Universiẗat, Diss., 2004--München.

A purification of pyruvic carboxylase from dried brewer's yeast is reported. The procedure consists of an initial extraction of one part yeast with three parts of a 5% glycerol-water mixture for two hours at room temperature, an acetone fractionation at -5° between the concentration (v/v) limits of 50% and 85%, an ammonium sulfate fractionation at 0° between the saturation limits of 0.50 and 0.60 and precipitation of inert material with 0.1 M lead acetate. After precipitation of the enzyme with ammonium sulfate, gel filtration on a Sephadex column at a pH of 6.8 is carried out. A mixture of equal amounts of Sephadex types G-100 and G-200 were used. The best purification obtained from the Sephadex column exhibited a single peak in the analytical ultracentrifuge. This represents a more highly purified preparation than the best purification previously reported (Holzer, H. and Beaucamp, K., Biochim. Biophys. Acta, 26, 225 (1961) ). Paper electrophoresis revealed a small amount of a second component. Therefore, homogeneity cannot, as yet be claimed for the preparation. A molecular weight of 250,000 to 300,000, based on the sedimentation coefficient (S_20o, W°) of 8.78 X 10^-13 cm.^2 sec.^-1, which was obtained by means of the ultracentrifuge, is estimated. This estimate is from two to three times previously reported molecular weight values. A conversion of 138.3 micromoles of pyruvate to acetaldehyde per minute per milligram protein has been calculated. A value of 35,000 moles of pyruvate converted to acetaldehyde per mole of pyruvic carboxylase per minute at 30° can be considered an approximate turnover number. Ultrasonic disintegration liberated 1.95 mg. of pyruvic carboxylase per g. of dried brewer's yeast. This yeast can therefore be estimated to contain the enzyme in the amount of 0.2% by weight. Preincubation of pyruvic carboxylase with thiamine pyrophosphate before assaying for activity caused a significant increase in the reaction rate, even with the initial extract. Both thiamine pyrophosphate and Mg(II) were present in the reaction mixture of the assay. It is suggested that dissociation of thiamine pyrophosphate (and probably some Mg(II) also) occurs throughout the purification procedure, which is carried out in the slightly acid pH range. A simple, rapid procedure for the preparation of apocarboxylase by gel filtration with Sephadex G-75 at a pH of 8.0 is also described.

No description available.

Graduation date: 1999

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on hepatic pyruvate carboxylase (PC) gene expression were investigated in C57BL/6J Ah[superscipt b/b male mice. A dose-dependent reduction of PC levels and activity occurred in animals given a single intraperitoneal dose of TCDD in a corn oil carrier. The dose ranged from 1 to 75 ug/kg body weight and the analysis performed 8 days postinjection. At the maximum TCDD level investigated, a 10-fold reduction in PC activity occurred. At doses beyond those required to initiate a reduction in PC, a lactate dehydrogenase isozyme patterns shift is observed. This is accompanied by increases in blood lactic acid levels. Northern blot analysis on RNA extracts from hepatic tissues indicated that at 8 days post TCDD treatment, a dose-dependent reduction of hepatic mRNA levels occurs. The aryl hydrocarbon receptor (AhR) is believed to mediate all responses to TCDD. Liganded AhR and the aryl hydrocarbon receptor nuclear translocator (ARNT) protein form a heterodimeric transcription factor which interacts with dioxin response elements (DREs). These are found in enhancer/promoter regions of many genes that respond transcriptionally to TCDD exposure. Cloning and sequencing a region approximately 1.4 kb upstream of the PC translational start site revealed an untranslated leader sequence of 124 nucleotides starting with adenosine. Primary structural analysis of the upstream region revealed an 1nr element in place of a TATA element. Additional transcription factor elements were identified including: Spl, GCF, UBP-1, GRE, CREB, NF-1, HNF-4, TFII-I and E-boxes; DRE elements were notably lacking. A tandem series of 10 evenly spaced E-boxes, which bind ARNT homodimers, are each juxtaposed to a TFII-I element, possibly forming composite elements. Tertiary structure analysis revealed the positioning of nine composite elements displayed as a trio of phased elements. Transient transfections into Hepa lc1c7 cells, using a luciferase reporter gene under the transcriptional control of the PC upstream region, unlike the animal studies, produced an induction in activity in the presence of 10 nM TCDD. Co-transfections with an ARNT encoding plasmid reduced induction indicating overexpression of ARNT protein partially overrides the TCDD-induced increase in activity. These results in relationship to whole animal experiments are discussed. / Graduation date: 1997

