|
|
Metabolism adaptation to excercise
Nencini, Ricky ; Šulc, Miroslav (advisor) ; Stiborová, Marie (referee)
Skeletal muscles are remodeled in response to chronic exercise training. Training induces adaptations, which can be reflected by changes in contractile protein functi- ons, mitochondrial amount and functions and also in specific enzyme activity. Due to extensive training muscle proteins and enzymes permeate through sarcoplasm and can be detected at blood plasma. Six male rowers, 19,6 ˘ 2,1 years old (trained), six sedentary men, 20,2 ˘ 1,8 years old (untrained) were included within this report. Except mentioned groups, two individual clinical cases: one male user of androsten, 28 years old, and one male user of erythropoetin (EPO), 32 years old, were also included at this theses. To report any species influence, the groups (five females per group) of two domestic animal species (cow, pig) with different treatment (housed, outdoor) were also included. Blood concentration of erythrocytes, blood plasma concentration of proteins, acti- vity of lactatedehydrogenase (LDH), malatedehydrogenase (MDH) and enoyl-CoA- hydratase were measured as well as quantitative pattern of LDH izoenzymes. The only one difference for man was observed: a significantly higher concentration of plasma protein for untrained group. On the other hand, the outdoors cows had a significantly higher concentration of erythrocytes and activity...
|
|
|
Determination of enzyme activity of cellobiase using capillary electrophoresis
Velvarská, Romana ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
This thesis deals with optimization of capillary electrophoresis conditions for the separation of unreacted substrate and products (cellobiose, glucose) generated by the enzymatic cleavage of cellotriose with enzyme cellobiase. The optimal separation electrolyte was shown to be a solution containing 30 mmol·dm-3 sodium hydroxide with addition of 1.0 mmol·dm-3 sodium phosphate and 1.0 mmol·dm-3 sodium phosphate dibasic (pH = 12.38). With this separation electrolyte calibration curves and repeatability were measured and the limits of detection and quantification were determined. The relative standard deviations of peak areas ranged up to 6.1 % and they did not exceed 0.2 % for the migration time. Limits of detection were determined in the range from 0.020 to 0.026 mmol·dm-3 and limits of quantification from 0.066 mmol·dm-3 to 0.085 mmol·dm-3 . Subsequently the enzyme reaction was monitored in offline setup. Furthermore, the offline procedure was automated and the relative standard deviation of cellodextrines after 30 minutes of hydrolysis was ranging up to 13.0 % of glucose and cellotriose and 3.0 % cellobiose using this automated method, which makes it suitable for semiquantitative and comparative measurements. The last part of the experiment was to carry out enzymatic reaction in online setup....
|
|
|
Digestive proteases of termites
Čermáková, Markéta ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
Digestive proteolysis in termites has not been studied yet. In this diploma thesis, proteolytic enzymes of the digestive tract of two significant pest species Reticulitermes santonensis and Coptotermes formosanus (Rhinotermitidae) were analyzed. Proteases were identified and quantified in gut compartments using a panel of specific substrates and inhibitors. Major proteases were localized in the midgut and were classified as endogenous serine proteases of trypsin type. Minor cysteine proteases were detected in the paunch and were most likely produced by symbionts. The trypsin protease from R. santonensis was chromatographically isolated and its N-terminal sequence was identified. The physiological importance of the digestive trypsin proteases was demonstrated using selective inhibitors tested in vivo with C. formosanus. Based on the analysis of proteases from additional 12 termite species, a general scheme of digestive proteolysis in the order Isoptera was proposed. (In Czech)
|
|
|
Applications of capillary electrophoresis in life sciences
Křížek, Tomáš ; Coufal, Pavel (advisor) ; Pacáková, Věra (referee) ; Tůma, Petr (referee)
Tomáš Křížek: Applications of Capillary Electrophoresis in Life Sciences (Dissertation thesis) ABSTRACT This thesis is focused on the applications of capillary electrophoresis in two important areas of life sciences, proteomics and enzyme assays. In the first part, Pluronic F-127 copolymer was studied as a sieving matrix for proteomic applications of capillary gel electrophoresis. The effect of thermoassociation of Pluronic F-127 on the separation selectivity was investigated and no difference in selectivity of the separation below, inside and above the thermoassociation temperature region was observed. The performance of Pluronic F-127 in capillary gel electrophoresis was compared with dextran as a commonly used sieving matrix. The results showed, that Pluronic F-127 offers superior performance for low-molecular-mass proteins because it provides higher separation power than dextran with significantly lower viscosity of the background electrolyte. The lower viscosity makes the polymer easier to replace after each analysis, which leads to remarkably higher repeatability of the experiments. On the other hand, dextran, due to its higher viscosity, was shown to be more convenient for separations of protein digests, where extremely high separation efficiency is required. The second part focuses on...
