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Untersuchungen zur Inaktivierung und Reaktivierung der Coenzym B12-abhaengigen Glycerin-Dehydratasen aus Citrobacter freundii und Clostridium pasteurianum / Studies on the inactivation and reactivation of the coenzyme B12-dependent glycerol dehydratases of Citrobacter freundii and Clostridium pasteurianumSeifert, Corinna 03 May 2001 (has links)
No description available.
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A new perspective on the importance of glycine N-acyltransferase in the detoxification of benzoic acid / Christoffel Petrus Stephanus BadenhorstBadenhorst, Christoffel Petrus Stephanus January 2014 (has links)
Despite being the first biochemical reaction to be discovered, the glycine conjugation pathway remains poorly
characterised. It has generally been assumed that glycine conjugation serves to increase the water solubility of
organic acids, such as benzoic acid and isovaleric acid, in order to facilitate urinary excretion of these compounds.
However, it was recently suggested that the conjugation of glycine to benzoate should be viewed as a
neuroregulatory process that prevents the accumulation of glycine, a neurotransmitter, to toxic levels. The true
importance of glycine conjugation in metabolism is therefore not well understood. However, no genetic defect of
glycine conjugation has ever been reported. This seems to suggest that glycine conjugation is a fundamentally
important metabolic process, whatever its function may be. Therefore, a major objective of this thesis was to
develop a deeper understanding of glycine conjugation and its metabolic significance. A review of the literature on
GLYAT and glycine conjugation suggested that the primary purpose of glycine conjugation is indeed to detoxify
benzoate and other aromatic acids of dietary origin. However, the commonly held assumption, that glycine
conjugation increases the water solubility of aromatic acids in order to facilitate urinary excretion, seems to be
incorrect. A better explanation for the detoxification of benzoate by means of glycine conjugation is based on
hydrophilicity, not water solubility. Because of its lipophilic nature, benzoic acid is capable of passively diffusing
across the mitochondrial inner membrane into the matrix space, where it accumulates due to the pH gradient
over the inner membrane. Although benzoate can be exported from the matrix by organic anion transporters, this
process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is
significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not transport out
of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine
conjugates to re-enter the matrix that is decreased.
The conversion of benzoate to hippurate is a two-step process. First, benzoate is activated by an ATP-dependent
acid:CoA ligase (ACSM2A) to form the more reactive benzoyl-CoA. Second, glycine N-acyltransferase (GLYAT)
catalyses the formation of hippurate and CoASH from benzoyl-CoA and glycine. Another major objective of this thesis was to gain a better understanding of the structure and function of the GLYAT enzyme. While the substrate
selectivity and enzyme kinetics of GLYAT have been investigated to some extent, almost nothing has been
published on the structure, active site, or catalytic mechanism of GLYAT. Furthermore, while interindividual
variation in the rate of glycine conjugation has been reported by several researchers, it is not known if, or how,
genetic variation in the human GLYAT gene contributes to this interindividual variation. To address these issues,
systems for the bacterial expression of recombinant bovine GLYAT and recombinant human GLYAT were
developed. Because no crystal structure of GLYAT has been reported, homology modelling was used to generate a
molecular model of bovine GLYAT. By comparing the molecular model to other acyltransferases for which the
catalytic residues were known, Glu227 of bovine GLYAT was identified as a potential catalytic residue. Site directed
mutagenesis was used to generate an E227Q mutant recombinant bovine GLYAT lacking the proposed catalytic
residue. Characterisation of this mutant suggested that Glu227 was indeed the catalytic residue, and the GLYAT
catalytic mechanism was elucidated. The molecular model was also used to identify Asn131 of bovine GLYAT as a
potential active site residue. Site-directed mutagenesis was used to generate an N131C mutant, which was
sensitive to inhibition by the sulfhydryl reagent DTNB. This suggests that the Asn131 residue of bovine GLYAT may
be situated in the active site of bovine GLYAT, but more work is needed to confirm this result. Finally, site-directed
mutagenesis was used to generate variants of recombinant human GLYAT corresponding to six of the known SNPs
in the human GLYAT gene. Expression and characterisation of the recombinant human GLYAT variants revealed
that the enzyme activity and KM (benzoyl-CoA) parameter of the recombinant human GLYAT were influenced by
SNPs in the human GLYAT gene. This suggests that genetic variation in the human GLYAT gene could partly explain
the interindividual variation in the rate of glycine conjugation observed in humans. Interestingly, the SNPs that
negatively influenced enzyme activity also had low allele frequencies, suggesting that there may be some selective
advantage to having high GLYAT activity. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014
