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Abstract The carbonic anhydrase (CA) gene family contains eleven active members, the basic physiological functions of which are linked to the interconversion of carbon dioxide and bicarbonate (CO2 + H2O ⇔ H+ + HCO3⇔ H2CO3-). They participate in a variety of physiological processes that involve pH regulation, CO2 and HCO3- transport and water and electrolyte balance, and some new functions have also been suggested recently. A novel tumour-associated antigen, MN, containing a CA-domain and named MN/CA IX, has been found to promote cell proliferation when transfected into NIH3T3 cells and has also been shown to be a potential biomarker for neoplasia in the uterine cervix. The present study examines the expression of MN/CA IX in the normal alimentary tract by immunohistochemistry and compares it with the expression of cytoplasmic CA I, CA II, apical plasma membrane associated CA IV and secretory CA VI. The distribution of mitochondrial CA V is examined by immunohistochemistry and Western blotting. The value of MN/CA IX as a potential biomarker of gastrointestinal tumours is assessed in a series of colorectal and hepatobiliary neoplasms. A positive immunoreaction for MN/CA IX was detected in the basolateral plasma membrane of the gastric, intestinal and biliary epithelium, but was confined to the proliferating cryptal enterocytes in the human gut, suggesting a role in cellular proliferation. In colorectal tumours, MN/CA IX immunoreaction was also located in the proliferative zone, indicating that it could be a useful marker of cellular proliferation. In the case of hepatobiliary tumours a positive signal was mainly associated with tumours of biliary epithelial parentage. These results demonstrate that MN/CA IX has a unique expression pattern in the alimentary tract relative to other CAs. Its localization and enzymatic properties suggest that it may have a dual function in the gastrointestinal epithelium. Through its CA activity it could participate in the regulation of carbon dioxide/bicarbonate homeostasis, while its localization to the basolateral surfaces of proliferating cryptal enterocytes suggests that it may serve as a ligand or receptor for one or more other proteins that regulate intercellular communication and/or cell proliferation. MN/CA IX may also serve as a new biomarker of gastrointestinal tumours.

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The effects of altered membrane fatty acid composition on the toxic interactions of heavy metals with Saccharomyces cerevisiae were examined. Saccharomyces cerevisiae was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Incorporation of the exogenous PUF As resulted in them comprising greater than 65% and 40% of the total fatty acids in whole-cell and plasma membrane lipids, and nuclear membrane lipids, respectively. Incorporation of the exogenous PUF As had no discernible adverse effects on cell division. However, inhibition of cell division in the presence of Cd(N03)2 was accentuated by growth in the presence of the di-unsaturated fatty acid linoleate. Furthermore, susceptibility to both Cd2+ - and Cu2+ -induced plasma membrane permeabilisation and whole cell toxicity was markedly accentuated in PUF A-enriched cells, and increased with the degree of fatty acid unsaturation. The increased sensitivity ofPUFA-enriched cells to membrane permeabilisation and whole-cell toxicity was correlated with increased levels of lipid peroxidation in these cells. Cu2+ - and Cd2+_ induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements. Levels of the lipid peroxidation products thiobarbituric acid-reactive substances (TBARS) and conjugated dienes were markedly higher in PUF A-enriched cells, compared with unsupplemented cells, following exposure to cadmium or copper. Thus, lipid peroxidation was demonstrated as a major means of heavy metal toxicity in a microorganism for the first time. In addition, the effects ofPUFA-enrichment on the interactions of heavy metals with cellular nucleic acids were examined. Exposure ofPUFA-enriched cells to the redox-active metals chromium and copper resulted in the uncoupling of DNA synthesis from cell division, leading to sequential S phases. For example, DNA levels of up to 8C were evident in 18:3-enriched cells after only 4.5 h exposure to 100 JJ.M Cu(N03h. Using flow cytometry, the heterogeneity in susceptibility to copper toxicity of exponential phase S. cerevisiae was also examined. Susceptibility towards copper toxicity was demonstrated to be cell cycle stage-dependent, whereby G2/M phase cells were found to be the most susceptible towards copper toxicity. Staining with the oxidantsensitive probe 2',7' -dichlorodihydrofluorescein diacetate (H2DCFDA) revealed that the greater copper sensitivity of G2/M phase cells correlated with elevated endogenous levels of reactive oxygen species in these cells.

Caloxin2A 1 is a novel peptide that inhibits the activity of Plasma Membrane Calcium ATPase (PMCA). PMCA is known to play a role in homeostasis of cytosolic calcium and cell signaling. There are 4 genes (PMCA1-4) that code for the various isoforms of the calcium pump. Based on hydropathy plots, PMCA proteins have 5 putative extracellular domains. We screened combinatorial peptide phage display library for binding to specific extracellular targets. Caloxin 2A1 was obtained as a peptide sequence that would bind to the 2nd putative extracellular domain of PMCA 1 isoform. Caloxin2A1 selectively inhibited the Ca2+-Mg2+ ATPase activity in human erythrocyte leaky ghosts that express mainly PMCA 4 isoform. It produced 50% inhibition of the pump activity at 0.4 mM. Caloxin2A1 inhibited the formation of the acid stable 140 kDa acyl phosphate in the reaction cycle of the calcium pump in the human erythrocyte leaky ghosts. It also produced endothelium dependent relaxation in the pig coronary artery. The random peptide phage display library was screened again with higher stringency to obtain caloxin with higher affinity in order to be cost effective and with greater therapeutic potential. This time, the targets were the 2nd putative extracellular domain of PMCA 1 and 2nd and 3rd putative domains of PMCA 4. The peptides selected for binding to the 2nd putative extracellular domain of PMCA 4 selectively inhibited the Ca2^+-Mg^2+ ATPase activity in human erythrocyte leaky ghosts but with a similar affinity as Caloxin2A1. The peptide selected for binding to the 3rd putative extracellular domain of PMCA 4 was hydrophobic and water insoluble. Substitution of its C-terminus amino acid with lysine residue made the peptide water-soluble and it did inhibit the Ca^2 +-Mg^2 + ATPase with slightly higher affinity. However, the inhibition was due to hydrophobicity of the peptide as the randomized version of the peptide also produced inhibition. We have obtained the first selective inhibitor of PMCA and shown that perturbing extracellular targets can affect protein activity even though most of the functional groups of this protein are in the cytosol. / Thesis / Master of Science (MS)

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