Optimale Bedingungen zur Bestimmung der Transaminasen und der Kreatin-Phosphokinase im Serum und ihre Konsequenzen für die Diagnostik

After discussion of the practical difficulties in establishing optimum conditions for the determination of enzyme activities in serum, improved methods are described for the assay of glutamic oxalacetic transaminase (EC 2.6.1.1), glutamic pyruvic transaminase (EC 2.6.1.2) and creatine phosphokinase (EC 2.7.3.2) in serum and the normal ranges of the 3 enzymes in serum of adults with these methods are presented. For both transaminases the ratios of improved assay to conventional assay methods and data on the effect of temperature and the addition of pyridoxal-5-phosphate are given. The satisfying in-vitro stability of the 3 enzymes is shown. Finally, the consequences of the improved conditions of assay for diagnostic use are discussed. The opportunity of combining an impending variation of methods with their standardization is. stressed.     

Chemical proteomic probes for profiling cytochrome p450 activities and drug interactions in vivo

The cytochrome P450 (P450) superfamily metabolizes many endogenous signaling molecules and drugs. P450 enzymes are regulated by posttranslational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here we describe a chemical proteomic strategy to profile P450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a "clickable" handle provided an activity-based probe that labels multiple P450s both in proteomic extracts and in vivo. This probe was used to record alterations in liver P450 activities triggered by chemical agents, including inducers of P450 expression and direct P450 inhibitors. The chemical proteomic strategy described herein thus offers a versatile method to monitor P450 activities and small-molecule interactions in any biological system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class.     

Enzymatic profiling system in a small-molecule microarray

[reaction: see text] We have developed a microarray-based strategy for detection of three major classes of hydrolytic enzymes on the basis of their catalytic activities. This enables the sensitive detection of proteins not merely by their bindings but rather by their enzymatic activities. This may provide a valuable tool for screening, identification, and characterization of new enzymes in a high-throughput fashion.     

Amino acids and peptides. XXIX. Synthesis of peptide fragments related to active center of eglin c and studies on the relationship between their structure and their inhibitory activity against cathepsin G and alpha-chymotrypsin

H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OMe, corresponding to the sequence 41-49 of eglin c, inhibited human leukocyte cathepsin G and alpha-chymotrypsin. In order to gain further insight into the relationship between the structure and the inhibitory activity against cathepsin G and alpha-chymotrypsin, peptide fragments related to the above nonapeptide were synthesized by a conventional solution method and their inhibitory activities were examined. The smallest peptide which exhibited inhibitory effects on the above enzymes was H-Pro-Val-Thr-Leu-OMe, corresponding to the sequence 42-45 of eglin c.     

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.     

Research activities in the field of enzyme engineering in the framework of the russian state scientific program “novel methods in bioengineering”

The article describes the research activities in the field of enzyme engineering in Russia. The discussion is focused on fundamental studies of biocatalytic processes that expand utilization of enzymes, biocatalytic synthesis of organic products from renewable raw mate rials, enzymes for hydrolysis of cellulose and lignocellulose materials, immobilized cells, new enzyme-based drugs, enzymes in fine organic synthesis, bioanalytic devices, biosensors, and biofuels.     

Enzymic activity of whole cells entrapped in reversed micelles

Studies have been conducted on the enzymic activity of Baker’s yeast and also of Brewer’s yeast entrapped into the reversed micelles formed by cetyl pyridinium chloride (CPC1) in n-hexane. The activities of α-amylase and invertase enzymes in the entrapped cells have been estimated and compared with those in the control experiments where there was no entrapment. The following significant observations have been made: 1. except for invertase, enzymes in Brewer’s yeast, the entrapped yeast cells showed enhanced enzymic activities; 2. when the yeast cells were entrapped inside the reversed micelles along with substrates of the two enzymes, α-amylase, and invertase, the activity of each of these enzymes showed a further enhancement in comparison to that showed in the experiments in which substrates of the individual enzymes alone were entrapped-the phenomenon of synergism; 3. when the yeast cells and the respective substrates were entrapped inside separate reversed micelles and the solutions containing entrapped cells and entrapped substrates were mixed, the activities of the individual enzymes, α-amylase and invertase, showed further enhancement in comparison to the case in which the cells and the substrates were entrapped inside the same reversed micelle (in this case also the phenomenon of synergism was observed); and (4) In the case of experiments in which there was no entrapment, it was observed that the presence of substrates induced more release of enzymes from the yeast cells. These observations on yeast cells, which to the best of our knowledge have not been reported before, should be biotechnologically relevant.     