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Efeito da coenzima Q10 no meio de fertilização in vitro de embriões bovinosRAMALHO, Flávia Camila Siqueira Pereira 09 February 2015 (has links)
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Previous issue date: 2015-02-09 / In vitro embryo production (IVP) in cattle became an important commercial tool in genetic improvement programs of the world herd, being widely used for this purpose. However in vitro fertilization (IVF) can cause generation of reactive oxygen species that can affect embryo viability. Coenzyme Q10, an important cofactor in the transport chain of mitochondria has antioxidant function on lipid membrane and it was proved a direct correlation between the presence of Coenzyme Q10 and normal spermatozoa parameters such as density, motility, morphology and volume. The objective of this study was to evaluate the effect of Coenzyme Q10 on sperm function in IVF using conventional or sexed semen; also if the addition of this cofactor can improve embryo IVP in bovine oocytes. In experiment 1 was evaluated the effect of sperm function during incubation periods of sexed and conventional semen samples. In experiment 2, IVF medium was supplemented with 0 (control group), 5 μM, 10 μM, 20 μM of Coenzyme Q10. Bovine oocytes were collected from a slaughterhouse located 20 minutes from the lab. It was observed a negative effect of Coenzyme with significant differences in the rates of cleavage or in the production of blastocyst (p< 0.05) at a concentration of 20 μM when compared with all other groups with either sexed as conventional semen. These results demonstrate that supplementation of the Coenzyme Q10 in the IVF medium, do not alter spermatozoa function. We can also infer that there is a tendency to improve embryo production in the concentration of 5 μM in IVF medium. / A produção de embriões in vitro (PIV) em bovinos tornou-se importante ferramenta comercial nos programas de melhoramento genético do rebanho mundial como técnica de multiplicação, sendo amplamente utilizada para esse fim. Entretanto a Fertilização in vitro (FIV) provoca geração de espécies reativas de oxigênio que podem afetar a viabilidade embrionária. A Coenzima Q10, um cofator de importância na cadeia de transporte das mitocôndrias tem função antioxidante na membrana lipídica e foi verificado uma correlação direta entre a Coenzima e os parâmetros normais de sêmen tais como, densidade, motilidade, morfologia e o volume. O objetivo desse trabalho foi avaliar o efeito da Coenzima Q10 na função espermática em FIV utilizando-se sêmen convencional e sexado; e se a adição desse cofator pode melhorar a produção embrionária in vitro de oócitos bovinos. No Experimento o meio de FIV foi suplementado com 0 (grupo controle), 5 μM, 10 μM, 20 μM da Coenzima Q10. Os Oócitos foram coletados de um abatedouro localizado a 20 minutos do laboratório. Foi observado um efeito deletério da coenzima com diferença significativa nas taxas de clivagem ou na produção de blastocisto (p<0,05) na concentração de 20 μM quando comparado com os demais grupos tanto com sêmen sexado como convencional. Estes resultados demonstram que a suplementação da Coenzima Q10 no meio FIV não altera a função espermática, entretanto tem um efeito deletério a partir da concentração de 20 μM. Podemos ainda inferir que na concentração de 5 μM no meio FIV há uma tendência na melhoria da produção embrionária.
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QPRTase : a wound-induced defence gene in NicotianaSinclair, Steven J. January 2003 (has links)
Abstract not available
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Theoretical Modeling of Enzyme Catalysis with Focus on Radical ChemistryPelmenschikov, Vladimir January 2005 (has links)
<p>Hybrid density functional theory (DFT) B3LYP method is applied to study the four diverse enzyme systems: <i>zinc-containing peptidases</i> (thermolysin and stromelysin),<i> methyl-coenzyme M reductase</i>, <i>ribonucleotide reductases</i> (classes I and III), and <i>superoxide dismutases</i> (Cu,Zn- and Ni-dependent enzymes). Powerfull tools of modern quantum chemistry are used to address the questions of biological pathways at their molecular level, proposing a novel mechanism for methane production by methyl-coenzyme M reductase and providing additional insights into hydrolysis by zinc peptidases, substrate conversion by ribonucleotide reductases, and biological superoxide dismutation. Catalysis by these enzymes, with the exception of zinc peptidases, involves radical chemistry.</p>
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Theoretical Modeling of Enzyme Catalysis with Focus on Radical ChemistryPelmenschikov, Vladimir January 2005 (has links)
Hybrid density functional theory (DFT) B3LYP method is applied to study the four diverse enzyme systems: zinc-containing peptidases (thermolysin and stromelysin), methyl-coenzyme M reductase, ribonucleotide reductases (classes I and III), and superoxide dismutases (Cu,Zn- and Ni-dependent enzymes). Powerfull tools of modern quantum chemistry are used to address the questions of biological pathways at their molecular level, proposing a novel mechanism for methane production by methyl-coenzyme M reductase and providing additional insights into hydrolysis by zinc peptidases, substrate conversion by ribonucleotide reductases, and biological superoxide dismutation. Catalysis by these enzymes, with the exception of zinc peptidases, involves radical chemistry.
