反胶团中的蛋白质分子受到周围水分子和表面活性剂极性头的保护，仍保持一定的活性，甚至表现出超活性[2]．由于蛋白质增溶于反胶团与蛋白质所带电荷及反胶团内表面电荷间的静电作用及反胶团的大小有关[3～5]，因而表面活性剂的种类、水溶液的PH值及离子强度等因素…     

The regulatory control of beta-receptor dependent adenylate cyclase.

The characteristics of the beta-receptor in turkey erythrocyte adenylate cyclase were studied using both kinetics of enzyme activation and direct binding measurement of the beta-agonists and antagonists to the beta-receptor. The regulatory ligands Gpp(NH)p and Ca2+ do not have any direct effect on the beta-receptor, but modulate the enzyme activity through the interaction with specific regulatory sites.     

Direct observation of substrate-enzyme complexation by surface forces measurement

The substrate-enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM) and a quartz crystal microbalance (QCM) in order to obtain new insights into the molecular mechanism of the enzyme reaction. This enzyme is composed of two dissociable subunits that exhibit a catalytic activity only when they are associated together in the presence of a cofactor, Mg2+, and a substrate, farnesyl diphosphate (FPP). The QCM measurement revealed that FPP was preferentially bound to subunit II in the presence of Mg2+, while the AFM measurement showed that the adhesive force between the subunits was observed only in the presence of both Mg2+ and FPP. This is the first direct demonstration of the specific interaction involved in the enzyme reaction. The dependence of the Mg2+ concentration on the specific interaction between subunits I and II well agreed with that on the enzyme activity of heptaprenyl diphosphate synthase. This indicated that the observed adhesive forces were indeed involved in the catalytic reaction of this enzyme. On the basis of these results, we discussed the processes involved in the substrate-enzyme complexation. The first, the substrate FPP bound to subunit II using Mg2+, followed by the formation of the subunit I-FPP-Mg2+-subunit II complex. Our study showed a very useful methodology for examining the elemental processes of biological reactions such as an enzyme reaction.     

Study on the interaction of copper-zinc superoxide dismutase with aluminum ions by electrochemical and fluorescent method

The interaction of superoxide dismutase (SOD) with aluminum (Al) ions was investigated by cyclic voltammetry, fluorescence spectroscopy and synchronous fluorescence spectroscopy. The electrochemical activity of the SOD enzyme electrode was inhibited irreversibly by the addition of Al. Meanwhile, the static fluorescence quenching mechanism further revealed the existing of molecular complex of SOD with Al(3+). The association constant was obtained from Lineweaver-Burk plot. The experimental results of voltammetry and fluorescence spectroscopy indicated that the conformation of SOD molecule was altered by the formation of Al-SOD complex. It may influence the activity of SOD enzyme since the optimum action of SOD depends upon a particular configuration of electrostatic charges in the enzyme molecule.     

铜锌超氧化物歧化酶与精氨酸钴（Ⅱ）的相互作用

采用ＶＩＳ,ＩＣＰ及酶活性测定等方法,研究了铜锌超氧化物歧化酶（Ｃｕ２Ｚｎ２ＳＯＤ）与精氨酸钴（Ⅱ）（Ｃｏ（Ａｒｇ）ｎ）的直接相互作用以及外加精氨酸钴（Ⅱ）的量、溶液ｐＨ值对此类相互作用的影响,结果发现：在水溶液中原酶活性中心处的Ｚｎ（Ⅱ）离子可被外加的精氨酸钴（Ⅱ）部分诱导、交换出来,而溶液中外加Ｃｏ（Ａｒｇ）ｎ中的Ｃｏ（Ⅱ）进行酶的活性中心,形成“Ｃｏ－ＳＯＤ”酶衍生物,并相应影响酶的催化活性,与此同时,外加精氨酸钴（Ⅱ）的量,溶液ＰＨ值对此类相互作用的进行有重要影响。     

Structure and catalytic activity of α-chymotrypsin in solutions of gemini surfactants

The regulatory effect of gemini alkylammonium surfactants (GSurf) with the hexamethylene spacer varying in the length of alkyl radicals on the structure and catalytic activity of a-chymotrypsin was studied. A correlation between the activity of a-chymotrypsin and the length of the alkyl radical of GSurf was found. Gemini surfactants enhance the enzyme activity below the critical micelle concentration (CMC) and inhibit that above the CMC. The results of IR spectroscopy and the data on tryptophan fluorescence show that the interaction of GSurf with a-chymotrypsin induces changes in the protein structure differed in intensity. The most probable enzyme complexes with GSurf were characterized by the molecular docking method.     

