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Photoacoustic Calorimetry Studies of the Earliest Events in Horse Heart Cytochrome-c FoldingWord, Tarah A. 16 September 2015 (has links)
The protein folding problem involves understanding how the tertiary structure of a protein is related to its primary structure. Hence, understanding the thermodynamics associated with the rate-limiting steps for the formation of the earliest events in folding is most crucial to understanding how proteins adopt native secondary and tertiary structures. In order to elucidate the mechanism and pattern of protein folding, an extensively studied protein, Cytochrome-c (Cc), was chosen as a folding system to obtain detailed time-resolved thermodynamic profiles for the earliest events in the protein folding process. Cytochrome-c is an ideal system for understanding the folding process for several reasons. One being that the system can unfold and refold reversibly without the loss of the covalently attached heme group. A number of studies have shown that under denaturing conditions, ferrous Cc (Fe2+Cc) heme group in the presence of carbon monoxide (CO) results in a disruption of the axial heme Methionine-80 (Met80) bond ultimately unfolding the protein. CO-photolysis of this ferrous species results in the formation of a transient unfolded protein that is poised in a non-equilibrium state with the equilibrium state being that of the native folded Fe2+Cc complex. This allows for the refolding reaction of the protein to be photo-initiated and monitor on ns - ms timescales. While CO cannot bind to the ferric form, nitrogen monoxide (NO) photo-release has been developed to photo-trigger ferric Cc (Fe3+Cc) unfolding under denaturing conditions. Photo-dissociation of NO leaves the Fe3+complex in a conformational state that favors unfolding thus allowing the early unfolding events of Fe3+Cc to be probed. Overall the results presented here involve the use of the ligands CO and NO along with photoacoustic calorimetry (PAC) to photo-trigger the folding/unfolding reaction of Cc (and modified Cc). Thus, obtaining enthalpy and molar volume changes directly associated with the initial folding/unfolding events occurring in the reaction pathways of both Fe2+ and Fe3+Cc systems that are most essential to understanding the driving forces involved in forming the tertiary native conformation. The PAC data shows that folding of proteins results from a hierarchy of events that potentially includes the formation of secondary structures, hydrophobic collapse, and/or reorganization of the tertiary complex occurring over ~ns – tens of µs time ranges. In addition, the PAC kinetic fits presented in this work is the first to report Cc folding exhibiting heterogeneous kinetics (in some cases) by utilizing a stretched exponential decay function.
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An investigation of heterologous expression of human steroidogenic cytochromes P450 in yeastsKolar, Norbert Wilhelm 04 1900 (has links)
Dissertation (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study: 1. Compares various heterologous expression systems for high-level expression of
cytochromes P450. Limitations of the existing cytochromes P450 expression
systems are discussed and possibilities to improve the expression yields of
human steroidogenic enzymes, are suggested. In addition the potential
applications of human steroidogenic cytochromes P450 expressed in Pichia
pastoris are illustrated.
2. Describes the cloning and extracellular expression of a recombinant full-length
human cytochrome P450 17a-hydroxylase (p45017a) enzyme in Saccharomyces
cerevisiae. After the optimisation of expression conditions, it was shown that this
system is not suitable for the expression of full-length human P45017a.
3. Describes the cloning and extracellular expression of the full-length human
cytochromes P45017a, aromatase, bs and truncated human cytochrome P45017a
in P. pastoris. The limitations using P. pastoris as an export system for expressed
P450 enzymes were pointed out.
4. Describes the cloning and intracellular expression of the full-length human
cytochrome P45017a in P. pastoris as well as the functional expression of
human P45017a in P. pastoris, showing progesterone conversion to 17ahydroxyprogesterone
and 16a-hydroxyprogesterone in vivo, for the first time.
5. Evaluates developed methods for the preparation of mierosomes from P. pastoris
expressing human P45017a and the spectral characterisation of detergent
solubilised human P45017a.
6. Describes the development of protocols for the purification of human P45017a
from P. pastoris microsomes. / AFRIKAANSE OPSOMMING: Hierdie ondersoek:
1. Vergelyk verskillende heterologiese proteïen uitdrukkings-sisteme vir die
preperatiewe produksie van sitochrome P450. Die tekortkomings van bestaande
sitochroom P450-uitdrukkings-sisteme word bespreek en moontlikhede om die
opbrengs van menslike steroïedogeniese ensieme te verbeter word voorgestel.