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A new perspective on the importance of glycine N-acyltransferase in the detoxification of benzoic acid / Christoffel Petrus Stephanus BadenhorstBadenhorst, Christoffel Petrus Stephanus January 2014 (has links)
Despite being the first biochemical reaction to be discovered, the glycine conjugation pathway remains poorly
characterised. It has generally been assumed that glycine conjugation serves to increase the water solubility of
organic acids, such as benzoic acid and isovaleric acid, in order to facilitate urinary excretion of these compounds.
However, it was recently suggested that the conjugation of glycine to benzoate should be viewed as a
neuroregulatory process that prevents the accumulation of glycine, a neurotransmitter, to toxic levels. The true
importance of glycine conjugation in metabolism is therefore not well understood. However, no genetic defect of
glycine conjugation has ever been reported. This seems to suggest that glycine conjugation is a fundamentally
important metabolic process, whatever its function may be. Therefore, a major objective of this thesis was to
develop a deeper understanding of glycine conjugation and its metabolic significance. A review of the literature on
GLYAT and glycine conjugation suggested that the primary purpose of glycine conjugation is indeed to detoxify
benzoate and other aromatic acids of dietary origin. However, the commonly held assumption, that glycine
conjugation increases the water solubility of aromatic acids in order to facilitate urinary excretion, seems to be
incorrect. A better explanation for the detoxification of benzoate by means of glycine conjugation is based on
hydrophilicity, not water solubility. Because of its lipophilic nature, benzoic acid is capable of passively diffusing
across the mitochondrial inner membrane into the matrix space, where it accumulates due to the pH gradient
over the inner membrane. Although benzoate can be exported from the matrix by organic anion transporters, this
process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is
significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not transport out
of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine
conjugates to re-enter the matrix that is decreased.
The conversion of benzoate to hippurate is a two-step process. First, benzoate is activated by an ATP-dependent
acid:CoA ligase (ACSM2A) to form the more reactive benzoyl-CoA. Second, glycine N-acyltransferase (GLYAT)
catalyses the formation of hippurate and CoASH from benzoyl-CoA and glycine. Another major objective of this thesis was to gain a better understanding of the structure and function of the GLYAT enzyme. While the substrate
selectivity and enzyme kinetics of GLYAT have been investigated to some extent, almost nothing has been
published on the structure, active site, or catalytic mechanism of GLYAT. Furthermore, while interindividual
variation in the rate of glycine conjugation has been reported by several researchers, it is not known if, or how,
genetic variation in the human GLYAT gene contributes to this interindividual variation. To address these issues,
systems for the bacterial expression of recombinant bovine GLYAT and recombinant human GLYAT were
developed. Because no crystal structure of GLYAT has been reported, homology modelling was used to generate a
molecular model of bovine GLYAT. By comparing the molecular model to other acyltransferases for which the
catalytic residues were known, Glu227 of bovine GLYAT was identified as a potential catalytic residue. Site directed
mutagenesis was used to generate an E227Q mutant recombinant bovine GLYAT lacking the proposed catalytic
residue. Characterisation of this mutant suggested that Glu227 was indeed the catalytic residue, and the GLYAT
catalytic mechanism was elucidated. The molecular model was also used to identify Asn131 of bovine GLYAT as a
potential active site residue. Site-directed mutagenesis was used to generate an N131C mutant, which was
sensitive to inhibition by the sulfhydryl reagent DTNB. This suggests that the Asn131 residue of bovine GLYAT may
be situated in the active site of bovine GLYAT, but more work is needed to confirm this result. Finally, site-directed
mutagenesis was used to generate variants of recombinant human GLYAT corresponding to six of the known SNPs
in the human GLYAT gene. Expression and characterisation of the recombinant human GLYAT variants revealed
that the enzyme activity and KM (benzoyl-CoA) parameter of the recombinant human GLYAT were influenced by
