Thèse (M.Sc.) -- Université Laval, 1998. / Bibliogr. Publié aussi en version électronique.

Thesis (M.Sc.)--York University, 2005. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 88-95). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss &rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR11743

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This thesis presents work designed to study the effects of acid secretagogues upon the parietal cell, in order to gain a greater understanding of their modes of action, and interaction and the role of circulating secretagogues in the mediation of parietal cell function, with the aim of increasing the understanding of the pathophysiology of the world-wide problem of peptic ulcer disease.

Adaptation to environmental fluctuations, through sensing and appropriate physiological responses, is crucial to homeostasis. Neuroepithelial cells (NECs) are putative chemoreceptors resembling mammalian Type I (glomus) cells. They have been shown to respond in vitro to changes in O2, CO2, NH3 and pH. Cytosolic carbonic anhydrase (Ca17a) is thought to be involved in CO2 sensing owing to its presence in NECs. A mutant line of zebrafish (Danio rerio) lacking functional Ca17a was generated using CRISPR/Cas9 technology and used to assess the role of Ca17a in initiating the cardiorespiratory responses to elevated CO2 (hypercapnia). Unfortunately, the homozygous knockout mutants (ca17a-/-) did not survive longer than ~12-14 days post fertilization (dpf), restricting experiments to early developmental stages (4-8 dpf). Changes in ventilation (fV) and cardiac (fH) frequency in response to hypercapnia (1% CO2) in wild type (ca17a+/+), heterozygous (ca17a+/-) and ca17a-/- fish were used to investigate Ca17a-dependent CO2 sensing and downstream signalling. Wild type fish exhibited hyperventilation during hypercapnia as indicated by an increase in fV. In the ca17a-/- fish, the hyperventilatory response was attenuated markedly, but only at 8 dpf. Hypercapnic tachycardia was observed for all genotypes and did not appear to be influenced by the absence of Ca17a. Interestingly, ca17a-/- fish exhibited a significantly reduced resting fH¬. This effect of knockout became more pronounced as the fish aged. Anesthesia did not contribute to the decreased fH in the ca17a-/- fish, nor did changes in cardiac adrenergic or cholinergic tone, which were probed using propranolol (-adrenergic receptor blocker) or atropine (muscarinic receptor blocker). The decrease in resting fH was prevented (“rescued”) when ca17a-/- embryos were injected with ca17a mRNA. Collectively, the results of this thesis support a role for Ca17a in promoting hyperventilation during hypercapnia in larval zebrafish and suggest a previously unrecognized role for Ca17a in determining resting heart rate.

Copper is an essential element for all living organisms. However, due to its low redox potential it can be involved in the production of reactive oxygen species; where excess amounts of copper can be exceptionally toxic.1 In humans, malfunctions in copper metabolism are linked to diseases such as Menkes syndrome, Wilson’s disease, prion disease, and Alzheimer’s disease.2 Maintenance of copper homeostasis requires a number of proteins, such as copper transporters and chaperones to deliver copper to the correct protein while limiting free copper in the cell.3 Therefore, understanding the thermodynamics of copper(II) coordination in proteins is critical to our understanding of copper homeostasis. Herein we report human carbonic anhydrase II contains a novel copper binding site with picomolar affinity.4 A full characterization of the structure and thermodynamics associated with the coordination of both Cu atoms into their respective sites is discussed. Techniques including paramagnetic nuclear magnetic resonance spectroscopy (NMR), and x-ray absorption spectroscopy (XAS) techniques provide insight into the high affinity CuA coordination environment. A detailed characterization of this high affinity binding site and related peptide-bound model complexes are included, with the results providing insights into the chemistry and physiological impact of copper binding in human carbonic anhydrase II.

Carbonic anhydrase (CA) is a well-studied, zinc dependent metalloenzyme that catalyzes hydrolysis of carbon dioxide to the bicarbonate ion. In the past, metal binding studies related to CA have continually relied on equilibrium dialysis measurements to ascertain an extremely strong association constant (Ka= approx. 1.2 x 1012) for Zn2+. However, new methodology has allowed us to collect data using isothermal titration calorimetry (ITC), which calls that number and the association constants for many other first row transition metal ions into question. Thermodynamic parameters associated with Zn2+, Cu2+, Ni2+, and Co2+ binding to apoCA are unraveled from a series of complex equilibria associated with the in vitro metal binding event. This in-depth analysis adds clarity to the complex ion chemistry associated with metal ion binding to carbonic anhydrase and validates thermochemical methods that accurately measure association constants and thermodynamic parameters for complex-ion and coordination chemistry observed in vitro. Additionally, the as-isolated and the reconstituted ZnCA and other metalsubstituted CAs were probed using X-ray absorption spectroscopy. Both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses indicate the metal centers in the reconstituted carbonic anhydrases offer new metal binding coordination sites that can be used as models to understand nonheme metal sites in vivo.

No description available.

Carbonic anhydrase (CA) from acetate-grown Methanosarcina thermophila strain TM-1 was purified> 10,OOO-fold (22% recovery) to apparent homogeneity and a specific activity of 4,900 units mg⁻¹.The gene encoding this CA was isolated fronl a partial genomic library on a 12-kb fragment and sequenced. Comparison of the deduced anlino acid sequence with the N-terminaI sequence of the purified protein shows that the gene encodes an additional 34 N-terminal residues with properties characteristic of signal peptides in secretory proteins. The deduced amino acid sequence has no significant identity to any known CAs, but has, among others, 35% sequence identity to the first 197 deduced N-terminal amino acids of a proposed CO₂-concentrating-mechanism protein from <i>Synechococcus</i> sp. strain PCC7942. / Ph. D.

Recently, plastidial carbonic anhydrase (CA, EC 4.2.1.1) cDNA clones encoding functional CA enzymes were isolated from a nonphotosynthetic cotton tissue. The role of CA in photosynthetic tissues have been well characterized, however there is almost no information for the role of CA in nonphotosynthetic tissues. A survey of relative CA transcript abundance and enzyme activity in different cotton organs revealed that there was substantial CA expression in cotyledons of seedlings and embryos, both nonphotosynthetic tissues. To gain insight into the role(s) of CA, I examined CA expression in cotyledons of seedlings during post-germinative growth at different environmental conditions. CA expression in cotyledons of seedlings increased from 18 h to 72 h after germination in the dark. Seedlings exposed to light had about a 2-fold increase in CA activities when compared with seedlings kept in the dark, whereas relative CA transcript levels were essentially the same. Manipulation of external CO2 environments [zero, ambient (350 ppm), or high (1000 ppm)] modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activities. On the other hand, regardless of the external CO2 conditions seedlings exposed to light exhibited increase CA activity, concomitant with Rubisco activity and increased chlorophyll content. Our data revealed that steady-state levels of CA and rbcS transcripts are regulated at the transcriptional level in response to external CO2 conditions, while CA and Rubisco activities are modulated at the post-transcriptional level by light. Thus CA expression in cotyledons during post-germinative growth may be to “prime” cotyledons for the transition at the subcellular level for the transition from plastids to chloroplasts, where it provides CO2 for Rubisco during photosynthesis. Furthermore, CA expression increased during embryo maturation similar to oil accumulation. Specific sulfonamide inhibitors of CA activity significantly reduced the rate of [14C]-acetate incorporation into total lipids in cotton embryos and tobacco leaves and cell suspensions in vivo and in vitro. Similar results were obtained in chloroplasts isolated from leaves of transgenic CA antisense-suppressed tobacco plants (5% of wildtype activity). Collectively, these results support the notion that CA plays several physiological roles in nonphotosynthetic tissues.