Abstract:
This thesis describes studies of the singular ion channel adenylyl cyclase (AC) from Plasmodium falciparum. At the beginning the exon determination is described, subsequently a synthetic gene was manufactured for optimized heterologous expression. The isolated AC-domain was generated as an active enzyme in an Sf9-baculovirus expression system and purified by affinity chromatography. The exclusive substrate was ATP, guanylyl cyclase side-activity was not detected. Mn2+ was required as metal-cofactor, Mg2+ did not support activity. From the kinetic data a homodimerisation of the catalyst could be infered. Forskolin, a potent stimulator of most mammalian ACs, had no effect on enzyme activity. The C-terminal tetratricopeptide (TPR) was indispensable for formation of a soluble active enzyme. It was not possible to express the holoenzyme as an active entity in Sf9-cells, fluorescence tagged constructs of the channel domain alone or the holoenzyme were retained intracellularly in CHO-cells. The lack of a measurable ion conductance upon expression of the holoenzyme in Xenopus oocytes also indicated retention problems. In contrast to this, preliminary results from expression experiments in Paramecium indicate some transport of the holoenzyme to cortical regions. Immunolocalisaton in Plasmodium parasites was performed with a purified peptide antibody against the C-terminus of the holoenzyme. In late trophozoites food vacuoles appeared to be labeled. Western blotting of this parasite stage revealed two specific bands, both with a higher than expected molecular weight. Additional AC-isoforms from the parvkingdom of alveolata (Tetrahymena, Cryptosporidium, Toxoplasma and several members of the species Plasmodium) were detected by homology cloning (Tetrahymena libary screen) and homology searches (BLAST), conserved domains are discussed by sequence alignments.