Th.--Sci. nat.--Aix-Marseille 2, 1980.

Thesis (M.Sc. (Physiology)) -- Mahidol University, 1971.

Heavy metal contamination from mining and other industrial operations is becoming an increasing problem with regards to the depleting water resources in South Africa. This study involved the investigation of the use of an algal biomass as a possible alternative to the traditional chemical means of removing these metals. When the toxic effects of metals were investigated, Spirulina was found to have a threshold level of about 30 μM for copper, zinc and lead. Copper and zinc appeared to have a direct effect on the photosynthetic pathway, thereby causing a rapid decline in cell growth. Lead on the other hand seemed to affect surface properties and hence took longer to cause deterioration in growth. Although relatively low concentrations of metal may have a toxic effect on the cyanobacterium, Spirulina may have potential as a precipitation agent. The role of Spirulina in the precipitation of heavy metals appears to be through its ability to maintain a high pH in the surrounding medium, possibly through the enzyme carbonic anhydrase. Subsequent studies therefore focused on the assay and isolation of this enzyme. Two different radiotracer assays, in which carbonic anhydrase converts radiolabelled bicarbonate to carbon dioxide, were investigated, but were found to have several problems. Results were insensitive and could not be reproduced. The standard Wilbur-Anderson method subsequently investigated also proved to be insensitive with a tremendous degree of variability. Although not quantitative, SDS-PAGE proved to be the most reliable method of detection, and was therefore used in subsequent procedures. Chlamydomonas reinhardtii was the subject of initial enzyme isolation studies as these procedures are well documented. Although the published protocols proved unsuccessful, affinity chromatography of a membrane stock solution from Chlamydomonas reinhardtii yielded two relatively pure protein bands. These bands were presumed to represent two subunits of carbonic anhydrase, although Western blot analysis would be required to confirm their identity. Purification of carbonic anhydrase from Spirulina, however, proved unsuccessful and results obtained were very inconclusive. Hence, further analysis of Spirulina is required. The possibility of cloning CA from a genomic library was also considered, but suitable primers could not be designed from the aligned sequences.

No description available.

Reproduction de : Thèse de doctorat : biophysiologie des organismes et des populations : Le Mans : 2007. / Titre provenant de l'écran-titre. Bibliogr. p. 133-146.

Carbonic anhydrases catalyze a central reaction in life – the inter-conversion between carbon dioxide and water. Consequently, there is an increasing interest in research in using carbonic anhydrases for industrial applications such as biofuel production and carbon capture, since current approaches for CO2 capturing are expensive, harsh and energy demanding. The proof of principle for using carbonic anhydrase in these applications for carbon fixation has been validated. However, the current known and tested carbonic anhydrases are not tolerating the harsh industrial conditions. An ideal carbonic anhydrase should display thermo-, salt, and solvent stability and exhibit a decent reactivity. Herein we present the characterization and activity assessment of a halophilic γ-carbonic anhydrase from the Red Sea brine pool Discovery Deep. Protein X-ray structure exhibited the molecular structure and allowed the successful engineering of a small, active mutant library. Stopped-flow measurements gave insights into the activity and evaluated the engineering principles.

Carbonic anhydrases (CAs) are fundamental and ubiquitous enzymes that catalyse the reversible hydration of CO₂ to form HCO₃^- and H⁺ ions. Evidence derived from heterologous expression systems has led to the proposal of a novel role for CA in intracellular pH regulation, where its physical and functional coupling to membrane H⁺ -equivalent transport proteins appears to enhance their activity. It has yet to be established whether such a functional association occurs naturally in wild-type cells. Additional evidence on CA activity in-vitro, has also suggested that certain CA isoforms are regulated by physiological changes of pH, an effect that may then affect their ability to enhance H⁺ -equivalent transport. No information, however, exists on the pH sensitivity of CA in intact cells. Finally, pharmacological inhibition of CA activity has been reported previously for various compounds, in addition to those designed specifically as CA inhibitors. It is possible that some compounds, currently used to inhibit membrane H⁺ transport, may also target CA. The present work has examined functional aspects of CA activity in ventricular myocytes isolated enzymically from rat heart, focusing on the potential role of C A in controlling sarcolemmal Na⁺/H⁺ exchange (NHE) and sarcolemmal Na⁺-HCO₃^- cotransport (NEC). NHE and NEC activity were estimated from the rate of recovery of intracellular pH (pH_i), following an intracellular acid load in myocytes loaded with carboxy-SNARF-1 (a pH-sensitive fluorescent dye, used to measure pH_i)). In other experiments, in-vitro CA activity was assessed from the time-course of pH change after addition of CO₂-saturated water to a buffered solution containing either CA II or a cardiac homogenate. In further experiments, intracellular CA activity was assessed from the rate of CO₂-induced fall of pH_i. Three major results emerged, (i) In intact myocytes, CA activity doubles acid extrusion on sarcolemmal NBC, but has no effect on NHE activity. Facilitation of NBC activity by CA is likely to be mediated by an intracellular CA isoform. (ii) In-vitro and intracellular CA activity displays strong pH-dependence within the physiological pH range, activity declining with a fall of pH. (iii) The NHE inhibitor, cariporide, the bicarbonate transport inhibitors DIDS (4,4'- diisothiocyanatostilbene-2,2'-disulphonic acid) and S0859 (an experimental compound from Sanofi-Aventis), and the aquaporin blocker, pCMBS (p-chloromercuribenzene sulphonate), all showed strong inhibitory activity towards CA in-vitro, but had no effect on intracellular CA activity. Overall, the work provides the first clear demonstration of a functional role of CA activity in H⁺-equivalent transport in a wild-type cell. CA thus represents an important regulatory mechanism of H⁺ -equivalent transport. The pH sensitivity displayed by in-vitro and intracellular CA activity may also have significant functional consequences for pH_i regulation. CA inhibition by various membrane transport inhibitors highlights the need for careful drug and experimental design, to avoid secondary inhibition of CA activity and its side-effects. The present work thus provides insight into the functional roles of CA, plus important new information on the enzyme's pharmacological properties.

Thèse (M.Sc.)--Université Laval, 2008. / Bibliogr. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Written for the Dept. of Chemistry. Title from title page of PDF (viewed 2009/06/11). Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1943. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves xv-xvii).