Mutagenesis approaches to deduce structure-function relationships in terpene synthases

This review highlights mutagenesis studies of terpene synthases, specifically sesquiterpene synthases and oxidosqualene cyclases. Mutagenesis studies of these enzymes have provided mechanistic insights, structure-function relationships for specific enzymatic residues, novel terpene structures and enzymes with novel activities. The literature through 2002 is reviewed and 113 references cited.     

Inhibition of apical membrane enzyme activities and protein synthesis by gentamicin in a kidney epithelial cell line LLC-PK1.

The mechanism of a gentamicin-induced decrease in apical membrane enzyme activities was investigated in LLC-PK1 cells. Increasing activities of apical membrane enzymes (alkaline phosphatase, aminopeptidase, and gamma-glutamyltransferase) were markedly suppressed by gentamicin during growth in culture. On the other hand, a lesser effect was observed when the activities of these enzymes were decreasing or relatively constant. Gentamicin treatment decreased the maximal enzyme activities of alkaline phosphatase and aminopeptidase, indicating that the number of active enzyme molecules in the apical membrane was decreased by gentamicin. [3H]Leucine incorporation in LLC-PK1 cells was inhibited by gentamicin in a dose-dependent manner, followed by a reduction of total protein. In addition, a well-known protein synthesis inhibitor, cycloheximide, also decreased the apical enzyme activities. These results suggest that the inhibition of protein synthesis by gentamicin is a possible cause of the decreased activities of apical membrane enzymes in LLC-PK1 cells. The inhibition of protein synthesis may be related to the nephrotoxicity induced by aminoglycoside antibiotics.     

The Effect of Iron and Copper as an Essential Nutrient on Mitochondrial Electron Transport System and Lipid Peroxidation in Trichoderma harzianum

Iron and copper are essential nutrients for all living organisms as cofactors of many enzymes and play important roles in electron transport system (ETS) enzymes which have heme and iron–sulfur centers. In the present study, ETS enzymes, namely, succinate dehydrogenase (SDH) and cytochrome c oxidase (COX), activities as well as adenine nucleotides and lipid peroxidation (LPO) levels of eukaryotic model Trichoderma harzianum grown in varied concentrations of iron (0–20 mg/l) and copper (0–25 mg/l) mediums have been examined. SDH and COX activities increased up to 10 mg/l of iron. COX and SDH activities increased significantly up to 15 and 1 mg/l of copper, respectively. ATP and ADP levels showed a positive correlation with SDH activity with respect to iron–copper concentrations. The trends of AMP were similar with those of ATP and ADP for iron concentrations, while AMP levels elevated until 5 mg/l of copper. As an indicative marker of membrane damage, LPO levels increased with iron and copper concentration. In conclusion, iron and copper concentrations are of critical importance on activities of the ETS enzymes besides adenine nucleotides and LPO levels by maintenance of this metal homeostasis.     

