The enzyme carbonic anhydrase (CA) contributes to multiple physiological processes by catalysing the reversible hydration of carbon dioxide. However, regulation of CA activity in response to homeostatic challenges remains poorly understood. The objectives of this thesis were to investigate whether CA is transcriptionally regulated by cortisol in fish and whether post-translational modification (PTM) of CA occurs in fish. The results of an in vivo reporter assay used to investigate potential transcriptional regulation of zebrafish, Danio rerio, cytoplasmic CA (CAc) were inconsistent, and it remains unclear whether zebrafish CAc is regulated transcriptionally by cortisol. Phosphorylation of rainbow trout, Oncorhynchus mykiss, CAc was predicted from in silico analysis of the putative amino acid sequence and confirmed by Western analysis of phosphoprotein levels following in vitro incubation of CA, purified from trout gill, under conditions designed to potentiate endogenous kinases. Again using in vitro incubations designed to potentiate endogenous kinases and phosphatases, changes to the phosphorylation state of CAc were found to modulate its enzymatic properties. These findings suggest that CA activity may be regulated by signalling pathways that activate cellular protein kinases, and future work should focus on identifying these pathways.

Thesis (Ph. D.)--Missouri University of Science and Technology, 2008. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed April 18, 2008) Includes bibliographical references.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (M.S.)--University of North Carolina at Wilmington, 2005. / Includes bibliographical references (leaves: 31-34)

Thesis (PH.D.)--Michigan State University. Biochemistry and Molecular Biology, 2008. / Title from PDF t.p. (viewed on Aug. 11, 2009) Includes bibliographical references. Also issued in print.

Abstract Secretory carbonic anhydrase isoenzyme VI (CA VI) catalyses the reversible hydration of carbon dioxide (CO2 + H2O ↔ HCO3- + H+). Low concentrations of salivary CA VI are associated with high decayed, missing or filled teeth (DMFT) index scores and a high incidence of acid injury in the upper gastrointestinal tract plus lowered taste and smell perception. Two mechanisms of action for CA VI have been proposed: acid neutralisation and growth factor function. In the present study the distribution and catalytic properties of CA VI have been examined in order to further clarify its mechanisms of action and biological significance. CA VI was found to be present and secreted by the alveolar epithelium of the mammary gland, serous acinar cells of lingual von Ebner’s glands, serous demilune cells of posterior lingual mucous glands and serous cells of submucosal tracheobronchial glands. CA VI was also found in the serous cells in the tracheobronchial mucosal epithelium, taste pore, taste bud, base of the tracheobronchial cilia, bronchiolar Clara cells and enamel pellicle. An immunofluorometric assay showed that the mean concentration of CA VI in colostral milk was eight times higher than that in mature milk (35 mg/l vs. 4.5 mg/l). Stopped-flow spectroscopy measurements revealed that the dehydration activity of CA VI is moderate (maximum kcat = 3.0 × 105 · s-1). The finding that CA VI is a potent catalyst of acid neutralisation emphasizes the possible role of the pellicle bound CA VI in local neutralisation of the acidic metabolic products of dental biofilm. The function of CA VI in von Ebner’s glands’ saliva is likely taste stimuli modification via CA activity although other functions may exist. Its role in milk or respiratory tract mucus remains open, however, as these secretions do not have significant acid predispositions that would need enzymatic catalysis for removal.

Abstract The carbonic anhydrases (CAs) catalyze the reversible hydration of carbon dioxide. In mammals, there are 13 active isoenzymes, which clearly differ in their cell localisation, tissue distributions and functions. CA IX, a unique transmembrane member of the CA gene family, is a tumour-associated protein which is thought to be involved in malignant cell invasion, adhesion and the regulation of cell proliferation. The main focus in the present study was on elucidating the function and expression of CA IX in normal and malignant tissues, especially in the alimentary tract. The functional studies also included CA II, which is regarded as another important CA isoenzyme in the alimentary tract. CA IX immunostaining showed a decrease in the staining intensity of gastric adenomas with increasing dysplasia grade. Well differentiated carcinomas of the intestinal type showed expression comparable to that in the normal mucosa, while expression was decreased in the less differentiated tumours. CA IX deficiency (Car9-/-) genotype and C57/BL6 strain were the main factors which increased the susceptibility of CA IX deficient mice fed on either a normal or high-salt diet to histological abnormalities, including foveolar hyperplasia and glandular atrophy in the gastric body mucosa, while CA II deficiency was associated with only minor histological abnormalities. In a physiological analysis, CA IX played only a minor role in duodenal bicarbonate secretion (DBS), whereas absence of CA II in mice completely abolished the stimulatory effect of E-type prostaglandin 2 (PGE2) on duodenal alkalisation. The results demonstrate that CA IX expression is diminished in most gastric tumours. The variations observed in its expression support the concept that gastric adenomas and carcinomas do not emerge as progressive steps on a single pathway but may instead represent distinct entities with heterogenic genetic backgrounds. In the stomach, CA IX is mainly involved in the regulation of tissue morphogenesis in the body mucosa, while CA II has a major role in maintaining the gastroduodenal acid/base balance.