Die potensiële aanwendings van menslike steroïedogeniese sitochrome P450, wat
in Pichia pas/oris uitgedruk word, word ook geïllustreer.
2. Beskryf die klonering en ekstrasellulêre uitdrukking van die rekombinante
vollengte menslike sitochroom P45017a-hidroksilase (P45017a) ensiem in
Saccharomyces cerevisiae. Na optimisering van die kondisies vir die uitdrukking
kon aangetoon word dat hierdie sisteem nie geskik is vir die uitdruk en sekresie
van vollengte menslike P45017a nie.
3. Beskryf die klonering en ekstrasellulêre uitdrukking van die vollengte menslike
sitochrome P45017a, aromatase, b5 en verkorte menslike sitochroom P45017a
in P. pas/oris. Die beperkinge van P. pas/oris as 'n uitvoersisteem vir die
uitdrukking van P450 ensieme word bespreek.
4. Besryf die klonering en intrasellulêre ekspressie van die vollengte menslike
sitochroom P450 17a. Die funksionele ekspressie van menslike sitochroom P450
17a in P. pas/oris is vir die eerste keer gekarakteriseer.
5. Evalueer die ontwikkelde metodes vir die voorbereiding van mikrosome van P.
pas/oris wat menslike P45017a uitdruk en karakteriseer die detergent
opgelosbare menslike P45017a t.o.v. spektroskopiese eienskappe.
6. Beskryf die ontwikkelling van protokolle vir die suiwering van die uitgedrukte
menslike P45017a vanuit P. pas/oris mikrosome.
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CREATION OF A BACTERIAL MUTAGENICITY ASSAY HIGHLY SENSITIVE TO DIALKYLNITROSAMINESCooper, Matthew Troy 01 January 2002 (has links)
Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds remains problematic. The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity. I have constructed several mutagenicity tester strains that co-express combinations of full-length human cytochrome P450 2E1, rat cytochrome P450 reductase, and human cytochrome b5 in S. typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt-; and YG7108ER, ogt-, ada-). These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction). Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver. Coexpressing human cytochrome b5 with cytochrome P450 2E1 and cytochrome P450 reductase potentiates the mutagenicity observed with dialkylnitrosamines. These strains were sensitive to nitrosamines with varying alkyl side chains, including dimethylnitrosamine, diethylnitrosamine, dipropylnitrosamine, and dibutylnitrosamine. Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts. These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples, and may serve as useful tools in investigating the molecular properties of proteins in the cytochrome P450 monooxygenase system.
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Steroid derivatives as probes of adrenal cytochrome P-450 structure and function.Stevens, Jeffrey Charles. January 1991 (has links)
Cytochromes P450 metabolize lipophilic substrates to water-soluble products that are readily excreted from the body. The result of the action of hepatic P450 forms is generally detoxification, whereas P450s of the mammalian adrenal gland are responsible for steroid biosynthesis. To better understand the structure and function of two microsomal P450s of the adrenal cortex, P450 17α and P450 C-21, we have designed potential mechanism-based inactivators. These compounds bind reversibly to the enzyme before being metabolized to reactive intermediates that can then bind covalently to the P450, resulting in enzyme inactivation. Our hypothesis is that alteration of the substrate at the known site of enzyme attack may target the P450 for inactivation. Specifically, replacement of the progesterone 21-methyl group with a difluoromethyl group produced a selective inactivator of bovine adrenal P450 C-21. In contrast, the rabbit adrenal progesterone 21-hydroxylase is selectively inactivated by 21,21-dichloroprogesterone. Whether the substitution at the 17-carbon is a dihalomethyl-keto group, an olefinic group, or an acetylenic group, each compound binds reversibly to P450 C-21 as shown by a type I spectral shift. Inactivation of bovine adrenal P450 C-21 by 21,21-difluoroprogesterone is NADPH-dependent, follows pseudo first-order kinetics, and is virtually eliminated by the addition of the physiological substrate progesterone, thereby fulfilling the criteria for mechanism-based inactivation. Metabolism of the dihalo compounds to 21-pregnenoic acid suggests that an acyl halide intermediate is the chemical species responsible for enzyme inactivation. Both 21,21-dichloro and 21,21-difluoroprogesterone inactivate P450 C-21 by the destruction of P450 heme and by protein modification as evidenced by the loss of spectrally detectable P450 relative to the loss of enzyme activity. In contrast, 17β-ethynylprogesterone inactivates P450 C-21 mainly by protein modification and produces an NADPH-dependent, irreversible type I spectrum. Studies to isolate and identify an active site peptide of P450 C-21 were therefore undertaken using proteolytic digestion and high performance liquid chromatography. These 17β-substituted steroids proved useful as probes of P450 structure and function to obtain unique information about P450 oxidative potential, retention of substrate regioselectivity, catalytic efficiency, and the enzyme active site.