Antioxidant properties,anti- SARS-CoV-2 study,collagenase and elastase inhibition effects,anti-human lung cancer potential of some phenolic compounds

Lung cancer is one of the main reasons for death worldwide. The natural compounds with anti-lung cancer potential are of main interest and are considered a very promising alternative to replace or raise the efficiency of conventional drugs. Diethylstilbestrol, Enterodiol, Enterolactone, Flavokawain A, Flavokawain B, and Flavokawain C compounds showed excellent to good inhibitory activities against studied these enzymes with IC₅₀ values in ranging between 9.66 ± 1.52 to 121.20 ± 15.87 μM for collagenase and 11.06 ± 1.87 to 27.31 ± 4.673 μM for elastase. Also, these compounds had In vitro anti-lung cancer activities. Comparison of the chemical and biological activities of the studied molecules was made by theoretical calculations. Gaussian sofware program was used for chemical activity. The Maestro molecular docking calculations were made to compare their biochemical activities. Afterwards, ADME/T calculations of the molecules were made.     

A specific gas chromatographic method for the determination of microsomal styrene monooxygenase and styrene epoxide hydratase activities.

A gas chromatographic (GC) method for the determination of the metabolite resulting from the activities of microsomal styrene monooxygenase (epoxide synthetase) and epoxide hydratase using styrene or styrene epoxide as substrates has been developed. The determination of the activities of both enzymes is based on the GC determination of phenylethylene glycol after its esterification with n-butylboronic acid. Kinetic parameters for both enzymes are given.     

纳米通道内酶组装及其催化反应研究进展

研究酶的组装和催化反应不仅有利于探索生命活动的本质,同时对开发酶在工业合成、分析检测、疾病治疗等领域的实际应用价值具有重要的指导意义. 研究发现,酶的有效固定和有序组装是保持酶活性、酶促反应的稳定性和对酶催化过程进行控制的重要途径,而在纳米通道内进行单酶或多酶的有序组装,利用纳米通道的限域效应可有效保持酶的构型进而提高酶催化反应的选择性和催化效率,增强酶级联反应的动力学进程. 本文概述了近年来基于纳米通道的酶反应器在生物传感领域的研究进展,着重描述纳米通道限域空腔内酶的组装方法、酶催化反应及其动力学机制,并展望了基于纳米通道的酶反应器的应用前景.     

胎儿肝、肾上腺微量元素的时序变化与抗氧化酶活性的关系

为探讨胎儿发育过程中组织器官抗氧化酶活性和微量元素的关系，分析了不同月龄胎儿肝、肾上腺中Cu、Zn、Fe、Mn、Se的含量和抗氧化酶活性，并分别与月龄进行相关分析。结果表明，肝、肾上腺中Cu、Zn、Fe、Mn、Se与抗氧化酶活性之间显示有依存关系，为揭示人体胚胎微量元素与酶的关系及其分子生物学机制提供了依据。     

Electrochemical insights into the ethanol tolerance of Saccharomyces cerevisiae

It is expected that intracellular redox activity may closely related to catabolic states of living cells, based on which a mediated electrochemical method has been proposed to measure the ethanol tolerance of the yeast Saccharomyces cerevisiae AS 3800. The couple menadione/ferricyanide was employed as a carrier mediator system, sensing intracellular redox activity. Microelectrode voltammetric method was introduced to assay the ferrocyanide accumulations arising from menadione mediated reduction of ferricyanide by the yeast. The mediated electrochemical study show that the maximal ethanol tolerance limit of S. cerevisiae is about 25% (v/v) ethanol, which is consistent with the result obtained by the conventional fermentative ability measurement. Moreover, the electrochemical method for the first time confirmed that the specific activities of the glycolytic and alcohologenic enzymes within intact living cells remained high by the presence of sublethal ethanol, which was only predicted by in vitro enzymatic assay and cannot be measured by conventional method. The new method can be used as an easy and rapid method to determine the maximal ethanol tolerance of yeast cells.     

Frontiers and opportunities in chemoenzymatic synthesis

Natural product biosynthetic pathways have evolved enzymes with myriad activities that represent an expansive array of chemical transformations for constructing secondary metabolites. Recently, harnessing the biosynthetic potential of these enzymes through chemoenzymatic synthesis has provided a powerful tool that often rivals the most sophisticated methodologies in modern synthetic chemistry and provides new opportunities for accessing chemical diversity. Herein, we describe our research efforts with enzymes from a broad collection of biosynthetic systems, highlighting recent progress in this exciting field.     