|
|
|
Prolyl endopeptidase from the tick Ixodes ricinus
Petrvalská, Olívia ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The ticks are important blood-feeding parasites and vectors of pathogens. The hard tick Ixodes ricinus is the most common species in the Czech Republic that transmits Lyme disease and tick-borne encephalitis. Proteases of the ticks are potential drug targets for the development of new vaccines against these parasites. This work is focused on biochemical analysis of a prolyl endopeptidase from I. ricinus, which has not been studied so far. The prolyl endopeptidase was identified in the extract from the tick gut tissue by the measurement of enzyme activity and by visualization on SDS-PAGE after labelling with activity-based probe. The tick prolyl endopeptidase is probably involved in the proteolytic digestion of host blood proteins based on the highest specific activity found in the gut tissue and its upregulation during the blood-feeding period. Biochemical analysis showed that the enzymatic activity of prolyl endopeptidase is (1) dependent on a free cysteine residue in a close proximity of the active site, (2) optimal at a pH range between 8 and 9, and (3) selectively inhibited by peptide inhibitors Z-Ala-Pro-CMK and Z-Pro-Pro-CHO. Key words: prolyl endopeptidase, proteolysis, enzyme activity, substrate specificity, tick (In Czech)
|
|
|
Characterization of N-demethyllincomycin-methyltransferase.
Poľan, Marek ; Najmanová, Lucie (advisor) ; Petříčková, Kateřina (referee)
Lincomycin is a naturally occurring member of a lincosamide group of antibiotics. The cluster of lincomycin biosynthetic gene was already decribed and the function of many of genes has been clarified. This work, "Characterization of N-demethyllincomycin-methyltransferase", is focused on the study of the final step of lincomycin biosynthetic pathway - the methylation of nitrogen atom from the pyrollo ring of the propylproline unit of the N-demethyllicomycin (NDL). The aim of this work was the characterization of the protein LmbJ, catalysing this final biosynthetic step. All the experiments were provided for the enzyme LmbJ with N-terminal histidine tag, which had been prepared by the heterologous expression in E.coli cells. The pH and temperature optimum was determined as well as the Michaelis constants for both substrates of the reaction - N-demethyllincomycin and S-adenosyl methionine (SAM - a methyl group donor). With the exception of the pH optimum, all specified parameters have markedly differed from the data published for the enzyme isolated from the natural source. Based on the comparison of electron microscopy, blue native gel electrophoresis and gel filtration results, the hypothetical model of the LmbJ quarternary structure was created. Majority of methyltranserases, so far described occure in...
|
|
|
Factors influencing the quality of red wine
Zechmeisterová, Lucie ; Vránová, Dana (referee) ; Omelková, Jiřina (advisor)
In my thesis, I focused on monitoring of microorganisms in the sample of red grape juice and on the interactions between yeasts, bacteria and filamentous fungi. Three different media were applied for the cultivation of microorganisms; firstly for monitoring of total volume of microorganisms, secondly for yeasts and third time for lactic acid bacteria. The indirect method was used for the determination of the amount of viable cells. This method consists in enumerating of visible macroscopic colonies grown up on agar plates. When the cells grew up, the forms of colonies were analyzed visually and the morphology of microorganisms was detected microscopically. The operating time of enzymes in grape juice in the production of red wine was monitored after application of commercial enzymatic preparation. The enzym action in grape juice was observed on the basis of the process of degradation of high – molecular substrate by enzymes through the use of Ubbelohd´s viscometer. The research findings provided a lot of knowledge about the occurance of microflora in the process of production of red wine. The commercial preparations added to grape juice played a significant role.
|
|
|
The study of enzyme activities in compost
Pernicová, Iva ; doc.Mgr.Pavlína Pelcová, Ph.D. (referee) ; Voběrková, Stanislava (advisor)
This thesis deals with the determination of enzyme activities in compost for consideration their changes during the composting. The changes in the activity of particular enzymes serve as the indicators of the activity of microorganisms, which are found in the compost. In the practical part of the work the changes in activity of proteases, cellulases, lipases, dehydrogenases and ureases within 21 days of composting under laboratory conditions were determined. The change in pH was observed as well. The changes in the pH-values and all enzyme activities except ureases present in the first week the same trend associated with the use of available substrates. The changes in the activities of the key enzymes in compost under laboratory conditions were compared with the changes in activities in compost under real conditions, which is the indicator of the different composting process under the given circumstances.
|
|
|
Characterization and stabilization of pancreatin
Wurstová, Agáta ; Němcová, Andrea (referee) ; Obruča, Stanislav (advisor)
This work focuses on a study of enzyme mixture pancreatin, its characterization and subsequent encapsulation into liposomes. As a reference proteins bovine serum albumin and trypsin were used. Characterization of pancreatin consisted of two parts. The first part focuses on optimization of methods for the concentration determination by absorption spectrophotometry using basic methods for identifying proteins (Biuret method, Hartree-Lowry method and Bradford method). Moreover, UV spectrums of the protein were measured. As a method for identification of protein´s molecular weight, SDS-PAGE was used. To identify components of pancreatin, LPLC was employed in two modifications, ion-exchange chromatography and size exclusion chromatography. The second part is dedicated to the characterization of pancreatin as enzyme in terms of pH and temperature optimum for the enzyme activities of protease (pH 9, 8 and 50 °C), amylase (pH 7 and 40 °C) and lipase (pH 7 and 50 °C). The last part of this work aimed at an encapsulation of pancreatin into liposomes and DLS analysis of distribution of particles and their zeta potential. Liposomes did not spontaneously release encapsulated enzyme. To confirm that proteins were successfully entrapped into liposomes, their structure was disrupted by application of phospholipase D. In conclusion, liposomes can be utilized as delivery systems for native enzymes.
|
|
| |
guest ::