Acid rmne drainage (AMD) is a major pollution problem througbout the world, adversely affecting both surface and groundwaters. AMD is principally associated with the mining of sulphide ores. The most commonly associated minerals being sulphur, copper, zinc, silver, gold, lead and uranium. As conventional methods for removing heavy metals from wastewater are often prohibitively expensive, the implementation of biological processes for the removal of heavy metals has become a realistic practice. The objectives of this project was firstly to establish the effect of copper, lead and nickel, heavy metals commonly found in AMD waters, on the enzyme carbonic anhydrase, which is an integral part of the carbon concentrating mechanism (CCM) and secondly, to determine the feasibility of using the alkalinity generated by Spindina for the precipitation of heavy metals from solution. Initially, batch flask experiments were performed and it was found that the algae were able to utilise the bicarbonate supplied in the medium, under CO, limiting conditions, through the induction of their CCM, resulting in the generation of carbonate. The effect of the inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), were also investigated in order to determine the importance of carbonic anhydrase (CA) in inorganic carbon accumulation and photosynthesis. Results obtained were consistent with those observed in literature and it was found that at IOOf.LM AZ and EZ, complete inhibition of photosynthesis and carbonic anhydrase occurred, with no oxygen being evolved. The results obtained from the inhibitor experiments substantiate the findings that carbonic anhydrase is an important part of the CCM, and that the dehydration of bicarbonate to carbon dioxide and hydroxide ions, is in fact an enzymatic process regulated by the enzyme carbonic anhydrase and is essential for efficient photosynthesis. The effect of heavy metals on Spirulina was also investigated. Lead, copper and nickel were all found to cause a reduction in the synthesis of chlorophyll a, which resulted in a decrease in photosynthetic efficiency and eventually death of the culture. The morphology of the algae was also severely affected by heavy metals, with degradation and aJmost complete disintegration of the algal filaments occurring. Using the Wilbur-Anderson assay method, carbonic anhydrase activity was found to be lower in the experimental flasks containing heavy metals, than the control flasks, reducing the algae's ability to utilise the bicarbonate in solution for effective photosynthesis. The Wilbur-Anderson assay method did not prove to be a reliable method for measuring changes in enzyme activity as results were found to be erratic. Therefore attempts were made to use an oxygen electrode as an alternative method for determining the effects of various parameters on enzyme activity and photosynthesis, this proved to be more successful. Because of the toxic effects of heavy metals on Spirulina it was decided that the use of the biogenic alkalinity generated by the algae for the precipitation of heavy metals may be successfully employed as an alternative method for bioremediation and metal recovery. Carbonate reacts readily with metals, therefore the carbonate produced by this algal system was used for the precipitation of metals. It was possible to categorise the precipitation reactions observed into three groups, namely those metals which, a) precipitate as hydroxides, b) precipitate as carbonates generated from the dissociation of bicarbonate and c) metals which can only precipitate if there is free carbonate present in solution.

The enzyme carbonic anhydrase (CA) contributes to multiple physiological processes by catalysing the reversible hydration of carbon dioxide. However, regulation of CA activity in response to homeostatic challenges remains poorly understood. The objectives of this thesis were to investigate whether CA is transcriptionally regulated by cortisol in fish and whether post-translational modification (PTM) of CA occurs in fish. The results of an in vivo reporter assay used to investigate potential transcriptional regulation of zebrafish, Danio rerio, cytoplasmic CA (CAc) were inconsistent, and it remains unclear whether zebrafish CAc is regulated transcriptionally by cortisol. Phosphorylation of rainbow trout, Oncorhynchus mykiss, CAc was predicted from in silico analysis of the putative amino acid sequence and confirmed by Western analysis of phosphoprotein levels following in vitro incubation of CA, purified from trout gill, under conditions designed to potentiate endogenous kinases. Again using in vitro incubations designed to potentiate endogenous kinases and phosphatases, changes to the phosphorylation state of CAc were found to modulate its enzymatic properties. These findings suggest that CA activity may be regulated by signalling pathways that activate cellular protein kinases, and future work should focus on identifying these pathways.