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TRANSIENT KINETICS OF ELECTRON TRANSFER REACTIONS OF FLAVODOXIN (CLOSTRIDIUM, PASTEURIANUM).SIMONDSEN, ROYCE PAUL. January 1983 (has links)
Electron transfer reactions between Clostridium pasteurianum flavodoxin semiquinone and various oxidants (horse heart cytochrome c, ferricyanide, and ferric EDTA) have been studied as a function of ionic strength using stopped-flow spectrophotometry. The cytochrome c reaction is complicated by the existence of two cytochrome species which react at different rates and whose relative concentrations are ionic strength dependent. Only the faster of these two reactions is considered here. At low ionic strength, complex formation between cytochrome c and flavodoxin is indicated by a levelling-off of the pseudo-first order rate constant at high cytochrome c concentration. This is not observed for either ferricyanide or ferric EDTA. For cytochrome c, the rate and association constants for complex formation were found to increase with decreasing ionic strength, consistent with negative charges on flavodoxin interacting with the positively charged cytochrome electron transfer site. Both ferricyanide and ferric EDTA are negatively charged oxidants and the rate data respond to ionic strength changes as would be predicted for reactants of the same charge sign. These results demonstrate that electrostatic interactions involving negatively charged groups are important in orienting flavodoxin with respect to oxidants during electron transfer. The effects of structural modifications of the FMN prosthetic group of C. pasteurianum flavodoxin on the kinetics of electron transfer to the oxidized form (from 5-deazariboflavin semiquinone produced by laser flash photolysis) and from the semiquinone form (to horse heart cytochrome c using stopped-flow spectrophotometry) have been investigated. The analogs used were 7,8-dichloroFMN, 8-chloroFMN, 7-chloroFMN and 5,6,7,8-tetrahydroFMN. The ionic strength dependence of cytochrome c reduction was not affected by chlorine substitution, although the specific rate constants for complex formation and decay were appreciably smaller. On the other hand, all of the chlorine analogs had the same rate constant for deazariboflavin semiquinone oxidation. The rate constants for tetrahydroFMN flavodoxin semiquinone reduction of cytochrome c were considerably smaller than those for the native protein. The results for the chlorine analogs indicate the important roles that the polarity of the exposed flavin edge and the substitution of the 8 position play in electron transfer. The data obtained with the tetrahydroFMN analog indicates that the (pi) electron system of the flavin is necessary for rapid electron transfer. These implications are discussed for the electron transfer mechanism of flavodoxin.
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Biochemical mechanisms of apoptosis : ordering of the biochemical events in chemical-induced apoptosisZhuang, Jianguo January 1999 (has links)
No description available.
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Mechanistic studies on CYP17 (17#alpha#-hydroxylase-17,20-lyase)Lee-Robichaud, Peter January 1995 (has links)
No description available.
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The NADPH oxidase in human neutrophil cell-free systemsHope, Elizabeth Lee January 1993 (has links)
No description available.
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Cloning, mutagenesis and expression studies of the pazS gene, encoding pseudoazurin from Paracoccus denitrificansPearson, Isobel V. January 1999 (has links)
No description available.
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The maintenance of cytochrome P4̲5̲0̲-related activity in rat hepatocytes in primary cultureWarren, M. January 1988 (has links)
No description available.
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