PEPC [PEP(phosphoenolpyruvate) carboxylase] is an essential and tightly controlled enzyme located at the core of plant C-metabolism. It fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and N-assimilation. In plants, a small multigene family encodes several closely related plant-type PEPC (PTPC) isozymes along with a distantly related bacterial-type PEPC (BTPC) isozyme. The PTPCs are well studied ~110-kDa subunits that typically exist as a homotetramer (Class-1 PEPC). By contrast, little is known about the larger ~118-kDa BTPC isozyme except that it occurs in developing castor (Ricinus communis) endosperm in tight association with PTPC subunits as a ~900-kDa hetero-octameric complex (Class-2 PEPC) that is greatly desensitized to metabolic effectors compared to Class-1 PEPC. This thesis elucidates the physiological purpose of the BTPC subunits by examining their structure/function relationship within Class-2 PEPC and identifying mechanisms of post-translational control. Recombinant expression and purification of the castor bean BTPC revealed unusual physical and kinetic properties including a remarkable insensitivity to metabolic effectors and a dependence upon PTPC subunits for structural stability. The first purification of a non-proteolyzed plant Class-2 PEPC complex was performed, and the kinetic analysis determined that the BTPC and PTPC subunits have complimentary catalytic properties. The BTPC subunits’ high Km(PEP) and desensitization to metabolic effectors may function as a metabolic overflow mechanism for sustaining flux from PEP to malate when PTPC subunits become feedback inhibited. An anti-PTPC co-immunopurification strategy was utilized to highly enrich non-proteolyzed BTPC from developing castor endosperm for downstream immunological and mass spectrometric analysis. BTPC was in vivo phosphorylated at multiple novel sites, identified by mass spectrometry as Thr4 or 5, Ser425 and Ser451. Phosphosite-specific antibodies towards Ser425 and Ser451 confirmed the existence of these sites in vivo and comparisons of Ser425 phosphorylation patterns established that the castor BTPC and PTPC phosphorytation sites are regulated independently. Phosphomimetic mutants of Ser425 caused BTPC inhibition by increasing its Km(PEP) and sensitivity to feedback inhibition. These results establish a novel mechanism of PEPC control whose implications within plant carbon metabolism are discussed. / Thesis (Ph.D, Biology) -- Queen's University, 2011-09-04 16:46:22.024

Thesis (Ph.D.) - University of Glasgow, 2008. / Ph.D. thesis submitted to the Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 101-118).

No description available.

The primary focus of this dissertation is the metabolic enzyme pyruvate carboxylase (PC). The structure and function of this fascinating enzyme has been studies and characterized by many laboratories over many decades. This extensive background is reviewed in Chapter 1, with an overview of the biotin-dependent carboxylase family and a particular focus on PC. In this dissertation, we primarily use X-ray crystallography to study PC at a structural level. This dissertation is divided into two overarching sections, with the first section (Chapters 2-5) focusing on the bacterial second messenger cyclic-di-AMP (c-di-AMP). This project was initiated by our collaborators in the laboratory of Josh Woodward at the University of Washington, who performed the first screen to identify c-di-AMP binding proteins in the bacterium Listeria monocytogenes. In Chapters 2 and 3, the regulation of PC by c-di-AMP in L. monocytogenes as well as the bacterium Lactococcus lactis is discussed. Crystal structures of the PC from each of these species in complex with cyclic-di-AMP reveal the binding site and give insights into the molecular mechanisms of this regulation. In Chapters 4 and 5, structural studies of other c-di-AMP binding proteins identified in the screen are discussed. The second section (Chapters 6) focuses on a second class of PC enzymes called the two-subunit PCs, which are found in a subset of Gram-negative bacteria. In Chapter 6, the first crystal structure of a two-subunit PC from the bacterium Methylobacillus flagellatus is determined. In collaboration with the Lars Dietrich laboratory at Columbia University, we investigate the physiological function of the two-subunit PC in the pathogen Pseudomonas aeruginosa. A theme which emerges from these studies is that PC is an incredibly diverse enzyme which has been adapted for the peculiar physiological needs of each organism it inhabits. Because PC is found throughout nature in every kingdom of life, further studies of its unique properties and role in each organism are sure to provide more surprising insights in the years to come.