SNPs in the human GLYAT gene. This suggests that genetic variation in the human GLYAT gene could partly explain
the interindividual variation in the rate of glycine conjugation observed in humans. Interestingly, the SNPs that
negatively influenced enzyme activity also had low allele frequencies, suggesting that there may be some selective
advantage to having high GLYAT activity. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014
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Chaîne respiratoire et pore de transition de perméabilité mitochondriale dans la cardioprotectionLi, Bo 14 December 2009 (has links) (PDF)
Le pore de transition de perméabilité mitochondriale (PTPm) joue un rôle majeur dans la mort cellulaire et dans la cardioprotection. Notre hypothèse est que le complexe I de la chaîne respiratoire est impliqué dans la régulation de l'ouverture du PTPm. Sur des mitochondries isolées de cœurs des rongeurs, nous avons pu démontrer que le PTPm est désensibilisé par la cyclosporine A, un inhibiteur de la cyclophiline D (CyP-D), et cet effet est largement amplifié en présence de la roténone, un inhibiteur du complexe I. Ces résultats ont été confirmés chez la souris CyP-D déficiente. L'étude de plusieurs types cellulaires a aussi confirmé l'effet de la roténone dans la régulation du PTPm. Ainsi, nous avons pu montrer que le flux d'électrons travers le complexe I est capable de réagir sur un site de régulation du PTPm cardiaque masqué par la CyP-D. De plus, les analogues de l'ubiquinone, élément de la chaîne respiratoire impliqué dans le transfert d'électrons entre les complexes I, II et III, modulent la susceptibilité du PTPm vis-à-vis du Ca2+. Par ailleurs, dans un modèle de cœur isolé du rat, le postconditionnement par le perindoprilate, un inhibiteur de l'enzyme de conversion, diminue la taille de l'infarctus après l'ischémie-reperfusion d'une façon NO-dépendant. L'ensemble de nos résultats ouvre de nouvelles perspectives thérapeutiques dans la cardioprotection et montre l'importance du complexe I et de la CyP-D comme cibles moléculaires incontournables dans la cardioprotection
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Purification of HMG-CoA Reductase and Regulation by Protein-Lipid InteractionsBrent, Lynn G. (Lynn Gran) 12 1900 (has links)
The enzyme 3-Hydroxy-3- Methylglutaryl Coenzyme A Reductase catalyzes the rate limiting step of hepatic cholesterol biosynthesis and is unique among the enzymes in the early part of the pathway in that it is membrane bound. This gives rise to potential regulation of the enzyme through interactions with the endoplasmic reticulum membrane. A purification procedure has been developed which consistently produces enzyme of high specific activity. In order to fully characterize the interactions between HMG-CoA reductase and the lipids in its immediate environment, HMG-CoA reductase was purified to homogeneity and shown to be a protein-lipid complex.
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Optimalizace kultivace karotenogenních kvasinek na směsných odpadních substrátech / Optimization of cultivation of carotenogenic yeasts on mixed waste substratesHolub, Jiří January 2020 (has links)
The master thesis addresses the issue of cultivation of selected strains of carotenogenic yeasts on waste materials of the food industry using a laboratory bioreactor. Carotenogenic yeasts are able to produce highly valuable metabolites during cultivation, which are located predominantly in the lipid part of the cells. Particularly, they are carotenoids, ergosterol, coenzyme Q and fatty acids. The thesis is divided into two main parts, the theoretical part and the practical part. The theoretical part describes individual yeast strains, types of waste materials, produced metabolites and methods of their analysis. The experimental part deals with the processing of waste materials of the food industry, specifically animal fat, whey and spent coffee grounds into the form of substrates usable as nutrition sources for yeast cultivation. Furthermore, cultivations focused on the recovery of the monitored metabolites and their analysis by using HPLC/PDA and GC/FID assemblies were studied as well. The yeast strains Rhodotorula mucilaginosa (CCY 19-4-6), Rhodotorula kratochvilae (CCY 20-2-26), Rhodosporidium toruloides (CCY 062-002-001), Sporidiobolus pararoseus (CCY 19-9-6) a Cystofilobasidium macerans (CCY 10-1-2) were used in this work. As one of the best producing strains Sporidiobolus pararoseus (CCY 19-9-6) was found, which achieved very high productions of carotenoids, coenzyme Q and ergosterol.
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