The principal research goal of this proposal is the identification and characterization of a protease of Plasmodium falciparum which is involved in hemoglobin degradation within the parasite's food vacuole. In preliminary experimetns, I observed that incubation of the protease inhibitor leupeptin with P. falciparum trophozoite inhibited both parasite differentiation and hemoglobin degradation. I also identified and partially purified a 25-28 kD protease from trophozoite infected erythrocytes. The protease had a pH optimum of 6, was inhibited by leupeptin and N- ethylmaleimide, was not inhibited by pepstatin, PMSF, or 1,10 phenanthroline, and was stimulated by cysteine. My hypothesis is that this enzyme is a cysteine protease of parasite origin which is localized to the parasite's food vacuole and is necessary for the initial stages of hemoglobin degradation. To test this hypothesis I will first continue to purify the malarial protease so that it can be characterized with respect to protease class (by inhibitor profile), substrate specificity (with fluorogenic peptide substrates), and its capacity to degrade hemoglobin. Second, I will identify and isolate cDNA clones encoding portions of the protease by screening a lambda gtll P. falciparum trophozoite cDNA library with (1) monospecific antibody prepared against the purified enzyme, (2) a heterologous cDNA probe for cysteine proteases, and (3) an oligonucleotide probe based on the N-terminal or active site amino acid sequence of the protease. Third, selected cDNAs will be sequenced in order to determine the protease's amino acid sequence. Fourth, monospecific antibody to the protease or a fusion peptide will be used to localize the enzyme by immunoelectron microscopy, and the subcellular localization of 3H-leupeptin will be determined by EM autoradiography. These studies will provide information about the mechanism of hemoglobin degradation by malaria parasites. Structural characterization of the protease may facilitate the identification or design of protease inhibitors for the treatment of malaria.
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