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Choline acetyltransferase (ChAc), the enzyme responsible for the synthesis of acetylcholine (ACh), has been extensively purified from chicken brains. Purification procedures included ammonium sulfate fractionation, DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150 column chromatography, and affinity chromatography on agarose-hexane-Coenzyme A column. ChAc activity was measured radiochemically. Due to the instability of the enzyme in the course of purification, the most active fraction obtained after agarose-hexane-Coenzyme A chromatography showed a specific activity of only 0.34 μmoles ACh formed/ min./mg protein which corresponded to a 773 fold purification from homogenate. However, on non-denaturing polyacrylamide gel electrophoresis at pH 8.8, the highly purified ChAc preparation showed two distinct bands, and ChAc activity was recovered by slicing and assaying the gel. ChAc activity corresponded to the position of the faster moving band. The same preparation showed one major band and two minor bands on SDS gel electrophoresis. The estimated MW of chicken brain ChAc by gel filtration and SDS gel electrophoresis was 42,500 daltons and no subunit was observed. Two forms of chicken brain ChAc with different Km values for the substrates were eluted from agarose-hexane-CoA column. The pH optimum was estimated to be between pH 7.6-8.0. NaCl, KC1, Ca²⁺ and EDTA stimulated, while Cu²⁺, N-ethylmaleimide and CoA inhibited the enzyme preparation. The apparent Km values for acetyl-CoA and choline were, studied and were found to be similar to those of other mammalian species. The ChAc preparation also showed species specificity by the Ouchterlony double immuno diffusion test. Effect and mechanisms of salt and EDTA activation of ChAc activity are discussed. / Medicine, Faculty of / Graduate

Human African trypanosomiasis (sleeping sickness) is a devastating disease which is endemic in parts of sub-Saharan Africa. It is caused by the protozoan parasite T. brucei, which are transmitted by the bite of infected tsetse flies. Although the disease is fatal if left untreated, there is a lack of safe, effective and affordable drugs available; therefore new drugs are urgently needed. The aim of the work presented in this thesis is to develop novel trypanocidal compounds. It is divided into two parts to reflect the two distinct strategies employed to achieve this aim. The first part focuses on the inhibition of glycophosphoinositol (GPI) anchor synthesis by inhibiting the Zn2+-dependent enzyme, GlcNAc-PI de-N-acetylase. Trypanosomes have a variable surface glycoprotein (VSG) coat, which allows them to evade the human immune system. The GPI anchor attaches the VSG to the cell membrane; therefore inhibiting GPI synthesis should expose the parasite to the immune system. Initially, large substrate analogues were synthesized. These showed weak inhibition of the enzyme. Zinc-binding fragments were screened, and small molecule inhibitors based on salicylhydroxamic acid were then synthesized. These compounds showed modest inhibition, but the excellent ligand efficiency of salicylhydroxamic acid indicates this may be a promising starting point for further inhibitors. The second part details the P2 strategy. The P2 transporter is a nucleoside transporter unique to T. brucei, which concentrates adenosine. The transporter also binds and selectively concentrates compounds that contain benzamidine and diaminotriazine P2 motifs, which can enhance the potency and selectivity of these compounds. The sleeping sickness drugs melarsoprol and pentamidine contain P2 motifs. Compounds comprising a P2 targeting motif, a linker and a trypanocidal moiety were synthesized. Initially, a diaminotriazine P2 motif was attached to a trypanocidal tetrahydroquinoline (THQ) protein farnesyl transferase (PFT) inhibitor, with limited success. The P2 strategy was also applied to a non-selective, trypanocidal, quinol moiety. The quinol moiety was attached to diaminotriazine and benzamidine P2 motifs, and an increase in selectivity for T. brucei over MRC5 cells was observed.

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