Characterization of heterologous and native enzyme activity profiles in metabolically engineered Zymomonas mobilis strains during batch fermentation of glucose and xylose mixtures

Zymomonas mobilis has been metabolically engineered to broaden its substrate utilization range to include d-xylose and l-arabinose. Both genomically integrated and plasmid-bearing Z. mobilis strains that are capable of fermenting the pentose d-xylose have been created by incorporating four genes: two genes encoding xylose utilization metabolic enzymes (xylA/xylB) and two genes encoding pentose phosphate pathway enzymes (talB/tktA). We have characterized the activities of the four newly introduced enzymes for xylose metabolism, along with those of three native glycolytic enzymes, in two different xylose-fermenting Z. mobilis strains. These strains were grown on glucose-xylose mixtures in computer-controlled fermentors. Samples were collected and analyzed to determine extracellular metabolite concentrations as well as the activities of several intracellular enzymes in the xylose and glucose uptake and catabolism pathways. These measurements provide new insights on the possible bottlenecks in the engineered metabolic pathways and suggest methods for further improving the efficiency of xylose fermentation.     

Preorganization boosts the artificial esterase activity of a self-assembling peptide

The creation of artificial enzymes to mimic natural enzymes remains a great challenge owing to the complexity of the structural arrangement of the essential amino acids in catalytic centers. In this study, we used the phosphatase-based enzyme-instructed self-assembly(EISA) to supervise artificial esterases' final structures and catalytic activities. We reported that peptide precursors containing different phosphorylation sites could preorganize into alternated nanostructures and undergo dephosphorylation in the presence of alkaline phosphatase(ALP) with variation in kinetic and thermodynamic profiles. Although identical self-assembly compositions were formed after dephosphorylation, precursors with more enhanced preorganized states tended to better promote ALP dephosphorylation, facilitate further self-assembly, and strengthen the catalytic activities of the final assemblies. We envisioned that our strategy would be useful for further construction and manipulation of various artificial enzymes with superior catalytic activities.     

Phospholipase A2-Catalyzed Hydrolysis of 1,2-DimyristoyI-sn-Glycero-3-Phosphocholine in Organic Solvents

The phospholipase A2-catalyzed hydrolysis of phosphatidylcholine in organic solvents is described. The effects of various sources of enzymes are discussed; among five phospholipases A2, bee venom enzyme has the greatest activity, Naja naja venom and Naja mocambique enzymes have moderate activities, and pancreatic enzymes have the least activities. The effects of adducts such as alcohols, ether, and bovine serum albumin in chloroform on this catalysis are discussed; adducts, such as ether or alcohol, in small proportions increased the hydrolysis rate, but in large proportions inhibited hydrolysis. Bovine serum albumin increased slightly the phospholipase A2 catalysis rate. From the effect of temperature on this catalysis in chloroform, the substrate conformation at the α-memylcne region of acyl chains is a major factor for activation of phospholipases A2, confirming our previous conclusion that the substrate conformation is an important factor in the activation of phospholipase A2.     

Luminescent Strategies for Label‐Free G‐Quadruplex‐Based Enzyme Activity Sensing

By catalyzing highly specific and tightly controlled chemical reactions, enzymes are essential to maintaining normal cellular physiology. However, aberrant enzymatic activity can be linked to the pathogenesis of various diseases. Therefore, the unusual activity of particular enzymes can represent testable biomarkers for the diagnosis or screening of certain diseases. In recent years, G‐quadruplex‐based platforms have attracted wide attention for the monitoring of enzymatic activities. In this Personal Account, we discuss our group's works on the development of G‐quadruplex‐based sensing system for enzyme activities by using mainly iridium(III) complexes as luminescent label‐free probes. These studies showcase the versatility of the G‐quadruplex for developing assays for a variety of different enzymes.