蛋白-脂质体复合物毛细管电泳筛选单胺氧化酶抑制剂

基于酶与底物间的相互作用,建立了蛋白-脂质体复合物毛细管电泳筛选单胺氧化酶(MAO)抑制剂的新方法.分别将不同浓度的4种N-炔丙基胺类化合物添加至含有蛋白-脂质体复合物的毛细管电泳缓冲液中,抑制MAO活性.考察MAO底物犬尿胺(Kyn)在含有不同浓度N-炔丙基胺类化合物缓冲液中的迁移时间比率(RMTR).结果表明,化合物N-炔丙基-N-甲基-R-2-庚胺(R-2-HMP)和N-炔丙基-R-2-庚胺(R-2-HPA)能够明显抑制MAO活性,导致MAO与Kyn的相互作用减弱,Kyn的RMTR值随着R-2-HMP和R-2-HPA的增加呈现出明显增加的趋势.化合物N,N-二炔丙基-R-2-己胺和N,N-二炔丙基-R-2-辛胺对MAO活性抑制不明显,Kyn的RMTR随这两种浓度增加变化不大.此结果与柱外孵育测定化合物活性结果一致.与传统MAO筛选剂筛选方法相比,本方法快速,成本低,酶消耗量少且不受分离电压等于扰因素的影响.     

Multiplexed enzyme assays in capillary electrophoretic single-use microfluidic devices.

We describe a method of performing multiple enzyme assays in a single reaction vessel. The resolving power of capillary electrophoresis enables several enzyme assays to be analyzed at high speed in microfluidic arrays. Multiplexed measurement can increase throughput significantly without requiring highly dense microfluidic arrays. Enzyme assays in a multiplexed format for selected kinases in this work show essentially identical performance to assays performed individually. This establishes an approach for screening one compound against multiple enzyme targets simultaneously. Another potential application for performing multiplexed enzyme assay is to study protein-protein (especially enzyme-enzyme) interaction by monitoring the enzymatic activity changes.     

A new proposed model of aldose reductase enzyme inhibition on the basis of an artificial intelligence approach: A computer automated structure evaluation (case) study

A large number of inhibitors of aldose reductase enzyme were submitted to the CASE (computer automated structure evaluation) program in order to ascertain the topological features relevant to activity. On the basis of the twenty-six biophores (activating fragments) and one biophobe (inactivating fragment), a new proposed interaction model was suggested for an aldose reductase enzyme with the chemical inhibitors. The critical relationship between enzyme inhibition and the structure of inhibitors is believed to depend on the relative positions of subordinate regions within the inhibitor structure.     

Interaction between organophosphorous hydrolase and paraoxon studied by surface chemistry in situ at air-water interface

Subphase conditions have been optimized to obtain stable organophosphorous hydrolase (OPH-EC 3.1.8.1) as Langmuir films. The Langmuir film was characterized by surface pressure and surface potential-area isotherms and UV-Vis spectroscopy in situ. The interaction of an organophosphorous compound, namely Paraoxon, with the OPH film was investigated for various surface pressures. The stability of the monolayer and the evidence of the enzyme activity at air-water interface support the use of enzyme LB films as biosensor.     

Detection of Hydrogen Peroxide and Glucose Based on Immobilized C60‐Catalase Enzyme

The interaction between fullerene C60 and catalase enzyme was studied with a fullerene C60‐coated piezoelectric (PZ) quartz crystal sensor. The partially irreversible response of the C60‐coated PZ crystal sensor for catalase was observed by the desorption study, which implied that C60 could chemically react with catalase. Thus, immobilized fullerene C60‐catalase enzyme was synthesized and applied in determining hydrogen peroxide in aqueous solutions. An oxygen electrode detector with the immobilized C60‐catalase was also employed to detect oxygen, a product of the hydrolysis of hydrogen peroxide which was catalyzed by the C60‐catalase. The oxygen electrode/C60‐catalase detection system exhibited linear responses to the concentration of hydrogen peroxide and amount of immobilized C60‐catalase enzyme that was used. The effects of pH and temperature on the activity of the immobilized C60‐catalase enzyme were also investigated. Optimum pH at 7.0 and optimum temperature at 25 °C for activity of the insoluble immobilized C60‐catalase enzyme were found. The immobilized C60‐catalase enzyme could be reused with good repeatability of the activity. The lifetime of the immobilized C60‐catalase enzyme was long enough with an activity of 93% after 95 days. The immobilized C60‐catalase enzyme was also applied in determining glucose which was oxidized with glucose oxidase resulting in producing hydrogen peroxide, followed by detecting hydrogen peroxide with the oxygen electrode/C60‐catalase detection system.     

