Hypoxanthine phosphoribosyltransferase (EC 2.7.2.8) of P.falciparum has been studied for its biological properties and cellular location. The enzyme plays an important role in the parasite's life, and therefore is a putative target for chemotherapy against malaria. Due to the difficulty in obtaining large amounts of the enzyme from the parasite, it was over-expressed in E.coli, first as a fusion protein with E.co/i-j8-galactosidase. This facilitated the one step purification of the protein, using /3-galactosidase substrate affinity chromatography, for making antibodies against the enzyme. The antibodies thus made were used to investigate the cellular location, a prerequisite for a successful drug design against the parasite enzyme. Im-munofluorescent microscopy (IFA) and immunogold electron microscopy revealed that; 1. the enzyme is expressed at all the stages of the parasite's life, 2. the enzyme is concentrated in some vesicular bodies of unknown origin, 3. in the sporozoite, it may be released into the intrapellicular spaces. Subsequently, the enzyme was over-expressed directly in E.coli, as a non-fusion protein and retained its enzymatic activity. This opened the way to study the enzyme for its biochemical properties and structure-activity relationship. The active E.co/i-expressed P.falciparum HPRT compensated a S.typhimurium hpt mutation. The strain, named S.lyphimurium SH4 can now be used to screen large numbers of putative antimalarial drugs which might act against the parasite HPRT. This will be both simple and inexpensive. The cell free extract of induced SH4 was used to study some biochemical properties of the enzyme. Such a study confirmed the finding that unlike any other Plasmodia studied so far, P.falciparum HPRT can use xanthine as its substrate in addition to hypoxanlhine and guanine. However, competitive inhibition studies revealed that hypoxanthine is the most favourable substrate. The possible biological significance of such properties is discussed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:661769 |
| Date | January 1990 |
| Creators | Shahabuddin, Mohammed |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14382 |
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