• MeSH
• enzymy MeSH
• kinetika MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie
• biochemie

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie

• MeSH
• beta blokátory MeSH
• enzymy imobilizované MeSH
• inhibitory enzymů MeSH
• teoretické modely MeSH

Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.

• MeSH
• chromatografie micelární elektrokinetická kapilární metody   MeSH
• elektroforéza kapilární metody   MeSH
• enzymatické testy metody   MeSH
• isoelektrická fokusace metody   MeSH
• izotachoforéza metody   MeSH
• kapilární elektrochromatografie metody   MeSH
• kinetika MeSH
• lidé MeSH
• miniaturizace metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie

Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize β-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of β-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.

• MeSH
• automatizace MeSH
• beta-glukosidasa chemie   MeSH
• katalýza MeSH
• kinetika MeSH
• kukuřice setá enzymologie   MeSH
• rostlinné proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie

Recent investigation of somatic variations of allosterically regulated proteins in cancer genomes suggested that variations in glucokinase (GCK) might play a role in tumorigenesis. We hypothesized that somatic cancer-associated GCK variations include in part those with activating and/or stabilizing effects. We analyzed the enzyme kinetics and thermostability of recombinant proteins possessing the likely activating variations and the variations present in the connecting loop I and provided the first experimental evidence of the effects of somatic cancer-associated GCK variations. Activating and/or stabilizing variations were common among the analyzed cancer-associated variations, which was in strong contrast to their low frequency among germinal variations. The activating and stabilizing variations displayed focal distribution with respect to the tertiary structure, and were present in the surroundings of the heterotropic allosteric activator site, including but not limited to the connecting loop I and in the active site region subject to extensive rearrangements upon glucose binding. Activating somatic cancer-associated variations induced a reduction of GCK's cooperativity and an increase in the affinity to glucose (a decline in the S0.5 values). The hotspot-associated variations, which decreased cooperativity, also increased the half-maximal inhibitory concentrations of the competitive GCK inhibitor, N-acetylglucosamine. Concluded, we have provided the first convincing biochemical evidence establishing GCK as a previously unrecognized enzyme that contributes to the reprogramming of energy metabolism in cancer cells. Activating GCK variations substantially increase affinity of GCK to glucose, disrupt the otherwise characteristic sigmoidal response to glucose and/or prolong the enzyme half-life. This, combined, facilitates glucose phosphorylation, thus supporting glycolysis and associated pathways.

• MeSH
• glukokinasa chemie   genetika   MeSH
• kinetika MeSH
• lidé MeSH
• nádory enzymologie   MeSH
• rekombinantní proteiny chemie   MeSH
• stabilita enzymů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• adenosindifosfát MeSH
• energetický metabolismus MeSH
• enzymy MeSH
• kinetika MeSH
• sarkolema MeSH
• srdce MeSH