Synthesis and anticholinesterase activity of S-alkynyl diethyl thiophosphates containing hydrophobic or onium groups in the alkylthiol radical

Conclusion A study has been carried out of the anticholinesterase activity of S-alkynyl dialkyl thiophosphates containing hydrophobic or onium groups in the thioalkyl radical. It has been shown that interaction of the acetylenic bond with the corresponding region of the enzyme is not in accordance with the hydrophobic interaction of the thioalkyl radical. The presence of an acetylenic bond in the onium compounds results in a decrease in their inhibitory activity as compared with the analogous uncharged compounds.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 8, pp. 1864–1868, August, 1986.     

Activity of carboxypeptidase a bound to a modified cellulosic matrix

Carboxypeptidase A immobilized on acid chloride of oxidized cellulose showed the following features: (a) as indicated by the linearity of reaction kinetics, the immobilized enzyme action is not diffusion controlled; (b) greater flow rates are achievable with less clogging during continual usage since the enzyme is attached to a porous screen; (c) ease of handling; and (d) no apparent electrostatic interaction with the support material that is uncharged. The immobilized enzyme retained 60% of the original activity. The half-life of free enzyme was only 20 min, whereas for immobilized enzyme it was enhanced up to 2 h 48 min. It could be recovered and repeatedly used.     

Activity of Subtilisin Carlsberg in macromolecular crowding

Enzymatic activity of a proteolytic enzyme Subtilisin Carlsberg (SC) in anionic sodium dodecyl sulfate (SDS) micellar medium has been explored and found to be retarded compared to that in bulk buffer. Circular dichroism (CD) study reveals that SDS, which is a potential protein denaturant, has an insignificant denaturation effect on SC. The structural integrity of the protein offers an opportunity to study the functionality of the enzyme SC in a macromolecular crowding of micelles. Dynamic light scattering (DLS) data indicates no sandwich-like micelle-SC complex formation ruling out the possibility of interaction of the enzyme with the hydrophobic core of the micelle. However, steady state and time resolved emission studies on specific and nonspecific fluorescent probes indicate the proximity effect at the surface of the enzyme due to macromolecular crowding of the micelles. The agreement of retarded enzymatic activity in the micellar crowd with a theoretical model ascribed to the facts that substrates are compartmentalized in the micelles and enzyme interacts with the micelle through stern layer.     

Fluorescence labeled and cross-linked subtilisin: kinetic characteristics and binding to Streptomyces subtilisin inhibitor

In the preceding paper, the preparation of fluorescent cross-linked subtilisin was described. In this paper we present the catalytic and binding properties of the modified enzyme. Kinetic analysis showed that the cross-linked dimeric subtilisin retained both catalytic activity and binding affinity toward synthetic substrates. These kinetic characteristics of the modified enzyme were nearly identical to those of the native enzyme. The modified enzyme also exhibited a specific interaction with Streptomyces subtilisin inhibitor (SSI) with 1 to 1 stoichiometry. The formation of a polymeric complex, which is the expected product with cross-linked subtilisin, was demonstrated.     

Immobilization of invertase on porous membranes made from modified polysulfone

The activity of invertase that was immobilized within asymmetric ultrafiltration membranes was determined as a function of the immobilization mode such as adsorption, ionic interaction and covalent bonding. For these different bonding possibilities adequate membranes were prepared from chemically modified polysulfone fitted out with the corresponding substituents. The amount of enzyme was lowest for adsorption and highest for chemical fixation. In the latter case spacers were useful because substituents adjacent to the polymer backbone caused a relatively large loss of activity which can stem either from chemical reaction or from the hampered development of the appropriate conformational structure. Compared to the activity of the native invertase the fixation yielded a remarkable reduction of activity which was compensated for by the very extended stability of the bonded enzyme.